Androgen receptor (AR) expression is emerging as a prognostic biomarker in breast carcinoma (BCa). The study aimed to determine the prevalence of AR expression by immunohistochemical analysis among a cohort of Sri Lankan women with early BCa and to evaluate its association with clinicopathological features including immunohistochemical molecular subtype and early survival.We studied the clinical and pathological features and immunohistochemical profile of 141 women undergoing primary surgery for early BCa, followed by standard adjuvant therapy. AR status was assessed by immunohistochemistry in all cases. Overall survival (OS) and disease-free survival (DFS) was determined. The relationship between AR expression and clinical and pathological parameters and immunohistochemical molecular subtype was analyzed using Student T test and chi-square tests. Cox regression analysis was used to analyze the prognostic impact of AR expression.p > 0.05). Ductal carcinoma in situ(p = 0.003), oestrogen receptor (ER) (p = 0.001) and progesterone receptor (PR) (p = 0.001) positivity and luminal IHC molecular subtype(p = 0.016) were significantly associated with AR-positive status. AR-negative status was significantly associated with tumour necrosis > 50%(p = 0.031), moderate to extensive lymphocytic infiltrate at the tumour margin(p = 0.025) and basal triple negative breast carcinoma(p = 0.016).AR expression was seen in 40.8%(95%CI 33.10–49.07%) of the BCa study cohort. None of the clinical data studied showed a significant association with the AR status(p = 0.047), specifically in postmenopausal women (p = 0.030). In ER-negative tumours, AR positivity was associated with worse DFS (p = 0.036). On multivariate analysis, TNM stage and ER/AR status were predictive of survival. ER-positive/AR-positive (ER+/AR+) tumours demonstrated better OS than ER-positive/AR-negative (ER+/AR-) tumours(p = 0.015). ER-negative/AR-positive (ER−/AR+) tumours (p = 0.014) had a worse DFS than ER-negative/AR-negative (ER−/AR-) tumours.The mean duration of patient follow-up was 46.70(95% CI 46.495–46.905) months (3.89 years). On univariate analysis, AR-positivity was associated with better OS among ER-positive tumours(AR prevalence obtained was low. AR positivity was associated with positivity for ER and PR. On multivariate analysis, apart from TNM stage only ER/AR status were predictive of OS and DFS, with concordant expression of ER/AR demonstrating a better, early survival. Breast carcinoma (BCa) is the commonest malignancy among women in Sri Lanka and worldwide. In 2010, 27% of women diagnosed with cancer in Sri Lanka had BCa with a lifetime risk of 2.5% in the population [BCa can be categorized into several molecular subtypes based on the surrogate immunohistochemical marker expression. Oestrogen receptors (ER), progesterone receptors (PR), HER2, Ki67 and basal markers  are analyzed by immunohistochemistry (IHC). Based on these results the tumours are categorized into Luminal A, Luminal B, HER2 enriched, triple negative and basal like subtypes [AR gene is located in the X chromosome at position Xq11–12. It is known to produce its effects through 3 major pathways which contribute to AR function in BCa: genomic signaling, non-genomic signaling and signaling via cross talk with growth factors and cytokines [Expression of androgen receptors (AR) in BCa has generated considerable interest in the recent past as a prognostic biomarker. The human androgen receptor ytokines . Differeytokines . This exytokines –10. It iytokines , 11.Systematic reviews and meta-analysis have shown AR positivity to be associated with favorable clinical outcomes , 13. HowData regarding the prevalence and the impact of AR expression in BCa and survival are mostly from the west and none from Sri Lanka. Only a few studies have been done in Asia; in Iran, India, Thailand and China –17.We aimed to determine the prevalence of androgen receptor expression by immunohistochemical analysis among a cohort of Sri Lankan women with early breast carcinoma, and to evaluate its association with clinical and pathological features including immunohistochemical molecular subtype and early survival.Ethical approval for the study was obtained from the Ethical Review Committee of the Faculty of Medicine, University of Colombo (EC-17-088). The study population comprised all women undergoing surgery for invasive breast carcinoma at the National Hospital of Sri Lanka from June 2012 to December 2014. Inclusion criteria were women undergoing mastectomy or wide-local excision for an invasive breast carcinoma of early stage. Early stage breast carcinoma was defined as cancer confined to the breast with or without regional lymph node involvement, and absent distant metastasis . PatientThe study population comprised 301 patients. One hundred and forty one cases were selected from 179 cases fulfilling the inclusion and exclusion criteria.The clinical parameters evaluated included ethnicity, age, body mass index, parity, history of breast feeding, menopausal status, family history of BCa and use of oral contraceptives and hormone therapy. The clinical data was collected by the investigators through patient interviews. The following information was retrieved from the pathology reports: type of surgery, tumour size, margin involvement and lymph node status. Slides of cases that had been reported as having a positive margin were reviewed to confirm the presence of margin involvement. Only cases showing the presence of tumour on the ink were classified as having a positive margin. The haematoxylin and eosin stained slides of the tumour were evaluated by the investigators for the following parameters: histological type, histological grade, necrosis, ductal carcinoma in situ (DCIS), lymphovascular invasion, tumour margins, degree of lymphoid infiltrate at the tumour-host interface and within the tumour, degree of desmoplasia/hyalinization at the tumour-host interface and within the tumour, vascular density at the tumour-host interface, cell margins and calcification. Histological subtyping was done according to the 4th World Health Organization Classification of Tumours of the Breast . The ElsImmunohistochemical staining for ER, PR, HER2 and AR were performed by investigators on representative paraffin embedded tissue in all selected cases. CK5/6, EGFR, 34ßE12 and CK14 were evaluated in the cases found to be triple negative . Relevant negative and positive control slides were included with each batch of slides stained with immunohistochemical markers.The antibodies, dilution and the method used and interpretation of IHC stained sections are shown in Table The tumours were broadly categorized into luminal, HER2 and triple negative breast carcinoma (TNBC) groups based on ER, PR and HER2 status. A proportion of the luminal breast carcinomas could have been further subclassified as luminal B, based on positivity for HER2 . HoweverAll patients had been treated and managed according to standard protocols  with 119p value of < 0.05 was taken as significant. Univariate Cox regression analysis was used to analyze the prognostic impact (OS and DFS) of AR expression on the entire sample as a whole and also when stratified according to histological grade, ER status and menopausal status. Multivariate Cox regression analysis was performed to analyse factors affecting OS and DFS. Statistical Package for the Social Sciences SPSS  was used for data analysis.Proportions and 95% confidence intervals were calculated to estimate prevalence. The relationship between clinicopathological parameters, IHC molecular subtype and the AR expression was analyzed using Student T test and chi-square tests. A 2–95% CI 25.7–27.7 kg/m2). Eleven (7.8%) had a family history of breast carcinoma.The characteristics of the study sample are summarized in Table Positive AR expression Fig.  was seenp > 0.05) (Table p = 0.003) and positivity for ER (p = 0.001) and PR (p = 0.001) were associated with AR positivity. AR-negativity was significantly associated with necrosis > 50% (p = 0.031) and a moderate to extensive lymphocytic infiltrate at the tumour margin (p = 0.025)  Table . Of the 5) Table .Table 3p = 0.016) and AR-positivity was associated with the luminal subtype. Basal triple negative breast carcinomas showed an association with AR negativity.AR was expressed across all IHC molecular subtypes, with 50.6% of luminal, 29.4% of HER2 and 21.6% of TNBC subtypes showing AR-positivity. The association of AR status and the IHC molecular subtype is shown in Table The mean duration of follow up was 46.70 (95% CI 46.495–46.905) months (3.89 years). One hundred and seven patients were alive and free of disease. There were 34 cases of early relapse. Twenty five patients died during this period. Of the nine that were living with disease, six had developed metastases and three had local recurrences.Positive margin involvement was seen in ten cases (7.1%), of which eight were mastectomies and two were wide local excisions in which no subsequent mastectomy was performed. Three of the patients who died, had positive margin involvement at the time of primary surgery. All three had undergone mastectomy. The remaining seven patients were disease free at follow up.p = 0.351) and DFS (p = 0.840). Post-hoc calculations of the power of the study showed that the power of the study to detect a difference in DFS and OS between the two groups (AR+ vs AR-) with an alpha value of 0.05 was 4.6 and 11.2% respectively.Androgen receptor status showed no significant association with OS (p = 0.030)  tumours showed significantly better OS than postmenopausal women with ER-positive, AR-negative (ER+/AR-) tumours (30) Fig. . DFS didp = 0.035) and DFS (p = 0.022) of this subgroup was significantly different from the grade 1/2/AR-positive tumours. Additionally among grade 1/2 tumours, AR-positive tumours showed a significantly better OS (p = 0.042) than AR-negative tumours  and ER-negative, AR-negative (ER−/AR-) tumours showed significantly better DFS than ER-negative, AR-positive (ER−/AR+) tumours (p = 0.036)  or disease-free survival (p = 0.826) when the tumours were categorized by molecular subgroup.The AR status of the tumour had no impact on either overall (p = 0.015) and ER−/AR+ tumours had significantly worse DFS than ER−/AR- tumours (p = 0.014) . However, many studies including a metanalysis of 22 studies involving 10,004 women  found thThe prognostic effect of AR expression may vary according to the age, menopausal status, and hormonal status of the tumour. Although the mean age was 55.46 years and most participants were in the post-menopausal age group, their age ranged widely from 29 to 77 years. The cases included both ER positive and ER negative cancers. This clinical heterogeneity needs to be taken into consideration, when interpreting the results of the group as a whole. Subsequent subgroup analysis was done according to menopausal status, tumour grade and ER status.Postmenopausal women with ER+ tumours comprised a subgroup with limited clinical heterogeneity. We found that AR-positivity was associated with a better OS among postmenopausal women with ER-positive tumors. This same association was not seen among premenopausal women with ER-positive tumours. Androgen is thought to act as an anti-oestrogen in premenopausal women, but as an oestrogen agonist in postmenopausal women. Therefore it is possible that the prognostic implications of AR status may be dependent on the menopausal status. However the smaller numbers of women in the premenopausal group  is a limitation that must be considered when interpreting the findings. Hu et al. who studied postmenopausal women also found AR expression to be associated with a more favorable prognosis in ER-positive tumors . HoweverAmong grade1/2 tumours, AR positive tumours showed a better prognosis than AR negative tumours. However this may be due to the fact that low grade tumours were more likely to be ER positive. ER+/AR+ tumours showed better OS than ER+/AR- tumours and ER−/AR+ tumours showed worse DFS than ER−/AR- tumours on both univariate and multivariate analysis. Other studies too have found that breast cancers with discordant ER/AR expressions (ER+/AR- or ER−/AR+) demonstrated a worse prognosis in comparison to breast cancers with concordant expression (ER + AR+ or ER−/AR -) in multivariable models . As discThe implications of AR expression in HER2 amplified BCa and TNBC is uncertain. Some studies show no effect with AR-positivity , 34, somNone of the patients studied had been treated with AR agonists or antagonists. Therefore the predictive value of AR status on treatment was not studied. The small sample size in the current study is a limitation. The power of the study is low due to the small sample size. The effects being investigated are also small. These factors increase the likelihood of both false negative and false positive results. This has to be taken into consideration in the interpretation of the results. Additionally the effect of adjuvant therapy also needs to be considered when assessing the impact on prognosis.However despite these limitations, the finding that AR positivity was associated with a better prognosis in ER positive tumours, especially in postmenopausal women is in keeping with other studies. AR positivity was associated with poorer outcomes in ER negative tumours. These findings are supportive of the use of androgen agonists in ER+/AR+ tumours and AR antagonists in ER−/AR+ tumours , 11. LarThe histological and IHC molecular tumour characteristics of this BCa patient cohort from Sri Lanka, were mostly similar to the findings from other regions of the world including Europe and United States of America , 36, 37.Currently, where the androgen receptor (AR) is emerging as a new biomarker and a potential new therapeutic target in the treatment of BCa , the stu


Background: Although cognitive decline (CD) is associated with increased post-operative morbidity and mortality, routinely screening patients remains difficult. The main objective of this prospective study is to use the EEG response to a Propofol-based general anesthesia (GA) to reveal CD.Methods: 42 patients with collected EEG and Propofol target concentration infusion (TCI) during GA had a preoperative cognitive assessment using MoCA. We evaluated the performance of three variables to detect CD (MoCA < 25 points): age, Propofol requirement to induce unconsciousness (TCI at SEF95: 8–13 Hz) and the frontal alpha band power (AP at SEF95: 8–13 Hz).Results: The 17 patients (40%) with CD were significantly older (p < 0.001), had lower TCI (p < 0.001), and AP (p < 0.001). We found using logistic models that TCI and AP were the best set of variables associated with CD (AUC: 0.89) and performed better than age (p < 0.05). Propofol TCI had a greater impact on CD probability compared to AP, although both were complementary in detecting CD.Conclusion: TCI and AP contribute additively to reveal patient with preoperative cognitive decline. Further research on post-operative cognitive trajectory are necessary to confirm the interest of intra operative variables in addition or as a substitute to cognitive evaluation. Cognitive Decline (CD) is characterized by an impairment or a gradual weakening in cognitive functions such as memory, language, or judgment  and therefore the importance of research on specific measures of precocious and accelerated aging. In the past decade, the advent of machine learning in neuroimaging and biomedical research has led to burgeoning interest in proxy measures for individual aging. One such measure is the brain age, which is defined as the difference between the passport age and the age algorithmically predicted from population-level brain images  . We propose an intraoperative model assessing indirectly cognitive decline under general anesthesia that we termed HELP: Between November 2018 and May 2019, patients eligible for interventional neuroradiology or orthopedic surgery performed under general anesthesia were selected to participate in this prospective, observational, mono-centric study. Inclusion criteria were an elective surgery, a Propofol-based Total IntraVenous Anesthesia (TIVA), a French-speaking and adult (>18yo.) patient. Pregnant women, patients sedated under mechanical ventilation at the time of their management or with a BMI > 35 kg/m2 were excluded .Frontal EEG data  were recorded using the Sedline brain function monitor . The sampling frequency of analyzable signal was 179 Hz. The intra operative EEG signal was not collected if the electrode impedance was >5 kΩ. Data collected from standard monitoring included: Pulse Oxygen Saturation (SpO2), Heart Rate (HR), Systolic (SBP), Diastolic (DBP) Blood Pressure and Mean Arterial Pressure (MAP), temperature, Expired CO2 Fraction (EtCO2). Patient demographics were collected during the medical consultation with the anesthesiologist. This study has been approved by the SRLF (Société de réanimation de la langue française) Ethics Advisory Committee 11–356. To participate, all volunteers had to provide an oral consent before any study-related activities.95 within 8 and 13 Hz after the oral tracheal intubation by modulating Propofol TCI. All patients were intubated after muscular relaxation obtained with atracurium besilate injection (0.5 mg/kg). They were mechanically ventilated with a tidal volume of 6–8 ml/kg and a with a respiratory rate adapted to obtain an EtCO2 between 35 and 38 mmHg. The administration of fluids and vasoconstrictors was left to the discretion of the anesthesiologist present in the operating room. The mean arterial pressure was strictly controlled to stay at or above 70 mmHg and temperature controlled above 36°C.General anesthesia was induced using total intravenous anesthesia (TIVA)  the surgical intervention. We defined the cognitive decline (CD) based on a D-1 MoCA score <25 points. Patients with a cognitive decline where designate by CD+, CD– otherwise. Two anesthetists and one anesthetist nurse, trained to administer, interpret and score the MoCA tests, carried out all administrations. Prior to each evaluation, we checked for the absence of a significant pain that could affect attention and concentration, for this purpose we used the numerical rating scales and included patients with a score ≤ 4. We also assessed delayed neurocognitive recovery (dNCR) by estimating the drop in MoCA score (ΔMoCA) between the D-1 MoCA (baseline) and follow-up intervention after two days (D+2). A patient was considered with a delayed neurocognitive recovery (dNCR +) if ΔMoCA > 2 .95 corresponds to the frequency below which 95% of the signal total power is found. This index reflects the depth of the general anesthesia, such that an SEF95 in 8–13 Hz ensures that patient sedation is appropriate to generate an alpha rhythm. On the contrary, a SEF95 outside the stable region might be the sign of burst suppression (deep sedation) or beta activity (light sedation). We used an automatic procedure to detect 5 min long 8–13 Hz SEF95 region in the signal  remained in a  and 0 otherwise. This binary outcome was modeled as a random variable drawn from a binomial distribution: CD ~ Binomial, where using the age we had:We evaluated the risk of presenting a cognitive decline based on age, the combination of Propofol TCI and AP, and all variables considered. Statistical analysis focused on a time window where the SEF0 as well as coefficients  were optimized numerically based on the data. Models HELP, AGE and HELP2 where compared and evaluated based on the log likelihood. Models were first compared with regard to the data-fit using log likelihood ratio tests. To account for the risk of overfitting, we then compared models based on information criteria penalizing for model complexity in terms of numbers of parameters. We reported the Akaike information criterion (AIC) and Bayesian information criterion (BIC). To explore the data-fit at the level of medical decision-making, we considered receiver operating characteristic (ROC) curves and their areas under the curve (AUC) for each of the three models.Finally, to investigate whether or not the three variables together bring complementary information in predicting CD, we extended HELP to include the age such that:1 (resp. β2), thus for instance a null β1 (null hypothesis) will translate into a null ME (mTCI = 0). Finally, we investigated the average ME of one variable conditioned to a specific value of the other one. Such metric assesses the importance of one variable in predicting CD given the value of the second variable. This conditional ME was shown as a graph of the means and standard error.Estimating the relative impact of the TCI and AP variables on the HELP score, we computed for each variable, based on the probability from HELP, the marginal effect (ME) 2-test for qualitative variables. The significance level for all statistic tests was α = 0.05. Statistical analyses were performed using R version 4.0.The description of qualitative data was given in percentage while quantitative ones were reported as median and interquartile range (IQR). Patient characteristics were compared between CD+ and CD– groups using a Mann–Whiney test for quantitative variables and a Chi95 . 40% (17 patients) had CD+ (MoCA pre-operative <25 points). Fourteen (33%) and 28 (67%) have been admitted for a neuroradiology procedures and orthopedic surgeries, respectively. These patients Propofol TCI and AP were 3.4  μg/ml and 6.37 ± 3.9 dB, respectively. The median time of surgery was 2.6 h   did not meet one of the three following conditions: agreed for D-1 MoCA evaluation , had a collected EEG signal  and had a stable SEF.0, 3.3] .p < 0.001), and had neither significantly more cardiovascular nor neurological risk factors (p = 0.036). No significant difference between women and men distribution could be found. The type of surgery was not significantly different between the two groups. Concerning the intra-operative variables, we found that the alpha-band AP was significantly lower in the CD+ group . Additionally, these patients required a significantly lower Propofol TCI  to achieve the same anesthetic depth (SEF95: 8–13 Hz). Interestingly, the cumulative time spend in iso-electrical suppression (burst suppression) state was statistically similar between CD + and CD- patients , and was thus poorly associated with CD (AUC = 0.63).CD+ patients were significantly older . Additionally, the AIC (resp. BIC) dropped from 43.7 (47.25) for AGE to 40.84 (46.05) and 39.42 (46.37) for HELP and HELP2, respectively, suggesting that the HELP model was the best trade-off between prediction score and the number of parameters. In summary, the model HELP, including TCI and AP, better predicted CD than age and was more parsimonious than HELP2. Furthermore, at this stage adding age to TCI and AP did not add obvious information to predict CD. Therefore, we focused on the HELP model. We, hence, extrapolated the probability of CD for TCI ranging from 1 to 7 μg/ml and AP from 1 to 15 dB , TCI+AP (HELP), and the all three variables together (HELP2). The ROC AUC for the AGE was 0.83, then 0.88 for HELP and 0.90 for HELP2 see . We foun5 dB see . The CD+p = 0.0012) compared to AP . These findings are coherent since TCI modulates AP, however they also reveal the additional effect of these two variables  could be used to reveal pre-existing cognitive decline. These two variables performed, in our patient group, better than the chronological age. The effect of AP on the patient probability to have CD was the most prominent at TCI at 3 μg/ml, and, in a more interesting way, TCI had a much greater effect than alpha band on the likelihood of pre-operative cognitive decline. We further confirmed these finding via HELP model generalization analysis performed on a secondary data set of 56 patients. These results highlight the importance of interpreting cognitive EEG biomarkers (AP) accounting for the hypnotic concentration (TCI). Such information, in daily practice, could help clinician to access patient's biological brain age and then a possible fragility. As more and more studies underline the influence of the preoperative cognitive state on the post-operative outcome  and a weaker frontal alpha-band power (AP) observed during a general anesthesia  or low (<2.5 μg/ml), then it is sufficient to stratify the risk, otherwise we have to evaluate the alpha-band power. There, the lower the alpha-band power, the greater the probability of cognitive decline. Nevertheless, one should keep in mind that AP is influenced by TCI  for predicting post-operative chronic cognitive disorder by comparing or implementing it to preoperative neuro-cognitive scales. This approach might allow anesthesiologists to play an active role in detecting, predicting and preventing neurocognitive disorders.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the SRLF (Société de réanimation de la langue française) Ethics Advisory Committee 11–356. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.CT and JC: study design, data analysis, patient recruitment, data collection, and redaction of the manuscript. CL and JS: patient recruitment and data collection. DS: data analysis. EM: patient recruitment and data collection. JJ and JM: data collection. EG: data analysis. DE: data analysis and manuscript redaction. FV: study design, patient recruitment, data collection, and manuscript redaction. 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.


Avicennia marina (Forsk.) Vierh. (Acanthaceae) is used as a Chinese herb. Studies have found that it contains marinoid J, a novel phenylethanoid glycoside (PG) compound, but its neuroprotective functions are largely unknown. Fruit of  This study evaluated the effects of marinoid J on vascular dementia (VD) and determined its potential mechanisms of action. The VD model was established by the ligation of the bilateral common carotid artery in Sprague–Dawley rats, who received daily intragastrically administration of saline, marinoid J (125 or 500 mg/kg body weight/d), or oxiracetam (250 mg/kg body weight/d) for 14 days (20 rats in each group). The Morris water maze (MWM) was used to evaluate cognitive performance. The hippocampus was subjected to histological and proteomic analyses.p < 0.05). It also decreased malondialdehyde (MDA) (27.53%) and nitric oxide (NO) (20.41%) while increasing superoxide dismutase (SOD) (11.26%) and glutathione peroxidase (GSH-Px) (20.38%) content in hippocampus tissues. Proteomic analysis revealed 45 differentially expressed proteins (DEPs) in marinoid J-treated VD rats, which included angiotensin-converting enzyme (ACE), keratin 18 (KRT18), cluster of differentiation 34 (CD34), and synaptotagmin II (SYT2). Marinoid J shortened the escape latency of VD rats (31.07 ± 3.74 s,  Marinoid J played a role in protecting hippocampal neurons by regulating a set of proteins that influence oxidative stress and apoptosis, this effect may thereby alleviate the symptoms of VD rats. Thus, pharmacological manipulation of marinoid J may offer a novel opportunity for VD treatment. Structural changes in the brain caused by cerebrovascular disease and other risk factors are considered to cause VD. Studies have shown that reduced cerebral blood flow results in oxidative stress, causing an increase in the levels of reactive oxygen species (ROS). A considerable number of unsaturated fatty acids are damaged by ROS, which consequently deteriorates the neural structure and function associated with learning and memory, leading to cognitive dysfunction  Vierh. (Acanthaceae), have been used in folk medicine for contraception and treatment of abscesses and diarrhoea , and other compounds. The main components of PGs are caffeic acid, phenylethanin, and glycosyl groups. In recent years, with the increased attention on PGs, research on these compounds has rapidly progressed. Currently, approximately 200 types of PGs have been isolated from different plants, and novel PGs are continuously being discovered. Numerous studies have shown that PGs have a variety of biological activities, such as enhancing immunity and exerting antibacterial and antioxidative effects. In addition, some studies have shown that PGs can improve neurocognitive function. For example, glycosides from Cistanche deserticola Y.C. Ma (Orobanchaceae) have been shown to improve learning and memory in a rat model of VD and to play a critical role in protecting hippocampal neurons by decreasing tau phosphorylation and increasing the expression of collapsin response mediator protein-2  was deposited in the Institute of Marine Drugs, Guangxi University of Chinese Medicine, China. HPD750, DA-201, DM-130, and AB-8 macroporous adsorption resins were purchased from Zhengzhou Qinshi Technology Co., Ltd. ; echinacoside was purchased from Chinese Pharmaceutical Organisms Inspection Institute ; carbon-18 reverse-phase silica gel  was purchased from Fuji Silysia Chemical Ltd. ; trypsin was purchased from Promega Corporation ; Calbiochem protease inhibitor was purchased from Shanghai Yubo Biotechnology Co., Ltd. ; and a BCA Kit was purchased from Biyuntian Biotechnology Co., Ltd. . The Thermo Labelling Kit, acetonitrile, and triethylammonium bicarbonate buffer were purchased from Thermo Fisher Scientific ; oxiracetam capsules were purchased from Shiyao Group Ouyi Pharmaceutical Co., Ltd. ; and penicillin potassium for injections was purchased from Harbin Pharmaceutical Group Veterinary Pharmaceutical Factory . SOD, GSH-Px, NO, and MDA Kits were purchased from Nanjing Jiancheng Bioengineering Research Institute . The Waters-2695 High-Performance Liquid Chromatography System, using the Sunfire C18 column  coupled to a Waters 2998 photodiode array detector , was used in the present study.Fresh ripe ad libitum. The experiment began after seven days of adaptive feeding. This study was conducted in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals and was approved by the Ethics Committee for Animal Experimentation and Use Committee of Guangxi University of Chinese Medicine .Adult male Sprague–Dawley rats, weighing 240 ± 10 g, were purchased from Guangxi Medical University . Animals were housed under 12 h light and dark conditions with food and water available n = 20), VD (n = 20), VD + olracetam (n = 20), VD + marinoid J high-dose (n = 20), and VD + marinoid J low-dose (n = 20). The high-dose and low-dose groups were intragastrically administered marinoid J at 500 and 125 mg/kg/d, respectively, for dose selection, we refer to some work of others   to prepare a 10% homogenate. After centrifugation, the supernatant was removed for further analysis. SOD activity, GSH-Px activity, MDA content, and NO content were determined according to the instructions of each corresponding kit as previously reported  in lysis buffer was adjusted to a final volume of 100 μL. Then, 50 mM of DTT solution was added to each sample, which was then placed in a sterile incubator for 30 min at 56 °C. Next, IAM solution was alkylated for 15 min at 37 °C in the dark. The urea concentration of each sample was diluted to less than 2 M after alkylation. Trypsin was added at a ratio of 1:50 and digested at 37 °C for 16 h. Then, trypsin was added again at a ratio of 1:100, digestion was continued for another 4 h, and the supernatant was collected for analysis of digested peptides. Third, for identification of proteins via nano LC-MS/MS, peptides were labelled with a TMT Labelling Kit and subjected to nano LC-MS/MS. Peptides were loaded onto an EASY-n LC 1000 Ultra High Liquid Phase System maintaining a flow rate of 800 nL/min. Then, the eluted peptide was loaded in a 2.0-kV NSI ion source for ionisation, with a capillary temperature set to 200 °C. Peptide ions were detected using a high-resolution Orbitrap Fusion Tribrid Mass Spectrometer. First-order MS was obtained in the MS mode at 350–1800 m/z with a resolution of 70,000. The secondary MS scan started at 100 m/z with a resolution of 35,000. The 20 most intense precursor ions were selected for collision-induced fragmentation with a normalised collision energy of 30% in a linear ion trap. Dynamic exclusion was used within 30 s to prevent repeated scanning of the parent ions.For proteomic analysis, data detection and analysis were commissioned by Hangzhou Jingjie Biotechnology Co., Ltd . The isobaric tag for relative and absolute quantification (iTRAQ) with high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses was performed as previously reported . Trypsin/P was designated as the cleaving enzyme. The mass tolerance of the precursor ion was set to 20 ppm in the first search and 5 ppm in the main search, and the mass tolerance of the fragment ion was set to 0.02 Da. The carbamoyl group on cysteine was designated as a fixed modification, and the oxidation on methionine was designated as a variable modification. The false discovery rate was adjusted to <1% and the lowest score of the peptide was set to >40.The resulting MS/MS data were processed using Maxquant (v.1.5.2.8). MS/MS with a reverse database connection was searched in the UniProt database . Group differences in the escape latency in the Morris water maze task were analysed by using two-way ANOVA with repeated measures, the factors being group and time. Otherwise, data were analysed by one-way ANOVA followed by the Tukey’s Avicennia marina were smashed and immersed in 95% ethanol, the extract was concentrated and dried, and the macroporous adsorption resin and carbon-18 reversed-phase silica gel were used to isolate and purify PGs. When eluted with 40% ethanol at a flow rate of 6 mL/min, PGs with a purity of 76.68% were obtained. PGs were subjected to Sephadex LH-20 column chromatography with CHCl3–MeOH (1:1) and were then separated via HPLC using mixtures of MeOH–H2O (45:55) to isolate marinoid J  and there was a significant difference among the groups . Compared with that of the VD group, the escape latencies of marinoid J groups  were significantly shortened. As can be seen from n = 10, p < 0.01). On the third day, the escape latency was significantly decreased in both marinoid J treated groups , on the fifth day after the start of MWM training, the escape latency of the high-dose marinoid J group was shortened to 31.07 ± 3.74 s. In the probe trial, the VD group exhibited a shorter time in the target quadrant compared to that of the sham group . After treatment with marinoid J, the time spent in the target quadrant was also increased , NO levels was decreased by 20.41% , while SOD activity was increased by 11.26% , SOD activity was increased by 20.38%  , MDA levels were decreased by 27.53% ( < 0.05) . These rp < 0.05 were identified as differentially expressed proteins (DEPs). Comparison of the VD group and the Sham group identified 34 DEPs, among which 22 were up-regulated and 12 were down-regulated. Comparison of with VD group and MJ group identified 45 DEPs, among which 25 were up-regulated and 20 were down-regulated. In the marinoid J group, the upregulated DEPs included ACE and KRT18, whereas the downregulated DEPs included CD34 and SYT2 .Through proteomic analysis, a total of 5902 proteins were identified, of which 5183 had quantitative information and the total number of peptides was 42,755. After the MS data were searched, the quantitative proteins were screened based on analyzable proteins, and a protein-change ratio ≥1.2-fold between groups was set, and proteins with To investigate the biological effects of DEPs identified by proteomic analysis, we performed Gene Ontology (GO) analysis. In the VD group, 34 DEPs were involved in binding, catalytic activity, and other processes . In the Figure S3) and the advanced glycation end product (AGE)-receptor for AGE signalling pathway (ko04933) . A total of 118 signalling pathways were retrieved from 45 DEPs in the marinoid J group. The main enrichment pathways were the TRP pathway (ko04750)  and pathways associated with Alzheimer’s disease (ko05010) , Parkinson’s disease (ko05012) , and Huntington’s disease (ko05016) .Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis showed that a total of 34 DEPs in the VD group were mapped to 47 pathways. The main enriched pathways were the transient receptor potential pathway (TRP) (ko04750) (Verbascum sinuatum L. (Scrophulariaceae) reduced the production ROS and inhibited cytochrome C release and caspase-3 clearance, thereby inhibiting the cytotoxicity in SH-SY5Y cells caused by amyloid (Aβ)25-35. Currently, the effects of PGs from Avicennia marina fruits on VD have not been reported.PGs are a type of glycosidic compound consisting of caffeic acid, phenylethanin, and glycosyl, and they are widely found in the roots, bark, and leaves of some dicotyledons. PGs have been reported to exert antioxidative, antitumor, and immune-regulatory activities. Studies have shown that PGs are efficacious in the treatment of neurological diseases. For example, previous research  into Ang II. ACE has vasoactive and aldosterone-activated effects and plays an important role in maintaining fluid balance and blood pressure stability. In the brain, Ang II binds to two receptors: AT1 and AT2. AT2 receptors primarily mediate the recall of learning acquisition and passive avoidance behaviour, predominating in learning and memory, and protecting cognitive function Braszko . In addiKRT18 encodes the type-I intermediate filament chain, keratin 18. KRT18 is perhaps the most commonly found member of the intermediate filament gene family. It is expressed in single-layer epithelial tissues throughout the body. Aberrant expression or mutations in this gene have been linked to disease, such as kidney disease  have been recognised to play a role in the maintenance of microvasculature. Decreased levels of bone-marrow-derived circulating CD34+ cells correlate with vascular dysfunction , and PKCδ were upregulated. In contrast, in the marinoid J group, PKCδ, PKC, and adenosyl cyclase were downregulated. TRP pathway members are widely expressed, can be activated through many different mechanisms, and play important roles in various cellular functions. For example, TRP pathway members, including proteins such as transient receptor potential cation channel subfamily V member 2, PKC, phosphatidylinositol 3-kinase, and insulin growth factor 1, are critical for the pathogenesis of several human diseases such as type-I diabetes, Alzheimer’s disease, cardiomyopathy, and myasthenia gravis syndrome (Nilius and Voets PKC plays an important role in regulating many different biological processes. PKCδ is one of the isoenzymes of PKC and is a substrate for the pro-apoptotic protein, caspase-3 (Ghayur et al. Our present study had some limitations. The DEPs identified by the proteomic analysis were not verified by immunohistochemistry or Western blotting. In addition, some of the key proteins that mediate the effects of marinoid J during VD require further identification, and their functions should be investigated and clarified in future studies.Avicennia marina. Marinoid J effectively ameliorated cognitive deficits in VD rats. Proteomic analysis of rat hippocampal proteins identified 45 DEPs in the marinoid J group compared with those in the VD group. Functional protein-type annotation, functional-type enrichment, and KEGG analysis of these DEPs revealed that marinoid J upregulated the expression of ACE while downregulating the expression of PKCδ proteins, which correlated with amelioration of VD-induced oxidative stress, apoptosis of hippocampal CA1 neurons, and cognitive deficits.In this study, marinoid J was obtained from the fruits of Click here for additional data file.


In addition, the abundance of cyclooxygenase 2 and tumor necrosis factor α immunoreactivity was evaluated. Crocin treatment had renoprotective effects manifested by significant improvement in kidney function as well as a reduction in the abundance of biomarkers of oxidative stress markers and inflammatory mediators. In conclusion, crocin has a protective effect against doxorubicin-induced nephrotoxicity in rats by serving as an antioxidant and attenuating the expression of NF-κB, iNOS, COX2, and TNFα.Doxorubicin is a drug that belongs to the anthracycline antibiotics. Nephrotoxicity is one of the serious side effects of doxorubicin treatment. Crocin, which is one of the most bioactive components of saffron, has antioxidant, anti-inflammatory, and antitumor effects. The current study was aimed at investigating the possible protective effects of crocin against doxorubicin-induced nephrotoxicity to elucidate the underlying mechanism of this effect. The study included four groups, six rats in each group: normal control, crocin control, doxorubicin, and crocin/doxorubicin. Doxorubicin and crocin/doxorubicin groups received intraperitoneal injections of doxorubicin (3.5 mg/kg twice weekly for 3 weeks). Rats in the crocin control group and the crocin/doxorubicin group were treated with intraperitoneal injections of crocin (100 mg/kg body weight per day) for 3 weeks. Biomarkers of kidney function and oxidative stress as well as the abundance of mRNA for nuclear factor- Doxorubicin is a member of the anthracycline family of cytotoxic antibiotics and one of the most potent and commonly used chemotherapeutic agents for the treatment of several types of cancer . The antα (TNFα), which would further activate multiple signaling pathways, including the nuclear factor κB (NF-κB) inflammatory pathway [κB, TNFα, inducible nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX2) in renal tissue [The detailed mechanisms of doxorubicin-induced renal damage remain unknown , 6; howe pathway . Consistl tissue , 12. HenCrocin is one of the most bioactive components of saffron (Crocus sativus L.), a monocotyledon species of the Iridaceae family. Saffron is cultivated in many areas of the world including Egypt . Crocin α mRNA as well as iNOS expression and nitric oxide production via downregulation of NF-κB activity [Crocin treatment also reduced nephropathy in other pathological settings, including gentamicin-induced nephrotoxicity , diabeteactivity . To the This work was performed at the Medical Physiology Department, Faculty of Medicine, Suez Canal University, Ismailia, Egypt.Twenty-four adult male albino Sprague-Dawley rats, body weight 130-175 g, were purchased from the Egyptian Organization for Biological Products and Vaccines . This study was performed in accordance with the Guide for the Care and Use of Laboratory Animals (1985), NIH, Bethesda, and was approved by the Ethics Committee of the Suez Canal University Faculty of Medicine. All rats were allowed to acclimatize for one week prior to the experiment and were housed in plastic cages maintained at controlled room temperature (22-24°C) with a 12-hour diurnal (day and night change) cycle and free access to tap water and a standard rat chow diet. Rats were randomly allocated to four groups: normal control, crocin control, doxorubicin, and crocin/doxorubicin groups.Rats in the doxorubicin and crocin/doxorubicin groups received intraperitoneal injections of doxorubicin  twice weekly for 3 weeks . Rats ing for 15 min. Serum samples were separated, collected in clean tubes, and stored at -80°C until use. Anesthetized rats were sacrificed by decapitation, and laparotomy was done for kidney collection. Right kidneys were kept in 10% buffered formalin solution for subsequent processing for histopathological and immunohistochemical evaluation. Left kidneys were frozen and stored at -80°C prior to homogenization and subsequent assays.At the end of the study, 24-hour urine samples were collected to determine urine albumin and creatinine levels. Rats were then anesthetized with ether, and retrobulbar blood samples were collected and processed by centrifugation at 2000 × Serum albumin, blood urea nitrogen (BUN), creatinine, and urine albumin and creatinine were determined using commercial kits on the Cobas c311 analyzer . The standard conventional formula (urinary creatinine concentration (U) (mg/ml) × urine volume (V) (ml/min)/serum creatinine concentration (P) (mg/ml)) was used for creatinine clearance calculation .w/v) of Tris buffer  using a Teflon and glass homogenizer . The homogenate was sonicated and then centrifuged at 20,000 × g for 10 min. Lipid peroxides  and superoxide dismutase (SOD) activity were assayed in renal tissue homogenate by the colorimetric method using specific kits supplied by Biodiagnostic, Egypt, according to the manufacturer's instructions.Kidneys were homogenized in 10 volumes (μg) was used to prepare cDNA using a high fidelity reverse transcription kit . Real-time qPCR amplification and analysis were performed using an Applied Biosystems instrument with StepOne™ software (version 3.1). Reaction mixtures contained SYBR Green Master Mix (Applied Biosystems), gene-specific forward and reverse primers (10 mM), and cDNA and nuclease-free water. The sequences of PCR primer pairs used for each gene are shown in Total RNA was isolated from left kidney frozen tissue using the Qiagen tissue extraction kit  according to the manufacturer's instructions. Extracted RNA was quantified by spectrophotometry . The RNA integrity was assessed using agarose gel electrophoresis and ethidium bromide staining. The total RNA , and examined with an Olympus light microscope. About 20 sections for the H&E stained kidney sections were selected for the measurement of glomerular, proximal, and distal tubular areas [Right kidneys were dissected and immediately transferred to 10% buffered formalin solution for fixation. Kidneys were dehydrated using a series of solutions of increasing ethanol content and then embedded in paraffin. Sections of 4 ar areas  using Imα immunostaining was using a polyclonal rabbit anti-TNFα antibody diluted 1 : 400 . COX2 immunostaining used a polyclonal rabbit anti-COX2 antibody diluted 1 : 100 . Twenty sections were evaluated for each group. The DAB-stained cytoplasmic option in the IHC profiler ImageJ plugin was used, as described by Varghese et al. [Kidney sections were cut, rehydrated, deparaffinized, and mounted for immunostaining. TNFe et al. . Images p ≤ 0.05.Parametric data were expressed as mean ± standard error of mean (SEM), and nonparametric data were presented as median and range (minimum-maximum). For comparisons of quantitative variables among the study groups, one-way ANOVA followed by the Bonferroni post hoc test was used if data were parametric, while the Kruskal-Wallis (KW) test was used if data were nonparametric. Statistical analysis was done by the Statistical Package for Social Sciences (SPSS) program version 20. Data were considered statistically significant with p = 0.008 and p = 0.015, respectively), an increase in serum creatinine concentration in the doxorubicin group in comparison with the normal control group (p = 0.034), an increase in urine albumin concentration in the doxorubicin group in comparison with the normal control group (p = 0.036), a decrease in urine creatinine concentration in the doxorubicin group in comparison with both the normal control group and the crocin control group , an increase in urine albumin/creatinine ratio (ACR) in the doxorubicin group in comparison with the normal control, crocin control, and crocin/doxorubicin groups , and a decrease in the creatinine clearance rate in the doxorubicin group in comparison with the normal control group (p = 0.034). The biomarkers of kidney function in the crocin+doxorubicin group did not differ significantly from those of the normal control group . There was also a significant decrease in the antioxidant SOD in the doxorubicin group in comparison with the normal control group and the crocin control group . The administration of crocin in combination with doxorubicin decreased in MDA (p = 0.026) and increased SOD (p = 0.012) in renal tissues relative to the doxorubicin group . Crocin administration with doxorubicin decreased the abundance of NF-κB mRNA relative to the doxorubicin group (p < 0.01) . However, although the administration of crocin plus doxorubicin decreased iNOS mRNA relative to doxorubicin alone, it remained elevated when compared with both the normal control group and the crocin group (p < 0.01 for both comparisons)  . The abuarisons) .p = 0.048 and p = 0.035, respectively)   . In addictively) .α and cyclooxygenase 2 immunoreactivity was assessed in renal tissue sections immunohistochemically. Immunostaining for TNFα was negative in both normal control and crocin control groups. In contrast, the doxorubicin group showed intense staining, which was markedly decreased in the crocin/doxorubicin group  signal transducers and activators of transcription (STAT). In addition, the mitogen-activated protein kinase (MAPK) pathway most likely contributes to iNOS gene expression [κB represents one of these pathways and is inhibited by crocin. Hence, the crocin-mediated inhibition of the ability of doxorubicin to increase NF-κB mRNA resulted in a partial decrease in doxorubicin-induced iNOS expression while the other doxorubicin-activated pathways also leading to iNOS upregulation were not targeted by crocin. A limitation of the current study is that the observed changes in the renal expression of NF-κB and iNOS have not yet been confirmed at the protein level.Interestingly, despite the ability of crocin to prevent the doxorubicin-induced increase in NF-pression . Hence, κB, iNOS, COX2, and TNFα. Further studies are required to evaluate other possible mechanisms mediating the renoprotective effects of crocin and to address the safety of using crocin/doxorubicin cotreatment in humans.In conclusion, the present study shows that, as an adjuvant therapy for doxorubicin chemotherapy, crocin has a renoprotective effect in that it attenuated doxorubicin-induced nephrotoxicity in rats by serving as an antioxidant and suppressing the doxorubicin-induced increase in NF-


Proganochelys quenstedtii and Proterochersis robusta and the Late Jurassic Plesiochelys bigleri to test explicitly for a relationship between shell shape and ecology. 3D models were obtained using surface scanning and photogrammetry. The general shape of the shells was captured using geometric morphometrics. The habitat ecology of extant turtles was classified using the webbing of their forelimbs as a proxy. Principal component analysis (PCA) highlights much overlap between habitat groups. Discriminant analyses suggests significant differences between extant terrestrial turtles, extant fully aquatic  turtles, and an unspecialized assemblage that includes extant turtles from all habitats, mostly freshwater aquatic forms. The paleoecology of the three fossil species cannot be determined with confidence, as all three fall within the unspecialized category, even if Plesiochelys bigleri plots closer to fully aquatic turtles, while the two Triassic species group closer to extant terrestrial forms. Although the shape of the shell of turtles indeed contains an ecological signal, it is overall too weak to uncover using shell shape in paleoecological studies, at least with the methods we selected.Turtles are a successful clade of reptiles that originated in the Late Triassic. The group adapted during its evolution to different types of environments, ranging from dry land to ponds, rivers, and the open ocean, and survived all Mesozoic and Cenozoic extinction events. The body of turtles is characterized by a shell, which has been hypothesized to have several biological roles, like protection, thermal and pH regulation, but also to be adapted in its shape to the ecology of the animal. However, only few studies have investigated the relationships between shell shape and ecology in a global context or clarified if shape can be used to diagnose habitat preferences in fossil representatives. Here, we assembled a three-dimensional dataset of 69 extant turtles and three fossils, in particular, the Late Triassic  The clade Testudinata sensu is definThe shell is a common characteristic of all turtles but subject to substantial morphological variation from one species to the other . It is uChelonia mydas) are more typical for turtles with aquatic habits, especially those that live in open marine environments  are prevalent among turtles with terrestrial habitats (Graptemys geographica) or the rounded and greatly flattened shells of many trionychids . Given that correlations appear to exist between shell shape and ecology, paleontologists have historically been tempted to reconstruct the paleoecology of fossil turtles by reference to their shell shape, but studies have been lacking that explicitly tested this relationship.The turtle shell is thought to provide several evolutionary advantages, including protection, pH control, or thermal regulation . The preronments , while hhabitats . A largeA broad selection of studies have recently focused on finding correlations between the ecology of extant turtles and their cranial or post-cranial morphology, including morphometrics e.g., , histoloProganochelys quenstedtiiProterochersis robustaProganochelys quenstedtii was originally argued to have had been a fresh-water aquatic bottom walker based on its low shell and details in femoral anatomy , since no intact adult specimens were available for this study. Finally, sampling was limited to specimens with complete shells, including naturally articulated bridges, that lack scute abnormalities, shell deformations , or pronounced asymmetry. Sex was disregarded as a selection criterion, as most specimens housed in collections, especially skeletal specimens, are not sexed and as the sex of turtles is only known to influence the overall shape of the shell in a subtle manner . The curves were captured in a two-step process. For the first step, semilandmarks were manually set along the curves of the specimen using the “curve” function of Checkpoint. The resulting curves are not yet comparable to one another, as they utilize a different number of unevenly set semilandmarks. The primary semilandmarks curves were therefore resampled in R v3.6.3  was performed with 1,000 permutations.To test for the impact of allometric shape variation we used the log-transformed centroid size of the specimens of each dataset and produced a linear regression against Procrustes shape see . The reggm.prcomp in the R package geomorph.Then, we performed a Principal Component Analysis (PCA), which is a commonly used method to convert a set of data into a set of independent variables. The PCA was computed using the function a priori classification model and assigned specimens of unknown ecology to a specific category. The LDA was performed using the function lda from the package MASS , which distinguishes morphological differences between groups . LDA ideage MASS  and was Proganochelys quenstedtii and Proterochersis robusta were then added as stem-turtles following Proganochelys quenstedtii as the most basal turtle in the tree. Plesiochelys bigleri was placed as sister group to Cryptodira following λ), which varies from 0 to 1, with 0 denoting the lack of a phylogenetic signal and 1 denoting a strong phylogenetic signal under Brownian motion . In contrast to LDA, pFDA addresses the impact of phylogeny on the data to provide predictions . The phyn motion . This coR2 = 0.0235, P-value = 0.134; The results of the linear regression and the ANOVA indicate no correlation between shape and log-transformed centroid size (Proterochersis robusta groups with non-webbed (category 0) and poorly-webbed (category 1) turtles with domed-shells, while Proganochelys quenstedtii and Plesiochelys bigleri are closer to turtles with flattened shells.For SET1 , PC1 expSET2, which describes the outline of the carapace , PC1 expProganochelys quenstedtii and Plesiochelys bigleri plot on the negative site of PC1, while Proterochersis robusta is found on the opposite of PC1. Part of the overlap is explained by the presence of the outliers for various categories, in particular the pancake tortoise , which is a greatly flattened terrestrial turtle, or the leatherback turtle (Dermochelys coriacea), which is a marine turtle with a strongly convex plastron.PC1 of SET 3, which captures the transverse cross-sectional shape of the shell, explains 68.44% of the total variance, most of which pertains to the height of the dome, from flat (negative scores) to highly domed (positive scores) . PC2 carProterochersis robusta plots close to the terrestrial turtles (category 0), while Proganochelys quenstedtii plots in the “fully webbed” range (category 2). Plesiochelys bigleri plots towards the left of the graph  is variable depending on the subset (SET) used . The maiFor SET1 , three gPlesiochelys bigleri plots just at the limits between ”fully webbed“ (category 2) and ”extensively webbed“ and ”flipper-shaped” forehand (category 3 and 4) and Proterochersis robusta at the limit between ”poorly webbed” (category 1) and ”fully webbed” (category 2) turtles (Proganochelys quenstedtii plots within the “fully webbed” (category 2) turtles. For SET2, the fossil turtles are identified as either fully webbed (category 2) or poorly webbed (category 1), but plot further away from the extant groups, except for Plesiochelys bigleri, which groups with fully webbed (category 2) turtles but was determined to be ”poorly webbed” with a probability of 49% (see Proterochersis robusta is predicted to be ”fully webbed” (category 2), but only with a probability of 38%. On the other hand, Plesiochelys bigleri is predicted to have been ”extensively webbed” (category 3) with a low probability of 49% while Proganochelys quenstedtii groups with turtles that are ”poorly webbed” (category 1), also with a low probability (47%). Finally, for SET4, Proganochelys quenstedtii is predicted to have been ”poorly webbed” (category 1), while Proterochersis robusta and Plesiochelys bigleri are reconstructed as ”fully webbed” (category 2), which is consistent with what can be observed on the graph.The predictions of the webbing (and thus ecology) of the fossil turtles are variable between the SETs see ; Table 5 turtles . ProganoThe confusion matrix based on the phylogenetic flexible discriminant analysis (pFDA) shows good recognition of ecological variables (expressed by the degree of webbing in the forelimbs) for extant species . The anaPlesiochelys bigleri is resolved as having flippers (category 4) and plots with extant turtles for SET1, the Triassic fossil turtles are resolved as “fully webbed” (2) but plot further away from the extant group. Proganochelys quenstedtii and Proterochersis robusta do not group close together with any other turtle. For SET2, Plesiochelys bigleri is grouped again within the extant group, contrary to Proganochelys quenstedtii and Proterochersis robusta, which are found to be outliers. Plesiochelys bigleri is predicted as poorly webbed” (category 1), while Proterochersis robusta and Proganochelys quenstedtii are predicted to have “flippers” (category 4) and ”poorly webbed” (category 1). In SET3, all fossils plot outside of the extant groups, even if the algorithm gives predictions such as extensive webbing (category 3) for Proganochelys quenstedtii, ”fully webbed” (category 2) for Plesiochelys bigleri, and “flippers” (4) for Proterochersis robusta (Proterochersis robusta and Plesiochelys bigleri and as ”poorly webbed” (category 1) for Proganochelys quenstedtii.The pFDA results for extant turtles are similar to the LDA results for SET1 and SET2. The distribution, however, is variable for SET3 and SET4. In SET1, the graph is divided into three major groups. One is composed of turtles with not-webbed morphologies, one includes turtles with poorly webbed and fully webbed forelimbs, and a last one with turtles having extensive webbing and flipper-shaped forelimbs . In SET2 robusta . For theIt is notable that the categories poorly webbed (category 1) and fully webbed (category 2) overlap each other in both LDA and pFDA, just as the categories extensive webbing (category 3) and flippered (category 4). However, the pFDA is not very insightful concerning the webbing/ecology of fossil turtles. To investigate the impact of the categorization done herein, the LDA analysis was performed on the SET1 again using a different combination of categories. In particular, the five previously used categories were reclassified for this purpose into three novel categories, herein defined as “terrestrial” , ”semi-aquatic”  and “aquatic” . The results of this secondary analysis are provided in the update confusion table  and grapPlesiochelys bigleri falls between fully webbed (2) and extensive webbing (3) in the first classification model (Proganochelys quenstedtii and Proterochersis robusta plot further away from the extant groups. It appears that splitting the semi-aquatic category into two (poorly-webbed and fully-webbed) gives a more precise placement for the Triassic turtles such as they plot closer to the extant groups, even if there is poor discrimination between these two categories.The misclassification rate for the confusion matrix associated with the three new categories (18.4%) is lower than what is observed in the one with five categories (25.3%). For instance, semi-aquatic turtles are well recognized (38 of 39), but some aquatic (3 over 13) and terrestrial turtles (5 over 17) are still misclassified. However, in general, the dataset containing three categories  gives sion model  and remaon model , betweenIn order to determine the paleoecology of extinct species, paleontologists often draw from correlations found among the shape and ecology of extant organisms e.g.,  for unguλ = 0, none of the subsets of the data contain a phylogenetic signal under Brownian motion  and use of an artificial outgroup  with changing ecology . In the plots resulting from the use of an ultrametric tree, the Triassic fossils pool with extant turtles, even though their phylogenetic distance has actually increased see . This man motion . This maPlesiochelys bigleri was included in the study to test the impact of fossils on the study, but also because the paleoecology of plesiochelyids remains poorly resolved as either riverine . The analysis with three ecological categories on the other hand suggests “intermediate” habitat preference, though the species plots together with Proterochersis robusta towards the edge of the plot, but once again close to semi-aquatic turtles, such as the testudinoids Glyptemys insculpta, Heosemys spinosa, and Emys orbicularis. In our opinion, the analysis suggests that this turtle has a non-specialized shell shape broadly consistent with continental habitat preferences ranging from fully aquatic to fully terrestrial. The majority of independent sources of information nevertheless still point towards a more dry continental signal.The LDA using with five ecological categories predicts for The relative performance of 2D versus 3D data in geometric morphometrics has recently been discussed , but theChedighaii barberi or Taphrosphys sulcatus  Model with all collected data, SET1. (B) Outline of the carapace, SET2. (C) Transverse cross section, SET3. (D) Longitudinal cross section, SET4.The Click here for additional data file.10.7717/peerj.10490/supp-2Supplemental Information 2mshape” from the R package geomorph.(A) “not webbed” (category 0). (B) “poorly webbed” (category 1). (C) “fully webbed” (category 2). (D) “extensive webbing” (category 3). (E) “flippers” (category 4). Mean shapes were computed using the function “Click here for additional data file.10.7717/peerj.10490/supp-3Supplemental Information 3(A) The categories not-webbed and flippers are very distinct using 5 categories. The three intermediate categories are overlapping each other.(B) The categories terrestrial and aquatic are very distinct using 3 categories. The intermediate categories are overlapping the terrestrial range.Click here for additional data file.10.7717/peerj.10490/supp-4Supplemental Information 4(A) All landmarks and curves, SET1. (B) Outline of the carapace, SET2. (C) Transverse cross-section, SET3. (D) Longitudinal cross-section, SET4. Phylogenetic tree used in the analysis is available in Supplementary Files, Fig. S5Click here for additional data file.10.7717/peerj.10490/supp-5Supplemental Information 5Abbreviation: Car, Carettochelyidae; Che, Chelydridae; Pel, Pelomedusidae; Pla, Platysternidae; Pod, Podocnemididae.Click here for additional data file.10.7717/peerj.10490/supp-6Supplemental Information 6We tested ways to improve the results on fossil turtles by using an outgroup where all the PC Scores are equal to 0, 1, or randomized scores. The artificial outgroup was tested using two different categorizations, one identified as tnot webbed (category 1), and one as unknown, like the other fossils. Creating this outgroup did not improve the results of the analysis for the fossil turtles. Phylogenetic tree used in the analysis is available in Supplementary Files, Click here for additional data file.10.7717/peerj.10490/supp-7Supplemental Information 7Abbreviation: Car, Carettochelyidae; Che, Chelydridae; Pel, Pelomedusidae; Pla, Platysternidae; Pod, Podocnemididae.Click here for additional data file.10.7717/peerj.10490/supp-8Supplemental Information 8The number of genera for each major clade is based on the TTWG (2007). Abbreviations: Ntotal = number of known genera; Nsample = number of sampled genera.Click here for additional data file.10.7717/peerj.10490/supp-9Supplemental Information 9http://www.morphosource.org/Detail/ProjectDetail/Show/project_id/1028All specimens reconstructed by us were uploaded on the MorphoSource platform. Available at Click here for additional data file.10.7717/peerj.10490/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.10490/supp-11Supplemental Information 11The cases in green represent the well-recognized turtles. The last line presents the percentage of recognition.0: not webbed. 1: poorly webbed. 2: fully webbed. 3: extensive webbing. 4: flippers.Click here for additional data file.10.7717/peerj.10490/supp-12Supplemental Information 12This table consists of all the extant turtles training dataset.Click here for additional data file.10.7717/peerj.10490/supp-13Supplemental Information 13This table consists of all the extant turtles training dataset.Click here for additional data file.10.7717/peerj.10490/supp-14Supplemental Information 14Each table shows results for a different SET (SET1 to SET4). The second column represent the predicted class by the model. The five next columns are the prediction values (between 0 and 1) for each ecological category. The four next columns are the results of the discriminant analysis.Click here for additional data file.10.7717/peerj.10490/supp-15Supplemental Information 15Time-calibrated phylogeny of 72 species used in the study based on Click here for additional data file.10.7717/peerj.10490/supp-16Supplemental Information 16Click here for additional data file.10.7717/peerj.10490/supp-17Supplemental Information 17Click here for additional data file.10.7717/peerj.10490/supp-18Supplemental Information 18an sheet for each SET (SET1-SET4) is presented. These data were used to conduct statistical analyses.Click here for additional data file.


Snacks, while widely consumed in the United States (U.S.), do not have a standard definition, complicating research to understand associations, if any, with weight status. Therefore, the purpose of this study was to examine the association between snacking frequency and weight status using various snacking definitions that exist in the scientific literature among U.S. adults . Four event-based snacking definitions were operationalized including participant-defined snacks, eating events outside of meals, and operationally defined snacks based on absolute thresholds of energy consumed . Weight status was examined using body mass index (BMI), waist circumference, and sagittal abdominal diameter risk. Logistic regression models examined snacking frequency and associations with weight status. Outcomes varied by the definition of a snack employed, but the majority of findings were null. Mean energy from snacks was significantly higher among women with obesity compared to women with normal weight when a snack was defined as any event outside of a typical mealtime , regardless of whether or not it contributed ≥50 kcal. Further investigation into ingestive behaviors that may influence the relationship between snacking frequency and weight status is needed. Snacking  is a hallmark of the American dietary pattern, with more than 90% of adults in the United States (U.S.) reporting consumption of one or more snacks on a given day . AlthougThe ability to elucidate the relationship between snacking and weight status is impeded by the lack of a consistent definition of the term “snack” or behavior “snacking” . VariousGiven the limitations of each snack definition and the consequent low confidence in the conclusions that can be drawn from studies that use them , 20–23, Previous studies have shown that snacking frequency is associated with greater risk of overweight and obesity (OW/OB) in both younger children  and amonThe 2013–2016 National Health and Nutrition Examination Survey (NHANES) was used for the present analysis. NHANES is a nationally representative, continuous cross-sectional survey of the noninstitutionalized, civilian residents of the U.S., conducted by the Centers for Disease Control and Prevention, National Center for Health Statistics (NCHS) . NHANES Demographic, anthropometric, and dietary data from the NHANES 2013–2014 and 2015–2016 cycles were combined to form a sample of 20,146 participants. Participants who were < 20 years of age , did not complete either the household interview or MEC examination (n = 789), were missing BMI or race/Hispanic origin information (n = 136), pregnant and/or lactating (n = 217), or did not provide reliable dietary recall information that met the minimum criteria  (n = 932) were excluded, yielding a total sample of 9,777 U.S. adults. Data on WC and SAD were available for 9,473 and 9,312 participants, respectively. Few participants were underweight (58 men and 86 women), and thus excluded due to small sample size, resulting in a final analytic sample of 9,633 adults .Demographic and health-related characteristics, including data on sex, age, race/Hispanic origin, income, and smoking status were collected from participants using the Computer-Assisted Personal Interview system during the household interview. These covariates were included because of their association with BMI and other dietary behaviors –37. Age Dietary intake was self-reported in the MEC using the in-person, day one 24-hour dietary recall (24HR). The 24HR is collected by trained interviewers using the U.S. Department of Agriculture’s (USDA) automated multiple-pass method (AMPM) . The USDSelf-reported dietary intake data can be prone to systematic error, like energy underreporting . In ordeFour event-based definitions were operationalized based on energy content, participant definition of a snack, and ingestive events consumed outside of typical meals. These definitions included: (1) any consumption event defined by the reporter as a “snack” or Spanish language equivalent; (2) any consumption event defined by the reporter as a “snack” or Spanish language equivalent that contributed ≥50 kcal; (3) any consumption event outside of a typical meal time  including self-defined snacks; and (4) any consumption event outside of a typical meal time  including self-defined snacks that contributed ≥50 kcal. Two of the four definitions took into account a 50 kcal cut-point in order to evaluate each definition with and without occasions that may not be considered an ingestive episode, as some previous studies consider ingestive episodes with less than 50 kcal to be trivial . While iThree indicators of weight status were chosen for examination in this analysis, including BMI, WC, and SAD. BMI, obtained from height and weight collected by trained health technicians during the health examination in the MEC, was calculated as weight in kg divided by height in meters squared. Standard BMI categories were constructed: underweight , normal (18.5–24.9), overweight 25.0–29.9), and obese ≥30) . Overwei.9, and o . Overwep-value of <0.01 was considered statistically significant , elevated WC risk , and elevated SAD risk , relative to snacking frequency when compared to the referent group of 0 snacks per day   and SAS-callable SUDAAN  software programs to account for differential nonresponse and non-coverage, and to adjust for oversampling and post-stratification by use of the NHANES dietary day 1 sample weights. PROC DESCRIPT was used to compute prevalence estimates and means of a group for demographic characteristics; a nificant . Associanificant . Sattertf <0.001 . LogistiSeveral models comparing snacking frequency by definition with weight status were examined. First, we examined the crude, unadjusted relationship between snacking and weight status. Second, we built models including factors related to the exposure and outcome and derived fully adjusted models, including demographic, lifestyle, and dietary factors related to the research question and informed by previous analyses, as presented in the main tables , 17, 46.As representative of the adult U.S. population, this analytic sample was approximately 48.3 years of age, non-Hispanic white (65.3%), primarily women (50.7%), and had overweight (33.2%) or obesity (39.3%) . While mThe number of daily snacking occasions varied in magnitude by the snack definition employed, and generally ranged between 1.2 and 3.0 snacks per day among men and women . The freMean snack energy ranged from 67.4 to 134.0 kcal per snacking occasion among men, and from 54.1 to 96.2 kcal per snacking occasion among women, contributing approximately 15.3–24.2% and 16.9–24.3% of total daily energy among men and women, respectively . Snacks In general, the relationship between snacking frequency and adiposity was largely null across all indicators of weight status  Tables  and 5. IOverweight and obesity persist as significant public health issues in the U.S. Understanding modifiable factors that are related to unhealthy weight status in adults is critical for the development of public health interventions aimed at reducing the risk of overweight, obesity, and their related comorbidities. Snacking is one behavior that has been implicated in the etiology of overweight/obesity. Snacking incidence has increased markedly over the past four decades  and it iTwo important dimensions of snacking are its possible contribution to eating frequency and the energy content of snacking events. This analysis focused on the effects of frequency and size of snacking occasions on multiple metrics of adiposity in a nationally representative sample of U.S. adults. While BMI categories and WC are primarily used to define weight status, SAD is an additional proposed measure that may provide a more accurate indicator of adiposity associated with increased risk of adverse metabolic effects/metabolic syndrome , 29, 30.This analysis indicated snack frequency was not associated with weight status regardless of the event-based definition tested. However, snack definition did play a role in the associations between weight status, mean energy from snacks among women, and the percent of total daily energy from snacks. Most notably, mean energy from snacks among women with obesity was higher than in women with a normal weight when a snack was defined as any ingestive event between meals , but not when a snack was defined by the participant (Snacks). This difference in mean energy between definitions suggests that energy from other ingestive events between typical meal times that participants do not consider to be snacks may be underestimated or poorly compensated and contribute to excess energy intake. Snack frequency and percent of total energy from snacks were not significantly different between women with a normal weight and women with obesity. Because this association is not statistically significant in men, other psychosocial factors associated with sex may play a role, or may be related to the omission of reported snacks by men , 60. TotThe second dimension of snacking contributes to total energy intake and is related to the energy content of snacking events, which was not investigated in this analysis. An investigation into the association between total snack energy intake and weight status may provide insight into whether snack energy intake opposed to snack frequency promotes differences in weight status. It would also be useful to examine food form or snacks as beverages, which may evoke weaker energy compensation  than solEach event-based definition included in the analysis addresses a slightly different question regarding the association between snack frequency and weight status. Defining a snack based on the participant’s self-report is culturally relevant and provides insight into how snacks are defined by the general population. However, each subject has their own individual classification system to define their ingestive events that could be based on a variety of criteria including time of day, type of food consumed, or portion size. This variability in snack classification between subjects introduces error leading to an over- or under-estimation of snack frequency. In addition, sugar-sweetened beverages, which can contribute substantially  to totaAt this time, an association between snacking and weight status among adults continues to be debated, and evidence remains mixed. Murakami and colleagues  observedThese largely null associations also differ from our previous work in children. A recent study conducted by Tripicchio et al. among U.S. adolescents concluded that adolescents with overweight and obesity not only consumed a greater number of snacks per day than their normal weight counterparts, but also have a higher energy intake at each snacking occasion than those with a normal weight . These fNHANES is a nationally representative survey of the U.S. noninstitutionalized population. This analysis was designed to provide a framework to evaluate the association between snacking and body weight status at the population-level. The USDA’s AMPM is a state of the art method for dietary data collection as is the FNDDS database used to process dietary data. However, self-reported dietary data are prone to systematic errors, and tend to underestimate true energy intake . NeverthDespite hypotheses that the use of different event-based definitions of snacking may account for the mixed findings on the role of snacking in weight management, this analysis did not yield robust effects of the definitions tested. Confirmation of these findings based on cross-sectional survey data with longitudinal assessments and clinical trials is warranted given the high level of energy contributed by snacks and consequent potential challenge they pose to achieving a healthy weight.S1 Text(DOCX)Click here for additional data file.S1 Table1–4 Abbreviations: BMI, body mass index ; SE, standard error; PIR, family income-to-poverty ratio. 1 Values are percent (SE) unless otherwise indicated. 2 Different superscript letters  indicate significant differences within a row  at p-value of < 0.01. 3 BMI Categories (kg/m2): Normal weight, 18.5≤ BMI <25; Overweight, 25≤ BMI <30; Obese, BMI ≥ 30. Underweight  were excluded due to a small sample size. 4 Race/Hispanic Origin does not sum to 100 because the other race category is not shown.(DOCX)Click here for additional data file.S2 Table1–2 Abbreviations: Ref, Reference Value. 1 OW/OB: Odds ratio of overweight or obesity (BMI≥25); WC: Odds ratio of waist circumference > 102cm; SAD: Odds ratio of SAD>25cm; EER: Men EER = 662 –(9.53 x age [y]) + PA x [(15.91 x weight [kg]) + (539.6 x height [m])], Women EER = 354 –(6.91 x age [y]) + PA x [(9.36 x weight [kg]) + (726 x height [m])]. A p-value of < 0.001 was considered statistically significant. 2 An asterisk “*”denotes a snack defined as an event that contributed ≥ 50 kcal. 3 Snack defined as an event defined by the reporter as a “snack.” 4 Covariates included in Model 2: age (continuous), race , family income-to-poverty ratio , day of recall , typical intake , smoking status , and EI:EER. 5 Covariates included in Model 3: covariates included in Model 2 with the exception of EI:EER, and the addition of mean energy of a snack and mean energy density  of a snack. 6 Snack defined as an event outside of a typical mealtime.(DOCX)Click here for additional data file.S3 Table1,2 Abbreviations: Ref, Reference Value. 1 OW/OB: Odds ratio of overweight or obesity (BMI≥25); WC: Odds ratio of waist circumference > 102cm; SAD: Odds ratio of SAD>25cm; EER: Men EER = 662 –(9.53 x age [y]) + PA x [(15.91 x weight [kg]) + (539.6 x height [m])], Women EER = 354 –(6.91 x age [y]) + PA x [(9.36 x weight [kg]) + (726 x height [m])]. A p-value of < 0.001 was considered statistically significant. 2 An asterisk “*”denotes a snack defined as an event that contributed ≥ 50 kcal. 3 Snack defined as an event defined by the reporter as a “snack.” 4 Covariates included in Model 2: age (continuous), race , family income-to-poverty ratio , day of recall , typical intake , smoking status , and EI:EER. 5 Covariates included in Model 3: covariates included in Model 2 with the exception of EI:EER, and the addition of mean energy of a snack and mean energy density  of a snack. 6 Snack defined as an event outside of a typical mealtime.(DOCX)Click here for additional data file.


Effect of bilateral, optic neuropathy on the function of retinal pigment epithelium has not been investigated extensively to date. This study aimed to determine the effect of bilateral, optic neuropathy on light peak:dark trough ratio, light peak and dark trough values of electro-oculography. Thirty-seven patients with a clinical diagnosis of bilateral optic neuropathy and 40 control subjects were recruited in this observational, cross-sectional study, carried out at the Neurophysiology unit, Teaching Hospital, Peradeniya, Sri Lanka. Pattern reversal visual evoked potentials, pattern electroretinography and electro-oculography were recorded in all of them.Twenty-four patients (64.9%) had reduced light peak:dark trough ratio values. The median light peak:dark trough ratio ± inter quartile range of the patient group (1.50 ± 0.4) was significantly lower than that of the controls (1.80 ± 0.2), (p < 0.001). Median dark trough value was significantly higher in patients compared to the control value. These changes may be due to higher conductance of ion channels in the retinal pigment epithelium, sub retinal space creation, changes in choroidal circulation or secondary to photoreceptor abnormalities in the macular region. These results indicate that retinal pigment epithelium might be affected in bilateral optic neuropathy. Acute demyelinating optic neuropathy is a clinical diagnosis. The history points to the aetiology of the optic neuropathy . PatternOptic neuropathy is commonly associated with multiple sclerosis in patients reported in the Western literature and this is mostly unilateral . CompareGiven the scarcity of evidence of the RPE electrophysiology in optic neuropathy, and higher prevalence of bilateral optic neuropathy in Asia, the aim of this study was to evaluate the effect of bilateral, demyelinating optic neuropathy on the RPE as estimated by the LP:DT ratio, amplitudes of light peak (LP) and dark trough (DT) of EOG.This was a cross-sectional study in which we compared a test group of patients with optic neuropathy and a healthy control group. The study was carried out at the Neurophysiology Unit of the Teaching Hospital, Peradeniya in Sri Lanka between February 2018 to October 2019. The study design and protocols complied with the code of ethics of the World Medical Association Declaration of Helsinki .A sample of 36 subjects per each group was required to observe an effect size of 0.7, with a 90% power at α-error probability of 0.05. Thirty seven patients ≥ 18 years of age with a clinical diagnosis of bilateral optic neuropathy referred by consultant ophthalmologists within 21 days of onset of ocular symptoms were recruited. Patients were diagnosed by the sudden onset of visual impairment, positive relative afferent papillary defect (RAPD) and fundoscopic changes. One of the co authors, who is a specialist ophthalmologist (SS) clinically examined and conducted fundoscopic examination of the potential subjects. None of the recruited subjects had previous eye diseases, evidence of neurological diseases or fundoscopic features of other eye pathologies that might confound our results. The control group comprised 40 healthy subjects of age ≥ 18 years with normal or corrected-to-normal vision in neuro-ophthalmologic examination. All subjects had a neuro-ophthalmologic assessment which included; measurements of visual acuity, visual field, colour vision, pupillary reflexes, fundoscopy and ocular motor examination at the referring ophthalmology units. Then visual electrophysiological tests viz. PRVEP by Natus machine (USA), pattern electroretinography (PERG) by Nicolet Viking Quest machine (USA) and EOG were performed in each subject conforming to International society for clinical electrophysiology of vision (ISCEV) guidelines , 12.−2. The light phase recording lasted for another 15 min [The EOG waveforms were recorded using the Natus EMG/NCV/EP machine (USA). Pupils were not dilated as it was shown that application of pupillary dilatation does not influence the quality or the results of electro-oculograms . The bitr 15 min . The patThe average of the EOG amplitudes within each 10 s recording epoch was taken and plotted against time. Then the underlying physiologic curve was drawn using computer-based curve fitting algorithms to derive reliable dark trough (DT) and light peak (LP) amplitudes. Then the LP:DT ratio was calculated by dividing the smoothed light peak by dark trough value for each subject.Continuous outcomes measures, i.e. PRVEP latencies, PRVEP amplitudes, PERG latencies, PERG amplitudes, amplitudes of light peak and dark trough, LP:DT ratios showed skewed distributions, and hence are reported as medians and inter quartile ranges (IQR) and were compared between groups using Mann–Whitney U test. The comparisons were interpreted as significant at a cut-off p value < 0.05. IBM SPSS statistics for windows, version 22.0. was used to analyze the data and Graph Pad Prism version 5.03 was used to create the graphs.There were 37 patients with bilateral optic neuropathy (15 (40.5%) males; median age: 47.0 ± IQR 23; range 18–70 years) in the test group and 40 subjects in the control group . All patients had a clinical history of sudden onset functional visual disturbances, most frequently blurred vision. The median duration to presentation was 10 ± IQR 14 days, with a range of 2–21 days. Fourteen (37.8%) patients had normal fundoscopy and 23 (62.1%) showed optic disc edema. Sixteen had magnetic resonance imaging (MRI) and 4 showed features of multiple sclerosis, 12 had a normal MRI. Twenty three (62.1%) had ocular pain at the onset of the clinical symptoms, but none had pain at the time of the visual electrophysiological examination. RAPD was positive in 24 (64.9%) patients. Median visual acuity by logarithm of the minimal angle of resolution (logMAR) units was + 0.50 ± 0.8 in the right eye and + 0.20 ± 0.5 in the left eye.Median N75, P100 and N145 latencies in both eyes were significantly prolonged in patients with optic neuropathy when compared with the control group  of the optic neuropathy patients, had a LP:DT ratio below 1.7, the ISCEV cut-off level. The group difference in the LP:DT ratio seems to be primarily contributed by the greater dark trough amplitudes in the optic neuropathy group.The RPE forms a barrier between the retina and the systemic circulation, and it performs many vital functions to support the neural retina , 16. As However, based on the structural and functional abnormalities of optic neuropathy and the unique anatomical arrangement of RPE, alternative mechanisms can potentially contribute for EOG changes that we observed. First, Brudet-Wickel and van Lith reported evidence of partial, temporary disturbance of the choroidal circulation in anterior ischemic optic neuropathy that may cause for the abnormalities of light rise in EOG. Since the choroid is the principal vascular structure nourishing the outer retinal layers and the RPE, disturbance of choroidal circulation could have caused hypoxia of the retinal tissues . This inSecond, disc edema associated with optic neuropathy could lead to accumulation of fluid creating sub retinal space above the RPE , 25. SucWe conclude that RPE exhibits abnormal function as indexed by reduced LP:DT ratio primarily contributed by the increased dark trough amplitudes in EOG in the majority of patients with bilateral optic neuropathy.MRI and optical coherence tomography (OCT) facilities were not available readily for every patient due to financial constraints. Also, further studies using a large cohort of patients would add more conclusive evidence.Additional file 1: Table S1. Median latencies and amplitudes of PRVEP between optic neuropathy group and the controls. Table S2. Median latencies and amplitudes of PERG between optic neuropathy group and the controls.


Pomacentrus moluccensis Bleeker, 1853 (Pomacentridae) is a bright yellow damselfish. Here, we assembled a complete 16,801 bp genome for P. moluccensis mitochondrion by employing the Illumina MiSeq platform. The complete mitochondrial genome contained 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and one putative control region. The overall genomic structure of P. moluccensis mitochondrion was conserved with the gene arrangements of mitogenomes published in subfamily Pomacentrinae, and phylogenetic analysis confirmed the sister relationship among the genus Amblyglyphidodon, Amphiprion, and Chrysiptera. This complete mitochondrial DNA information of P. moluccensis will provide essential molecular reference to elucidate the geographical distribution, the phylogenetic relationship, and evolutionary history of the damselfish genus Pomacentrus. Pomacentrus Lacepéde, 1802 comprises 77 species and is the second most diversified damselfish  are one of the most abundant and diverse families of coral reef fish taxa Allen . They arPomacentrus moluccensi, also known as the lemon damsel, is widely distributed in the Indo-Pacific region (Drew and Barber P. moluccensis (Accession no. MT410992). An individual of P. moluccensis was sampled at East China Sea (30°45′33.0″N 126°22′10.2″E) in July 2013. The voucher specimen was deposited in the Research Institute of Basic Sciences of Incheon National University (Specimen ID: 2008-Pomacentridae025). Genomic DNA was extracted from muscle tissue by using the DNeasy Blood and Tissue kit . Genomic DNA was quantified using a Qubit 4 Fluorometer . The library construction and Illumina MiSeq sequencing were performed by a commercial company . Genomic libraries were constructed from the total genomic DNA (1 μg) using the TruSeq RNA Sample Preparation Kit according to the manufacturer’s instructions . Additional PCR procedure was conducted to confirm the DNA sequence of the control region. Overall sequences were annotated using the MITOS web-based software .The coral-dwelling damselfish P. moluccensis was 16,801 bp in length and contained a typical set of 13 PCGs, 22 tRNAs, two rRNAs, and one control region, as shown in Pomacentridae mitogenomes (Nam and Rhee CO1) mitochondrial DNA sequence showed that P. moluccensis grouped together with other representatives of Pomacentrus species in the family Pomacentridae (P. moluccensis mitogenome with including 16 published mitogenomes from Pomacentridae and Mugilidae (outgroup)  . We usedutgroup)  to selecutgroup)  with 100


Spodoptera littoralis) is one of the most destructive herbivorous insects worldwide. The present study reports the complete mitochondrial genome of S. littoralis collected from Egypt. The circular-mapping mitogenome was 15,408 bp in length with an overall A + T content of 81.1%, encoding a common set of 37 genes, including 13 protein-coding genes (PCGs), 22 tRNA genes, and two rRNA genes. Most PCGs were found to use conventional ATN as the start codon and TAN as the stop codon. The phylogenetic tree based on the nucleic acid sequences of 13 shared PCGs of 29 Noctuidae species revealed that S. littoralis and Spodoptera litura are sister species. The data in this study will be helpful to understand geographical genetic variations, phylogenetic relationships, and species identification of S. littoralis.The polyphagous cotton leafworm ( Spodoptera littoralis  (Lepidoptera: Noctuidae), is considered one of the most destructive herbivorous pests because of its high reproductive rate and associated heavy losses to crops. S. littoralis can feed on more than 100 host plants, potentially resulting in yield losses of 50%, which is related to its larval foliage consumption activity  are often regarded as ideal markers because of their rich distribution, maternal inheritance, and high mutation rate, and thus they are widely used in species identification, genetic diversity, and molecular phylogeny  in Egypt in 2011. The voucher specimen’s genomic DNA was assigned with a unique code (AGIS-SL-EG-2011) and deposited at the Agricultural Genomics Institute, Chinese Academy of Agricultural Sciences, Shenzhen, China. Total genomic DNA was extracted using the Qiagen Genomic DNA kit . DNA quality and concentration were determined using NanoDrop One UV-Vis spectrophotometer (Thermo Fisher Scientific). A total of 0.5 μg of genomic DNA was used to construct a 350-bp insert library, which was then sequenced using the Illumina NovaSeq 6000 platform with 150-bp paired-end reads. The mitogenome was assembled using the software NOVOPlasty v2.5.6   is 15,408 bp in length and contains 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and one non-coding A + T-rich region (putative control region). In the genome, the content of A + T is 81.1% , which represents a typical high A + T content among insect mitogenomes  has a complete stop codon (TAG), and two genes (cox2 and nad4) end with an incomplete stop codon (T). Further, 22 tRNA genes ranged in size from 65 to 71 bp. All tRNAs harbor typical predicted secondary cloverleaf structures, except for trnN and trnY.The complete mitogenome of S. littoralis, we downloaded mitogenomes of 29 Noctuidae species and one outgroup species Cydia pomonella (Tortricidae) published in GenBank. Multiple nucleic acid sequence alignments of 13 PCGs were conducted using MAFFT v7.455 (Katoh and Standley http://tree.bio.ed.ac.uk/software/figtree/). The phylogenetic analysis showed that the Spodoptera genus was monophyletic, and S. littoralis and S. litura were sister species. In summary, the current mitogenome sequence will be useful for the phylogenetic analysis of this species (S. littoralis is native to Africa and Israel (Yones et al. To validate the phylogenetic position of  species . Further species . S. litt