Primary vaginal cancer is a rare disease and, consequently, evidence about the outcome of treatment is scarce. The aim of our retrospective, observational multicenter study was to assess the oncological outcome of the nowadays standing treatment for vaginal cancer, namely radio(chemo)therapy, followed by image-guided adaptive brachytherapy (IGABT). Our study confirms the results of the earlier published small monocentric IGABT studies, showing a high local control with acceptable morbidity. Notably, patients with large (T3/T4) tumors especially seem to benefit from volumetric (3D) image-guided brachytherapy, as compared to two-dimensional-based radiotherapy. In addition, although interpreted with caution, as for cervical cancer, a higher dose seems to lead to better local control. These results should, however, be further investigated in a prospective trial.α/β10 was delivered to the clinical target volume (CTV) at the time of brachytherapy. Conclusions: In this large retrospective multicenter study, IGABT for primary vaginal cancer resulted in a high local control with acceptable morbidity. These results compared favorably with two-dimensional (2D) radiograph-based brachytherapy and illustrate that IGABT plays an important role in the treatment of vaginal cancer.Purpose: This study assessed outcomes following the nowadays standing treatment for primary vaginal cancer with radio(chemo)therapy and image-guided adaptive brachytherapy (IGABT) in a multicenter patient cohort. Methods: Patients treated with computer tomography (CT)–MRI-assisted-based IGABT were included. Retrospective data collection included patient, tumor and treatment characteristics. Late morbidity was assessed by using the CTCAE 3.0 scale. Results: Five European centers included 148 consecutive patients, with a median age of 63 years. At a median follow-up of 29 months (IQR 25–57), two- and five-year local control were 86% and 83%; disease-free survival (DFS) was 73% and 66%, and overall survival (OS) was 79% and 68%, respectively. Crude incidences of ≥ grade-three urogenital, gastro-intestinal and vaginal morbidity was 8%, 3% and 8%, respectively. Lymph node metastasis was an independent prognostic factor for disease-free survival (DFS). Univariate analysis showed improved local control in patients with T2–T4 tumors if >80 Gy EQD2 Primary vaginal cancer (PVC) is a rare gynecological cancer with an annual incidence of 0.8–1.0/100,000 women, corresponding to less than 3% of all gynecological malignancies .Treatment of vaginal cancer includes an organ-sparing approach with pelvic external beam radiotherapy (EBRT), followed by a brachytherapy (BT) boost to the residual tumor, to achieve a high dose with maximum sparing of the organs of risk (OAR). In the During the last decades, new imaging techniques have been adopted in radiotherapy. For EBRT, these developments include the use of computer tomography (CT) and/or magnetic resonance imaging (MRI) for contouring of the target and organs at risk (OAR), for highly conformal treatment planning and image guidance during treatment. These techniques have led to a decrease in dose delivered to the organs at risk, and consequently reduction of acute and late morbidity ,7.For brachytherapy similar improvements have taken place with the introduction of image-guided adaptive brachytherapy (IGABT). The principle of IGABT is to apply repetitive three-dimensional (3D) imaging (preferably with MRI), at minimum before EBRT and at time of BT, and combine these images with gynecological examination and define different target volumes according to their presumed tumor density meanwhile taking the tumor regression during EBRT into account . In locaVaginal cancer is a rare disease, and therefore only retrospective and mainly monocentric studies with a limited number of patients are available . In the In 2013 the Gynaecological Working Group of the Groupe Européen de Curiethérapie and the European Society for Radiotherapy and Oncology (GYN GEC–ESTRO) formed a task group with the general aim to introduce a common target concept IGABT in vaginal cancer. In preparation of this task, the multicenter retrospective international image-based adaptive BRAchytherapy for Vaginal cancer (retroEMBRAVE) study was conducted to collect clinical data from patients with primary vaginal cancer that have been treated with 3D-image-guided adaptive brachytherapy.The present study reports on the outcomes of the RetroEMBRAVE study.RetroEMBRAVE is an international retrospective observational study involving five European centers that actively participate in the GYN GEC–ESTRO network. All participants have experience with state-of-the-art IGABT for gynecologic cancer and participate in the EMBRACE  studies .All patients treated in the five centers were consecutively included in the study if they fulfilled the following criteria: histologically confirmed primary vaginal cancer ; MRI at time of diagnosis; treatment with curative treatment intent with combined external beam radiotherapy EBRT (+/− concomitant cisplatin) and CT–MRI guided (Pulsed-dose rate (PDR) or High-dose rate (HDR)) IGABT, or in selected patients brachytherapy alone.Patient, tumor, and treatment characteristics as well as outcome data related to disease status, survival and morbidity were collected. Data collection started April 2014 and closed in July 2017. Due to variation in commencement of image-guided BT in the participating centers, patients were treated between July 2001 and September 2016 with the majority of the patients (72%) treated after January 2009.All patients were clinically staged according to the International Federation of Gynecology and Obstetrics (FIGO) criteria . Clinicaα/β10) dose of 60–64 Gy.The target volume for EBRT included the vagina, paravaginal space, cervix, parametria and pelvic lymph nodes, and, dependent on the location of the vaginal tumor and pathological lymph nodes, the para-aortic and inguinal lymph nodes. A total dose of 45.0–50.4 gray (Gy) was given in 1.7–2.0 Gy per fraction. In case of pathological lymph nodes, a nodal  boost was given to a total equivalent . For the MRI/CT approach, aMRI with applicator (cylinder) in situ was conducted a few days before the application and matched with the CT on the applicator, at the time of brachytherapy. The CTV was delineated on the MRI and the organs at risk on the CT scan. During the period of patient inclusion, no vaginal-cancer-specific consensus guideline regarding target-volume definition and dose prescription was available. However, all centers prescribed—in accordance to their joint experience in cervical cancer ,13 and iPatients were assessed for disease status and adverse effects, according to institutional guidelines, at regular intervals. In general, this was every 3 months in the first 2 years, every 6 months for the third year and annually thereafter.p ≤ 0.05) for the univariate analysis were entered in a multivariable Cox regression model. Late severe (grade 3 and higher) urogenital (GU), gastro-intestinal (GI) and vaginal morbidity were scored according to the common terminology criteria for adverse events version 3.0  and analyzed in crude numbers.Local control (LC), pelvic control (PC), disease-free survival (DFS), overall survival (OS) and severe late morbidity were evaluated. Follow up for disease status was calculated as time from diagnosis until an event or last follow-up. Patients were censored at last follow-up or at the time of any disease recurrence during follow-up. Survival analysis for disease status was calculated by the Kaplan–Meier method, with 95% confidence intervals (95% CIs). Univariable analyses of risk factors for LC and DFS were conducted by Cox regression analysis. Significant risk factors  was used for statistical analysis.A total of 148 patients were included. Patients characteristics are shown in Treatment characteristics are shown in 3 (IQR 6.8–32.1), and the median D90 of the CTV was 80 Gy EQD2α/β10 (IQR 73.0–85.2). The median doses to the D2cm3 of the bladder, rectum, sigmoid and bowel were 64 Gy EQD2α/β3 (IQR 55–73), 64 Gy EQD2α/β (IQR 57–68), 49 Gy EQD2α/β (IQR 46–54) and 49 Gy EQD2α/β (IQR 46–57), respectively. Doses for sigmoid and bowel were not reported in 40 (28%) and 87 (62%) of the patients. In the remaining patients, these OARs were located far from the target, resulting in no significant dose from the brachytherapy boost. In seven patients, information on dose-volume histogram (DVH) parameters could not be retrieved from older treatment planning systems.The median volume of the clinical target volume (CTV) was 17.6 cmAt a median follow-up of 29 months (IQR 25–57), 42 recurrences  had been reported, of which 16 were isolated vaginal recurrences a. PatienActuarial two- and five-year local control (LC) were 86%  and 83% , respectively . Ninety Twenty-five patients (17%) developed severe (grade ≥3) morbidity. In 23 patients, one grade-three/four event was diagnosed. In the remaining two patients, two severe events were diagnosed. The crude incidence of ≥ grade-three urogenital, gastro-intestinal and vaginal morbidity was 8.1%, 3.0% and 8.1%, respectively.Only one grade-four event occurred, namely a life-threatening vesico-vaginal fistula. This patient had a bulky (6 cm) T2N0 tumor located in the lower third of the vagina. She was treated in 2008, with a combination of EBRT and concurrent cisplatin, followed by CT-guided brachytherapy. Due to the good response on EBRT, she was treated with an intracavitary only approach (mould) at time of brachytherapy. The fistula was diagnosed 2.5 years after treatment. She had no comorbidities and did not smoke. However, at the age of 37, she was treated for a vulvar Bowen disease with radiotherapy, and was thus partially re-irradiated.n = 7) and complete vaginal obliteration (n = 10). Nine out of ten patients with complete vaginal obliteration had involvement of the lower  third of the vagina at the time of diagnosis.No grade-five morbidity was seen. The most frequent severe morbidities were vesico-vaginal fistulas  towards better five-year local control in patients with a T1/T2  versus T3/T4 tumor  (p = 0.013)  a. Univar, 59–90) . In the = 0.013) . Patient= 0.013) b.90  of the CTV showed a trend in better local control in favor of patients who received more than 80 Gy EQD2α/β10 This difference was, however, not statistically significant    a. When a= 0.099) b. In pat= 0.036) c. Due toα/β10 in the clinical target volume at time of brachytherapy . T-stage, maximum tumor diameter at diagnosis and lymph node status were associated with DFS in univariable analysis. However, only lymph node status remained an independent prognostic factor for DFS , with two-year DFS for lymph-node-positive patients 55% versus 82%, respectively. Crude incidence of overall  severe (≥ grade 3) morbidity was 17%, of which vaginal and urogenital morbidities were most frequently observed.This study investigated the outcomes of definitive radiotherapy with CT/MRI-based radiotherapy in a large retrospective, observational cohort of consecutively included patients with primary vaginal cancer. Two-year local and pelvic control were 86% and 83%, respectively. In patients with advanced (T2–T4) tumors, a significantly better local control was found if patients received more than 80 Gy EQD2Introduction of the GEC–ESTRO recommendations for MRI-guided IGABT in locally advanced cervical cancer ,13, resuThere are several explanations for the improved outcomes presented in our study, compared to historical series. First, in the majority of patients, MRI was used for delineation and planning at time of brachytherapy. Since MRI is the preferred imaging modality for visualization of pelvic tumors due to its superior soft tissue contrast, the chance of a geographical miss is reduced, as compared to radiograph-based brachytherapy . Second,Despite the large number of included patients for this rare tumor, this retrospective study has several limitations. First, there was no published target concept during the period of patient inclusion. However, the participating centers were active in IGABT for cervical cancer and all adopting these basic concepts into their treatment, with a presumed high agreement in the clinical target volume for dose prescription. Second, different volumetric imaging modalities at brachytherapy were allowed, as long as a diagnostic MRI was available. This very much reflects the clinical practice where a transition is made also with regard to availability of MRI compatible IC/IS applicators. Third, the median follow-up (FU) of 29 months may seem short. However, the majority of local recurrences take place in the first 24 months . An explThe presence of lymph node metastasis was an independent prognostic factor for DFS. A finding that is in line with experience in cervical cancer, where this led to incorporation of the nodal status into the new FIGO 2018 classification. This underscores the importance of adequate imaging at the time of diagnosis with MRI and (PET–)CT for staging purposes ,28. In ap = 0.055) towards improved pelvic control was found in patients that were treated with a total dose of at least 70 Gy [α/β10, which is also in agreement with the recommendations from the American Brachytherapy Society (ABS) for interstitial brachytherapy for vaginal cancer [α/β10 in the CTV D90, at the time of brachytherapy (92% versus 75% at two years). Despite the limitations of this study, this result is in line with the impact of IGABT demonstrated in cervical cancer. For the small group of patients (n = 28) with T1 tumors, a very high local control rate was found and, thus, no dose effect relationship could be established , according to the recently published GYN GEC–ESTRO/ACROP recommendations for a common target concept in primary vaginal cancer, is essential to be able to evaluate the results in a multicenter setting [At present, clear evidence regarding the optimal dose that should be prescribed in patients with primary vaginal cancer is lacking. Most evidence has come from the results of IGABT in locally advanced cervical cancer within the EMBRACE studies . In onlyst 70 Gy . The autl cancer . Notablyl cancer . In our ablished a. To dra setting . A prospα/β10) to the clinical target volume was associated with a higher local control in univariate analysis. Fortunately, these high local control rates did not appear to lead to higher severe late toxicity, presumably due to the possibility of maximal sparing of the organs at risk with 3D planning. These findings warrant, however, prospective validation using the recently published common target concept and methodology for dose reporting in primary vaginal cancer.Our large contemporary multicenter study confirms the findings of previous small, monocentric studies regarding the role of image-guided brachytherapy in primary vaginal cancer, demonstrating good local and pelvic control of 86% and 83% at two years, respectively. Especially in T2–4 tumors an improved local control was found compared to historic radiograph-based studies. In these larger T2–4 tumors a dose of ≥80 Gy (cumulative dose in EQD2


This study aimed to identify the clinical characteristics of Chinese patients with ovarian endometrioid carcinoma (EC) and clear cell carcinoma (CCC) and to assess the impact of concurrent endometriosis on this group.The present study reviewed the medical records of patients who received initial treatment and a postoperative pathological diagnosis of EC or CCC at our center in China between 1998 and 2018.p = 0.001).Of 211 patients, 73 had pure EC, and 91 had pure CCC, and the remaining 47 had mixed cancer. The proportion of EC and CCC remained stable over past 21 years. The proportion of EC declined with aging and the age of EC onset to incline to the young. And the age of CCC onset had two peaks, namely, 36 and 77 years. After review by the pathologist, the number of endometriosis cases found in the pathological section of the analysis increased to 114, accounting for 54% of patients. As the stage progressed, the appearance of endometriosis became increasingly scarce in pathological sections(p = 0.000). And more patients were in premenopausal state in EC group(p = 0.040).Compared with CCC, EC had a higher frequency of concurrent endometrial cancer  and estrogen and progesterone receptor expression(p = 0.041). In patients with mixed cancer, mixed endometrioid histology was associated with better survival than other subtypes, even with stage III or poorly differentiated tumors(p = 0.001).In the pure group, multivariate analysis showed that correlation existed between relevance to endometriosis and worse outcomes(CCC and EC which are common in ovarian cancer patients who have associated with endometriosis have distinct clinicopathological characteristics. Attention should be paid to ovarian cancer patients with a history of endometriosis and those with concurrent endometriosis in pathological sections.The online version contains supplementary material available at 10.1186/s13048-021-00804-1. Ovarian cancer is the second most common gynecologic malignancy, with over 90% of cancers arising from epithelial cell s, 2. The Endometriosis is a benign gynecologic disease that is characterized by endometrial glands and stroma occurring outside the uterus. Endometriosis is a risk factor for epithelial ovarian cancer , 7, and The prognosis of early ovarian CCC is good, but late-stage CCC is known to be less sensitive to platinum-based front-line chemotherapy and to be associated with a worse prognosis than serous adenocarcinoma or endometrioid adenocarcinoma during the same period –19. It iWith ethical approval (No. 2020PHB212–01) given for ovarian cancer specimen collection with annotated clinical information, all patients with primary EOC who were treated at Peking University People’s Hospital from 1998 to 2018 were reviewed. Individual informed patient consent was not sought, as these data were routinely obtained and used as part of these patients’ clinical care. The women were followed until December 2019 or until they died of ovarian cancer or other conditions, whichever happened first.Data from electronic medical records were retrospectively reviewed. Two pathologists reviewed the pathology details to determine whether there was concurrent endometriosis. We screened patients according to the established criteria , FIGO stage, fertility history, first symptoms, blood cancer antigen-125 (CA-125) level (before and after surgery), tumor size and side, surgical procedure, tumor grade, surgical debulking status, endometrial pathology , chemotherapy regimen, objective response to chemotherapy, progression-free survival and overall survival.Endometrial lesions included endometrial cancer and endometrial dysplasia. If there was endometrial cancer by uterine pathology, the Scully criteria were used to distinguish between dual primary and metastatic disease  was used. All p-values were 2-sided, and p-values less than 0.05 were considered statistically significant.Summary statistics were used to describe the data. Medians (ranges) or means (standard deviations) were used for continuous variables. After a normal distribution was confirmed with the Shapiro-Wilks test, the Mann-Whitney U test was used to compare median values, and Student’s t-test was used to compare mean values. Categorical variables are presented as frequencies (percentages). Fisher’s exact test or the χ2 test were used to analyze the distributions of characteristics according to their associations with endometriosis. Survival curve analyses were performed with the Kaplan-Meier method, and comparisons were performed using the log-rank test. A Cox proportional hazards model was used to perform univariate and multivariate analyses to evaluate the prognostic significance of the association with endometriosis and other clinicopathological features. Multivariate http://www.R-project.org).The Joinpoint Regression Program 4.6.0.0 provided by the National Cancer Institute was used to determine potential changes in the temporal trends in the incidence rate. The trends in the histological subtypes of ovarian cancer were examined for every calendar year or every patient age. Linear segmented regression analysis was utilized for the model. Statistical analyses were performed using SPSS 26.0 (IBM), GraphPad Prism 8.0 and R 3.0.6 , followed by clear cell , endometrioid , and mucinous . Figure One can interpret the data as indicating that serous ovarian cancer patients were getting older, while patients with endometrioid ovarian cancer were getting younger over time Fig.  C-D. AmoThe detailed data are presented in Tables p = 0.001).The common symptoms at initial presentation were sequentially palpable mass, abdominal pain, incidental finding, abdominal distension and irregular vaginal bleeding. A normal preoperative serum CA-125 value was observed in 27.5% (58/211) of patients. The distribution of FIGO stage was as follows: Stage I, 54% (114/211); Stage II, 18.5% (39/211); Stage III, 25.6% (54/211); and Stage IV, 1.9% (4/211). There were 16 patients with a previous history of endometriosis. In the initial records, endometriosis was found by pathology in 41 patients. After review by the pathologist, the number of endometriosis cases found in the pathological section of the analysis increased to 114, accounting for 54% of patients. From the early stage to the late stage, the frequency of endometriosis by pathology gradually decreased  patients received comprehensive staging surgery, and 97 (46.0%) patients received cytoreductive surgery. A total of 142 (67.3%) patients had no residual lesions, and 13 (6.2%) patients had residual lesions less than 1 cm in size. In total, 177 (83.9%) patients received chemotherapy, among which 174 (82.3%) received PBC.p = 0.000). According to the standard of Scully et al. [p = 0.000). The proportion of premenopausal women was higher in the EC group (p = 0.040). EC were predominantly positive for estrogen receptor (ER) and progesterone receptor (PR), but CCC exhibited lower ER and PR expression (p = 0.000) (Table The present study found that the age at diagnosis was 50.0 ± 11.7 range 24 to 79) years in the EC group and 52.6 ± 9.5 years range 32 to 77) in the CCC group  died, and 28 patients (17.1%) experienced relapses. The 5-year OS and PFS rates of CCC patients were 88.0 and 89.9%, respectively. The 5-year OS and PFS rates of EC patients were 92.1 and 78.4%, respectively. The difference in prognosis between EC and CCC was not significant (p < 0.05). Survival analysis revealed significant differences in PFS and OS between patients whose disease was related to endometriosis compared to those whose disease was unrelated to endometriosis. The Kaplan-Meier curve for PFS showed a survival advantage for patients whose disease was unrelated to endometriosis .There were 47 cases of mixed cancers of other histological types, including 31 cases of cancers mixed with ECs, 13 cases of cancers mixed with CCCs, and three cases of cancers mixed with ECs and CCCs  died, and 20 patients 25.5%) experienced relapses. The 5-year OS and PFS rates of mixed carcinoma were 77.9 and 57.4%, respectively. The difference in prognosis between mixed carcinoma and pure carcinoma was significant . The pure cancer group had a better overall prognosis than the mixed group, and among mixed cancers, EC was always associated with a better prognosis. In another single-center study, Dawn J. Story et al. found that despite their similar response rates to platinum-based chemotherapy (PBC), endometrioid ovarian cancer has a better survival outcome than ovarian serous adenocarcinoma .Two histologic types, CCC and EC, are common histological types of ovarian cancer in patients who have associated endometriosis. However, both tumor types have distinct clinicopathological characteristics and molecular phenotypes. The high incidence of combined endometrial disease in the EC group distinguishes EC from CCC. Studies have shown that EC subtypes have a much higher chance of concurrent endometrial cancer than other histological subtypes of epithelial ovarian cancer in the same period . Our datIn terms of morphological characteristics, ECs show broad morphological similarities to their endometrial counterparts. Overall, the most common molecular alterations in EC include mutations in CTNNB1 31–53.3%), PIK3CA (15–40%), ARID1A (30%), and PPP2R1A (7–16.6%) . This suggests that the proportion of EC and CCC coexisting with endometriosis was previously underestimated. The reasons for the differences before and after the pathological review may be as follows: 1) tumor cells are so aggressive that they invade and destroy most or all endometriotic tissues; 2) because pathology reports are used for the diagnosis of malignant tumors, the reporter focuses on malignant tumors rather than endometriosis; and 3) we focus more on the tumor site when taking pathological sections. In clinical work, it is very difficult to obtain specimens of endometriosis, tumor tissues and transitional tissues all at once.p = 0.001). The probability of endometriosis coexisting in early disease is higher, which is consistent with the conclusion drawn by Kim HS et al. [In this study, as the stage progressed, the appearance of endometriosis became increasingly scarce in pathological sections . Considering that the absence of endometriosis does not mean that it never existed, we consider a history of endometriosis and cooccurrence of endometriosis as a whole to be a clinical pathological factor. As a result, we found that patients with endometriosis had worse outcomes. This may be explained by the abnormal immune regulation system and aberrant pelvic microenvironment in patients with endometriosis [Several studies have reported that endometriosis has no effect on the prognosis of EAOC , 40, andetriosis . As a beetriosis , immune To our knowledge, this is the largest retrospectively collected pure and mixed ovarian clear cell and EC set in China. We have discussed the relationship between endometriosis and EC or CCC, aiming to provide some guidance for clinical work. Attention should be paid to ovarian cancer patients with a history of endometriosis and those with concurrent endometriosis in pathological sections. This study also has some limitations. Multicenter, large-sample prospective clinical research is necessary in the future.Additional file 1.


EA reduced the days in estrus caused by DHEA, improved the abnormal sex hormone receptor genes, and attenuated the DHEA-induced histomorphological changes in mouse ovaries. The average expressions of the androgen receptor (AR), estrogen receptor (ER), luteinizing hormone receptor (LHR), and follicle-stimulating hormone receptor (FSHR) genes in the ovary greatly increased after DHEA treatment and significantly decreased in the DHEA + EA group. After EA treatment, the cystic follicle (CF) number was reduced and corpora lutea (CL) increased in the DHEA + EA group compared to the DHEA group. EA improved glucose intolerance and insulin intolerance. Statistical analysis of intraperitoneal glucose tolerance test-area under curve (IPGTT-AUC) glucose levels revealed a significant decrease in DHEA group mice compared to the control and DHEA + EA groups. EA was found to restore fasting blood glucose, fasting serum insulin, and HOMA-IR. In summary, our study suggests that EA has a remarkable effect in the DHEA-induced murine PCOS model. Management of EA could improve estrous cycle, hormonal disorders, abnormal sex hormone receptors in ovaries, ovary morphology, and insulin resistance in PCOS mice.Polycystic ovarian syndrome (PCOS) is a common, complex, and heterogeneous endocrine and metabolic disorder. There is no standardized treatment, and it therefore requires individualized therapies according to the symptoms and pathogenesis of each patient. The present study aimed to determine the effect of electroacupuncture at the acupoints Zusanli (ST36), Sanyinjiao (SP6), and Neiguan (PC6) on reproductive disorders and insulin resistance in a murine model of PCOS induced by dehydroepiandrosterone (DHEA). Vaginal smear analysis was used to determine mice estrous cycle; intraperitoneal glucose and insulin tolerance tests were adopted to analyze metabolic characteristics; enzyme-linked immunosorbent assay was used to measure hormone levels; gene expression was quantified with real-time PCR; hematoxylin and eosin staining was used to observe ovarian morphology. We observed disordered estrous cycle, polycystic ovarian morphology, and higher levels of homeostasis model assessment-insulin resistance (HOMA-IR) and testosterone (T), indicating successful modeling of PCOS. DHEA increased levels of estrogen (E Polycystic ovarian syndrome (PCOS) is a common but complex and heterogeneous endocrine and metabolic disorder characterized by anovulation or oligoovulation, hyperandrogenism, and polycystic ovarian morphology –4. ApproAccording to histological analyses, the ovaries of PCOS patients have numerous antral follicles; however, the etiology needs further research. Reactive oxygen species (ROS) induced by PCOS could cause ovarian damage, and some reports have shown that antioxidants have a positive effect on ovaries , 9. RepoThere have been reports that both pharmacological and nonpharmacological therapies for PCOS may cause side effects such as cardiovascular disease and multifetation , 16. A gPrevious research has demonstrated that DHEA-induced PCOS models have various representative characteristics such as estrous cycle disorder, impaired sensitivity to glucose and insulin, disruption of hormones, and change in ovarian morphology –27. In tThirty 21-day-old female C57BL/6J mice were purchased from the Experimental Animal Center of Guangzhou University of Chinese Medicine (SCXK (Yue) 2018–0034). All animals were housed in South China Research Center for Acupuncture and Moxibustion , with sufficient food and water freely available. All animal studies described in this article were approved by the Ethical Committee of Guangzhou University of Chinese Medicine.n = 12) and the DHEA group (n = 18). The DHEA group was given DHEA subcutaneously across their back  for 20 consecutive days. The control group mice were injected with 90 μl sesame oil and 10 μl 95% ethanol daily for 20 consecutive days. Estrous cycle was monitored daily from day 10 after DHEA treatment until the end of the experiments. After 20 days of treatment, six mice were selected at random from each group to confirm the PCOS model, and the DHEA group was randomly divided into two groups: the DHEA control group (n = 6) and the DHEA + EA group (n = 6). The DHEA + EA group received EA stimulation at the acupoints Zusanli (ST36), Sanyinjiao (SP6), and Neiguan (PC6). Mice were stimulated with density waves from a Hwato SDZ-III machine at a frequency of 2 Hz and an intensity of 1 mA for 20 min per day for four weeks. When the EA treatment was finished, the mice were fasted overnight, anesthetized, and then sacrificed. Serum samples and left ovaries were collected and stored at −80°C for ELISA and real-time PCR. The left ovaries were dissected for hematoxylin and eosin staining.On day 25 after birth, the mice were randomly placed into two groups: the control group . Estrous stages were determined based on the predominant cell type as described previously , 28.After 20 days of DHEA treatment, the mice fasted for 12 h before the experiment. Blood was extracted from the tail vein following disinfection of the tail with 75% ethanol. After measuring the fasting glucose levels at 9 am by ACCU-CHEK Performa (YZB/GER 5513–2014), the mice were given D-glucose  through intraperitoneal injection. Blood glucose was then measured at 30, 60, 90, and 120 min.Three days after the intraperitoneal glucose tolerance test (IPGTT) experiment, mice were fasted for 6 h before intraperitoneal injection with insulin . Tail blood sampling was performed at 9 am and then at 30, 60, 90, and 120 min after intraperitoneal injection.At the end of the EA treatment, the mice were anesthetized and blood samples were collected from the orbit. After standing at room temperature for 2 h, blood samples were centrifuged at 2500 rpm for 15 min. The upper serum was extracted and stored at −80°C. Serum estrogen, progesterone, testosterone, follicle-stimulating hormone, luteinizing hormone, and insulin were determined with mice kits . The procedures were carried out in accordance with the manufacturer's instructions.−ΔΔCT method. The primer sequences are AR: forward: TAAAGACATTTTGAACGAGGCC, reverse: GTCAGATATGGTTGAATTGCCC; ER: forward: CTACTACCTGGAGAACGAGC, reverse: GCGTCGATTGTCAGAATTAGAC; PR: forward: TAGTCTCGCCTATACCGATCTC, reverse: CTTCCCTATGAGTGGCTTCTAC; GNRHR: forward: ATTCTCAGCATTGTCTTTGCAG, reverse: GCA-TTGCAGATTAGCATGATGA;GAPDH: forward: GCACCACCAACTGCTTAG, reverse: CAGTGATGGCATGGACTG.Ovaries were collected and stored immediately at −80°C. Total RNA was extracted from the ovaries using TRIzol reagent (Life Technologies) according to the manufacturer's instructions. cDNA was generated with the PrimeScript reverse transcriptase reagent kit . Real-time PCR (RT-PCR) was performed using TB Green Premix Ex Taq II . The program used for RT-PCR was as follows: 95°C for 30 s; 40 cycles of 95°C for 5 s and 60°C for 31 s; 95°C for 15 s; 60°C for 1 min; 95°C for 15 s; and 60°C for 15 s. Relative gene expression was calculated using the 2μm serial sections with a tissue slicer . To get the most considerable sections of each ovary, one out of every five sections was selected. The sections were stained using the hematoxylin and eosin staining kit  following the manufacturer's guidelines. Corpora lutea were counted using an optical microscope .The left ovaries were fixed with 4% paraformaldehyde at 4°C for 24 h. After tissue dehydration and paraffin embedding, the ovaries were cut and sliced into 5 p < 0.05 was considered statistically significant.All statistical analyses were performed in GraphPad Prism . One-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used to analyze differences among groups. T levels than the control mice  gene expression was decreased to the control level . The estTo assess EA efficacy on glucose tolerance and insulin tolerance of the PCOS mice, IPGTT and IPITT were performed. After glucose injection, the blood glucose level of the DHEA group was significantly higher than that of the control group at 30 min . Mice inBecause of the complexity of polycystic ovarian syndrome, there is no standardized treatment available; treatment is individualized according to the symptoms and pathogenesis of each patient. For example, acne and hirsutism caused by excess androgen often have psychological repercussions in women with PCOS. Bleaching, plucking, shaving, waxing, chemical treatment, laser hair removal, and electrolysis could improve hirsutism, and dermabrasion, laser therapy, and light therapy can effectively treat acne –31. WithPanax ginseng extract (100 μg/mL) as an antioxidant not only had a positive effect on growth of isolated preantral follicles from the ovaries of prepubertal mice but also increased the expression of PCNA and FSHR genes [Apium graveolens and Cinnamon zeylanicum could mitigate ovarian tissue injury in rats induced by PCOS. Serum levels of FBS, insulin, triglyceride, low-density lipoprotein, and cholesterol all showed obvious improvement [Oxidative stress has a pivotal impact on the biological functions of reproduction and metabolism and also plays a key role in the pathophysiology of PCOS. Excessive mitochondrial production of ROS can damage the function of mitochondria and even cause cell apoptosis. A previous study reported CoQ10 as an antioxidant compound could attenuate ovarian dysfunction induced by cyclophosphamide. CoQ10 reduced ROS levels and increased expression of the proliferation cell nuclear antigen (PCNA) and FSHR genes . AnotherHR genes . Fariba rovement .Although the pathological mechanism of PCOS has not been clarified, most evidence has indicated that it has a close relationship with obesity and insulin resistance . ConsideAcupuncture plays an essential role in traditional Chinese medicine, where it is widely accepted and used to treat many diseases, mainly because of the absence of side effects compared to many medicines. Electroacupuncture (EA) is a new treatment comprising traditional acupuncture combined with pulsed electrical stimulation. In recent years, the mechanism underlying the effect of EA has been widely studied. A randomized controlled trial found that, after 16 weeks of low-frequency EA treatment, body weight, body mass index, waist-hip ratio, oral glucose tolerance test, insulin release, and glucose and lipid metabolism indicators such as total cholesterol, triglycerides, and high-density lipoprotein cholesterol decreased compared with the baseline. The results show that EA improves insulin resistance in PCOS patients by regulating glucose and lipid metabolism . The thrIn this study, we successfully established a mouse model of PCOS with irregular estrous cyclicity; excessive estrogen, progesterone, and testosterone in serum; and increased plasma insulin, LH, and FSH levels. The pathological changes of ovaries were as follows: the number of corpora lutea and mature oocytes decreased, atresia follicles appeared, and expressions of AR, ER, PR, and GNRHR were increased. After EA treatment, excessive serum hormones and sex hormone receptors returned to normal levels, and ovarian morphology tended to improve. We also conducted IPGTT and IPITT experiments, and the results suggested that, after EA treatment, mice with PCOS were more sensitive to glucose and insulin. Thus, EA had a positive effect on IR caused by PCOS.The hypothalamus-pituitary-ovary axis regulates reproductive functions of female mice. In PCOS, the massive LH stimulated ovarian theca cells lead to production of more androgen. In ovaries, follicles are not sensitive to FSH, which may be a critical factor in PCOS. This further causes reduced conversion efficiency of androgens to estrogens, leading to hyperandrogenism.ß and serine/threonine-specific protein kinase (AKT) returned to normal in primary granulosa cells [However, the relationship between IR and PCOS is unknown. Some studies have reported the mechanism of EA in the treatment of IR in PCOS. For instance, EA treatment could activate the activated protein kinase (AMPK) pathway in PCOS rats, inhibiting high expression of sterol regulatory element-binding protein-1 (SREBP1). Phosphorylation of insulin-stimulated insulin receptor sa cells .ß-endorphin [α [ß-Endorphin in rats could inhibit GnRH and LH secretion [α-Melanocyte-stimulating hormone has an excitatory effect on the GnRH neuron by inducing central melanocortin receptors [γ-aminobutyric acid (GABA) and glutamate [GABAB1 knockdown mice had a disordered estrus cycle and decrease in fecundity pulse frequency [ß-endorphin concentration in both the hypothalamus and peripheral blood and increase ovarian blood and ovarian nerve growth factor (NGF) [ß-endorphin and improving the concentration of hormones secreted by the ovaries.Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARC) not only are a central regulator of energy homeostasis but also play a role in reproductive circuits. POMC neurons in mice with IR develop perturbations in maintenance of glucose metabolism and leptin sensitivity . It is pndorphin . Furtherrphin [α , and therphin [α . ß-Endorecretion . α-Melaneceptors . POMC nelutamate . GABA islutamate . GABAB1 requency . A meta-requency . It has or (NGF) , 55, 56.In the present study, we demonstrated that EA has a considerable effect on DHEA-induced PCOS mice. However, our study did not address the mechanism of acupuncture in treating PCOS. We did not examine the effect of acupuncture on neuronal sensitivity to glucose and did not examine changes in insulin receptor and leptin receptor on POMC neurons. In future studies, we will focus on how EA treatment improves IR through POMC neurons.In summary, our study suggests that EA has an ameliorative effect on PCOS model mice induced by DHEA. EA can improve estrous cycle, hormonal disorders, abnormal ovarian sex hormone receptors, ovarian morphology, and insulin resistance in PCOS mice. Therefore, EA may represent a valid therapeutic option for women with PCOS.


The diagnosis of community-acquired pneumonia (CAP) with chronic heart failure (CHF) is associated with objective difficulties. Our case–control study aims to establish whether established serum inflammatory biomarkers are relevant to the diagnosis of CAP in patients with CHF. Seventy inpatients with previously diagnosed CHF and suspected non-severe CAP were recruited and then stratified into two subgroups with confirmed and rejected diagnosis of CAP. C-reactive protein (CRP), procalcitonin (PCT), tumor necrosis factor α (TNFα), interleukin-6 (IL-6) and brain natriuretic peptide (BNP) were measured. The value of biomarkers was determined using logistic regression, and their discriminatory efficacy was assessed by analyzing receiver operating characteristic (ROC) curves. Significantly higher levels of CRP 50.0 (35.5–98.5) mg/L, PCT 0.10 (0.05–0.54) ng/mL and IL-6 46.1(21.4–150.3) pg/mL in cases were identified as compared to the control group—15.0 (9.5–25.0) mg/L, 0.05 (0.05–0.05) ng/mL and 13.6  pg/mL, respectively. The Area Under the ROC Curve (95% CI) was the highest for CRP—0.91 (0.83–0.98), followed by PCT—0.81 (0.72–0.90) and IL-6—0.81 (0.71–0.91). A CRP value of >28.5 mg/L had an optimal sensitivity and specificity ratio (85.7/91.4%). In conclusion, the measurement of serum CRP, PCT and IL-6 levels can be useful for the diagnosis of CAP in patients with CHF. CRP showed optimal diagnostic utility in this population. Streptococcus pneumoniae (S. pneumoniae), Influenza A, Mycoplasma pneumoniae and Chlamydophila pneumoniae, and the main risk factors are age, smoking and comorbidities. CAP disproportionately affects persons who are very young or very old, with an annual incidence of 9.2 to 33 per 1000 person-years [Community-acquired pneumonia (CAP) is one of the most common acute infections requiring admission to hospital. The main causative pathogens of CAP are S. pneumoniae remains the most commonly isolated pathogen in CAP, the relative frequency of other pathogens has increased. Clinical suspicion should be driven by comorbidities and other risk factors [Although the diagnosis and treatment of CAP are straightforward in most cases, they can be more complex, and recent data indicate that the mortality of CAP in the UK is surprisingly high. Although  factors . Commonl factors . The dia factors ,7. Impor factors .Symptoms and signs lack sensitivity and specificity, and chest radiography is not very accurate . The senThe overdiagnosis of pneumonia leads to the inappropriate use of antibiotics and may delay appropriate management of mimetic disease. Alternative imaging strategies, including CT or pulmonary ultrasound, may improve the diagnosis of pneumonia. CXR is distinguished by relatively low specificity for CAP verification in the population of patients with CHF .Patients with CAP should be assessed for disease severity using the CURB-65 score (or its derivative CRB65) . The CURBiomarkers may facilitate clinical decisions in order to guide antimicrobial treatment and the prediction of prognosis in community-acquired pneumonia. For optimal management and use of healthcare resources in CAP, early identification of the causative agent(s), recognition of disease severity and prediction of unfavorable outcome are of major importance . BiomarkInflammatory biomarkers are undoubtedly among the crucial molecules in the pathogenesis of CAP, where the most studied are C-reactive protein (CRP), procalcitonin (PCT), tumor necrosis factor α (TNFα) and interleukins, namely IL-6. The utilized markers of cardiac dysfunction include creatine kinase-muscle/brain, troponins, copeptin, pro-adrenomedullin, atrial natriuretic peptide and B-type natriuretic peptide (BNP). Parallel measurement of biomarkers of different profiles may prove useful in the cases of coexistent CHF and CAP ,17,18.Our research aims to establish whether established serum inflammatory biomarkers are relevant to the diagnosis of CAP in patients with CHF. One specific question is addressed as follows according to the PICO principles: In adults with CHF, does measuring established serum inflammatory biomarkers improve the diagnosis of CAP?This study was designed as a case–control study conducted at the two primary care hospitals in Smolensk, Russia. This research was performed in accordance with the STROBE guidelines  Table S1The study comprised 70 patients, aged over 40 years, from a hospitalized pool, between September 2013 and September 2014. The inclusion process is presented in Guidelines from the Infectious Diseases Society of America advise diagnosing CAP based on suggestive examination findings and characteristic infiltrate on chest radiography with or without microbiologic data . Patients’ heart failure classifications are determined by a doctor based on their heart failure symptoms and functional limitations. The measure of the severity is based on the New York Heart Association Class guidelines. New York Heart Association functional class II–IV CHF includes:Class II (Mild): Patients with cardiac disease resulting in slight limitation of physical activity. They are comfortable at rest. Ordinary physical activity results in fatigue, palpitation, dyspnea or anginal pain.Class III (Moderate): Patients with cardiac disease resulting in marked limitation of physical activity. They are comfortable at rest. Less than ordinary activity causes fatigue, palpitation, dyspnea or anginal pain.Class IV (Severe): Patients with cardiac disease resulting in the inability to carry on any physical activity without discomfort. Symptoms of heart failure or the anginal syndrome may be present even at rest. If any physical activity is undertaken, discomfort is increased.Chest CT was obtained upon admission and interpreted in a blinded fashion by 3 observers and classified as definite, normal or uncertain for CAP. Bayesian analysis was applied to assess the accuracy of chest radiograph in the diagnosis of pneumonia.g for 15 min and stored in several aliquots at −70 °C.Blood samples were obtained at hospital admission with serum and plasma samples drawn into pyrogen-free vacutainer tubes. Tubes for plasma samples contained EDTA as an anticoagulant. Serum or plasma was separated from whole blood within 60 min by refrigerated centrifugation at 2000× The CRP was measured using photometric turbidimetry immunoassay with semiautomatic analyzer Humalyzer 2000  and HUMAN diagnostic kits. PCT was measured using chemiluminescence method with automatic mini-VIDAS analyzer  and BioMerieux diagnostic kits (VIDAS BRAHMS). Cytokines (IL-6 and TNFα) were measured with the use of solid-phase ‘sandwich’ variant of enzyme linked immunosorbent assay (ELISA) by means of diagnostic kits Interleukin-6-IFA-Best and alfa-FNO-IFA-BEST  and Multiscan EX IFA-reader . BNP measurement was made using chemiluminescence method with automatic analyzer ADVIA Centaur XP , Siemens diagnostic kits (closed system) and assayed BIO-RAD controls (USA).Reference values of biomarkers were as follows: CRP ˂ 8 mg/L or equal, PCT less than 0.1 ng/mL, IL-6 < 10 pg/mL or equal, TNFα < 6 pg/mL or equal, BNP 0–80 pg/mL for patients under 60 and 0–100 pg/mL for those >60 years.p-value was less than the level of significance (0.05) in two-tailed tests.Statistical data processing was performed with the use of R v.3.3.2 free software environment for statistical computing and graphics . Based on the sample size, the distribution of numerical variables was considered to be abnormal. Summary statistical data are presented as the number of observations, median and interquartile range (IQR) with 25th–75th percentile values. Intra-group comparisons were carried out by means of Wilcoxon test for paired samples with Holm–Bonferroni adjustment for multiple comparisons. Mann–Whitney test was applied to study inter-group differences of continuous variables. Categorical variables are presented as absolute and relative rates that were compared using Fisher’s exact test. Sensitivity and specificity for each biomarker are presented. CRP cut-off >28.5 mg/L was chosen to achieve the optimal balance between sensitivity and specificity, while giving priority to sensitivity, since the risk of underdiagnosis of CAP and inadequate treatment in terms of clinical consequences is more serious than overdiagnosis, although this can lead to over-prescription of antibiotics. Deviations into higher or lower CRP value will significantly degrade either sensitivity or specificity. The statistical significance of biomarkers in the diagnosis of CAP was determined using logistic regression. First, the independent efficacy of each parameter was measured by constructing univariable predictor models. Discriminatory efficacy was assessed by analyzing receiver operating characteristic (ROC) curves and was numerically interpreted using Area Under the ROC Curve (AUC) values. All differences were considered to be statistically significant when the Our study included 35 cases and 35 controls—median (IQR) age 78 (64–82) and 77 (71–82) years old, respectively. Baseline demographic and clinical characteristics of patients are presented in p < 0.0001 for both comparisons).The results of the measurements of serum CRP, PCT, IL-6, TNFα and BNP concentrations are presented in For TNFα, there were no statistically significant differences in the concentrations of this biomarker during between-group comparisons with all median values in both groups being within the reference range.The concentration of BNP was 141.2 (51.6–362.1) pg/mL in cases vs. 117.6 (47.8–256.8) pg/mL in the control group. There were no differences in the values of this biomarker between the two groups.p = 0.0001), PCT (p = 0.042) and IL-6 (p = 0.003) demonstrated significant associations with the presence of CAP, while TNFα (p = 0.092) and BNP (p = 0.339) did not bear any relationship to this diagnosis. After performing regression analyses using five individual models for each predictor, CRP , and there were isolated cases of other bacterial pathogens, such as Haemophilus influenzae, Enterobacterales and Staphylococcus aureus.Despite the fact that the etiology of CAP was not a subject of the study, pathogens were identified in 15/35 (42.9%) cases. p < 0.001) [The hypersecretion of CRP is registered during other conditions including CHF . Serum C< 0.001) . Regression analysis confirmed the diagnostic value of CRP for the prediction of CAP in CHF patients with excellent discriminatory ability . Our results match those previously demonstrated by other authors ,23,25,26Alongside CRP, PCT is a more specific biomarker of acute generalized bacterial inflammation . The ovePCT readings in CHF patients are also variable. Our results concur with the latter findings: PCT measurements in CHF patients without CAP were lower than 0.1 ng/mL in almost all cases. Such contradictory findings can be attributed to the differences in baseline characteristics of CHF patients. Significant PCT elevation may be due to the predominance of patients with marked left ventricular (LV) systolic dysfunction in those samples . In our Regression analysis demonstrated high PCT efficacy for the diagnosis of CAP in CHF patients with AUC = 0.81 95% CI 0.72–0.90). According to the literature, the predicting value of PCT for the diagnosis of CAP in different patient populations is, nevertheless, relatively variable 2–0.90. A,26,27. IIL-6 is a typical proinflammatory cytokine, which is elevated in various infectious and non-infectious diseases. Its mean concentration increases in CAP cases in the range of 48 pg/mL to 3569 pg/mL, being highly variable due to different study designs, although not usually exceeding 300 pg/mL ,34. The TNFα is a multifunctional proinflammatory cytokine. In CAP patients, the published results ranged from being close to normal to greatly elevated ,39. In oThe role of TNFα in the pathogenesis of CHF has been demonstrated before, but the degree of its effect on the development and progression of the disease is not yet completely determined. Normal and elevated mean values of biomarkers in CHF were detected in relevant studies with similar frequency ,40. In oBNP is considered to be the ‘gold-standard’ biomarker of CHF development and progression, although its concentration could rise in other illnesses as well, while still being associated with cardiovascular injury in most cases. The variability of published results in CAP (mean BNP level from 26 ± 15 pg/mL to 273 ± 36 pg/mL) could be attributed to the presence of concomitant CHF in a certain proportion of patients, as they were not formally excluded. Komiya et al. evaluated the usefulness of BNP and CRP for distinguishing an acute lung injury/acute respiratory distress syndrome (ALI/ARDS) from cardiogenic pulmonary edema (CPE) in a cross-sectional study . AlthougDue to the inclusion of patients with CAP and confirmed CHF in the study, serum BNP concentrations were logically elevated and amounted to 141.2 (51.6–362.1) pg/mL. Similar results were obtained in the group of CHF patients without CAP. The literature data demonstrate the wide variation of this parameter in patients with CHF ,43. ThisThe BNP values obtained for CHF patients in our study, irrespective of the presence of CAP, did not deviate from the previously mentioned range of values. The lack of difference in BNP readings points to the futility of its additional use for CAP diagnostics in this patient category. A limitation of our study was the relatively small sample size, which allows us to regard the obtained results as preliminary, requiring verification in larger studies. The study would also be strengthened by using a validation cohort, although our algorithm based on proposed CRP cut-off >28.5 mg/L was validated further in a randomized prospective controlled study that recruited 76 patients in each arm . It provClinical features of CAP in CHF hamper the diagnostic process, providing the impetus for the search for affordable, effective and safe confirmatory diagnostic methods. Such serum biomarkers as CRP, PCT and IL-6 were significantly elevated in CAP in comparison to isolated CHF. The measurement of TNFα and BNP in this scenario had no practical value. CRP was proven to be the most useful diagnostic biomarker with an elevation of >28.5 mg/L, allowing us to verify the presence of CAP in patients with concomitant CHF with sufficiently high sensitivity (85.7%) and specificity (91.4%).


Several significant advancements in the field of bone regenerative medicine have been made in recent years. However, therapeutic options, such as bone grafts, have several drawbacks. There is a need to develop an adequate bone substitute. As a result, significant bone defects/injuries pose a severe challenge for orthopaedic and reconstructive bone tissue. We synthesized polymeric composite material from arabinoxylan (ARX), β-glucan (BG), nano-hydroxyapatite (nHAp), graphene oxide (GO), acrylic acid (AAc) through free radical polymerization and porous scaffold fabricated using the freeze-drying technique. These fabricated porous scaffolds were then coated with chitosan solution to enhance their biological activities. The complex structure of BG, nHAp, GO was studied through various characterization and biological assays. The structural, morphological, wetting and mechanical analyses were determined using FT-IR, XRD, XPS, SEM/EXD, water contact angle and UTM. The swelling (aqueous and PBS media) and degradation (PBS media) observed their behavior in contact with body fluid. The biological activities were conducted against mouse pre-osteoblast cell lines. The result found that BGH3 has desirable morphological, structural with optimum swelling, degradation, and mechanical behavior. It was also found to be cytocompatible against MC3T3-E1 cell lines. The obtained results confirmed that the fabricated polymeric scaffolds would be a potential bone substitute to regenerate defective bone with different loading bearing applications for bone tissue engineering. Bone is a connective tissue supporting and protecting the rest of the body systems and organs by storing bone marrow, minerals and ions. Bone constantly remodels to adapt to the mechanical environments and heal the minor lesions . HoweverA biodegradable scaffold is placed as a temporary implant into the impaired sites of defective bones to support and activate bone regeneration which is slowly degraded and replaced by the new bone in bone tissue engineering. Tissue engineering scaffolds were developed and tested using bioactive ceramics and polymers with similar chemical cosmetics of a natural bone, supporting osteogenesis and bonds with the host tissue ,7. BioceFor use as scaffolds, many types of natural and synthetics polymers were investigated. The scaffolds used in tissue repairing are bone, tendon, cartilage, skeletal muscle, skin, ligament, vascular tissues, and neural tissues. Natural polymers, which are comprised of proteins  or polysaccharides , have restricted mechanical characteristics, immunogenic prospect, and demand ,11,12. PPolysaccharides are the most common type of carbohydrate in foods. These are polymeric carbohydrates made up of monosaccharide units linked by glycosidic linkages that form long chains. Chitosan is a polysaccharide cationic biopolymer that belongs to the polysaccharide class of biodegradable biopolymers. It is non-toxic, biodegradable, and non-antigenic. Unlike synthetic polymers, chitosan has a hydrophobic nature that encourages cell adhesion, differentiation, and proliferation while causing only a minor inflammatory response when implanted . ChitosaHere we report a biodegradable porous composite scaffold, its synthesis and properties. Briefly, polymeric composites were synthesized using the free radical method from arabinoxylan (ARX), β-glucans (BG), nano-hydroxyapatite (nHAp), graphene oxide (GO), acrylic acid (AAc) and freeze-drying technique was used to fabricate porous scaffolds. These fabricated scaffolds were coated with chitosan to enhance their biological activities. X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM), water contact angle, and universal testing machine (UTM) were used to characterize the structural, morphological, wetting analyses, and mechanical testing of the fabricated scaffolds, respectively. The swelling (aqueous and PBS media) and degradation analysis (PBS media) were conducted to investigate their behavior. The biological activities were determined against mouse pre-osteoblast (MC3T3-E1) cell lines.Β-glucan is a natural polymer extracted from barley flour and supplied by DSP Gokyo Food and Chemical Co., Ltd., Osaka, Japan. Acrylic acid (monomer), N, N’-methylene bisacrylamide (crosslinker) and graphene oxide (CAS# 763713-1G) were supplied by Sigma Aldrich Selangor, Malaysia. Hydroxyapatite nanoparticles , phosphate buffer saline (PBS) solution and ethanol were supplied by Sigma-Aldrich, Selangor, Malaysia. All chemicals and reagents were used as received.The osteoblast cells were purchased from the American Type Culture Collection (ATCC), Manassas, VA, USA. ThermoFisher Scientific  supplied Alpha-MEM (α-MEM). L-glutamine penicillin/streptomycin and fetal bovine serum (FBS) were provided by Hyclone Laboratories Inc. .2CO3 . For 30 min the suspension was stirred at 45 °C and centrifuged at 18,000× g for 20 min (4 °C and pH 4.5) to isolate protein contents. The precipitated proteins were removed, and equal amounts of absolute ethanol were added to the suspension with slow stirring to precipitate β-G. Then, the suspension was centrifuged (4000× g) for 15 min to get β-G precipitates and kept at 4 °C overnight. These were freeze-dried to have fine powder of β-G and packed in an airtight glass jar.β-glucan was extracted using a standard method with a slight modification . BrieflyThe psyllium (Plantago ovata) seed husk extracted arabinoxylan with slight modification by a well-reported method by Saghir et al. . BrieflyThe polymeric composite materials were synthesized by free radical polymerization. Briefly, biopolymeric powder (ARX = 1 g and BG = 1 g) was dispersed in deionized water (25 mL) to have homogenized suspension. Different amounts of nHAp  were dispersed in deionized water (10 mL) to have homogenized suspension by sonication. These all suspensions were transferred into a round bottom flask (two-necked) and stirred for 45 min. Then, GO (0.3 mg) was added to the reaction media and stirred for 30 min at 65 °C. The amount of GO was optimized in our previous studies. After, 65 °C, the monomer (AAc = 0.50 mL) and crosslinker  were added into well homogenized mixture. The free-radical polymerization reaction was initiated by adding potassium persulphate (0.05 g) and heated for 3 h at the same temperature under an inert environment with constant stirring. The reaction was stopped heating, and nitrogen gas was removed. The reaction media was cooled and vacuum filtered, and the residue was washed 3 times with deionized water to ensure the unreacted species were from the product. The synthesized polymeric composite was dried in the oven at 50 °C overnight and packed for further use, and the actual yield was found to be approximately 63.82%. The composition of materials is mentioned in −1 was scanning range. The average scan was 150 with a resolution of 4.0 cm−1.The structural properties and chemical interactions were determined of fabricated scaffolds using FTIR . The analysis was conducted using ATR mode, and 4000–400 cm−1 and 0.03 o step size.The diffraction pattern of fabricated scaffolds was determined by X-ray diffractometry. The analysis was conducted using XRD ) using CuKα radiation (λ = 1.540 Å) using the voltage at 30 kV with a current of 30 mA. The XRD spectrum was obtained with a fixed range of 10 to 60°. The scan speed was 1.5 minThe morphological analysis of fabricated scaffolds was conducted by SEM . The SEM was coupled with EDS to analyze elemental composition. Before analysis, the fabricated scaffolds were gold-sputtered.The XPS analysis was performed by a Scienta-Omicron system coupled with a micro-focused monochromatic Al K-alpha X-ray source. The detailed survey scan analysis was performed by operating X-ray at 15 KeV with 700-micron spot size. The constant analyzer energy (CAE) was kept at 100 eV. The charging effects were avoided by applying a combined low energy/ion flood source. The Matrix software was used to perform data acquisition, and Igor pro was applied to analyze the XPS fitting procedure. The shrilly background was corrected, and Gaussian–Lorentzian line shape was used to study the Curve fitting of detailed spectra. The C1s were used as reference data by fixing binding energy at 284.8 eV.The mechanical testing was performed using a universal testing machine  to obtain strain–stress curves. The strain–each fabricated cylindrical scaffold determined stress curve with dimensions 1.5 cm (width) and 1.7 cm (height) at 0.5 mm/min speed.The hydrophilicity/hydrophobicity was determined of fabricated scaffolds through wetting analysis. The contact angle meter (XCA-50)  determines the wetting behavior of fabricated scaffolds. The wetting was investigated at a different time interval to analyze the effect of time.The swelling of well-dried fabricated scaffolds was determined in aqueous and PBS media at 37 °C. The fabricated samples of each scaffold were weighted (50 mg) and placed in corresponding media to determine swelling behavior. The samples were taken out after a specific interval, and the excess surface liquid was removed carefully by tissue paper to record actual weight. The scaffold sample was placed into media until equilibrium, and the swelling percentage was determined by Equation (1).tW = scaffold weight at “t” time, “iW” initial scaffold weight.The degradation studies of fabricated scaffolds were observed in PBS media (pH 7.4 at 37 °C) for 30 days. The fabricated scaffold was weighed (50 mg) carefully and placed in PBS media. The scaffold samples were taken out after a specific time interval. The scaffolds were rinsed with deionized water and oven-dried weight. The percentage degradation was determined by Equation (2).2) were seeded in a 24-well plate triplicate by cell culture media. The 24-well plates were incubated for 24, 48 and 72 h under in vitro conditions, i.e., 37 °C, 5% CO2 and 90% humidity. After removing cell culture media, the grown cells were fixed by absolute ethanol for 5 min at room temperature. The cell was gold-sputtered and SEM  to capture the photographs at different intervals of time.The cell viability of the polymeric composite was studied against MC3T3-E1 cell lines in triplicates. The 24-well plate was coated by a fine layer of polymeric scaffold and sterilized using a UV light for 1 h. The cell lines were maintained in culture media  L-glutamine, 10% FBS and α-MEM. The cell lines (5000 cell/cm2 at 37 °C with 90% humidity), as mentioned in our previous work [sOD = sample optical density and cOD = control optical density.The MC3T3-E1 cell lines were used to determine the scaffolds’ cell viability and optical density by taking gelatin (0.1%) as +ive control, and 1% dimethyl sulfoxide (DMSO) was taken −ive control. These well plates were incubated at different time intervals  under in vitro conditions (5% COous work . The culous work . The excn = 3), and p < 0.05.The statistical analysis was conducted by software (IBM SPSS Statistics 21) of the obtained data and presented in mean, standard error (S.E.) and have shown as Y-error bars in figures. (−1 and 1089 cm−1 present the triply degenerated P−O stretching and O−P−O bending (first two) of HAp. The characteristic bands from 560 to 600 cm−1 and 1000–1100 cm−1 exhibit moiety of calcium phosphate due to nHAp [−1, which are belonged to stretching vibrations of –C–O–C– and –C–O–, respectively, which is a clear indication that increasing nHAp amount decrease polymerization contents due to increased ceramic contents and decreased monomeric contents [−1. The band from 1750 to 1600 cm−1 is attributed to the C–O stretching of GO, NN-MBA and free hydroxyl (–OH) [−1 is typical of saturated aliphatic C–H stretching vibrations, and broadband at 3100–3500 cm−1 is attributed to inter and intra-hydrogen bonding due to biopolymers  [The structural analysis of the porous scaffolds was analyzed by FTIR spectral profile as shown in  to nHAp . It was contents . Howeveryl (–OH) . The peaXRD analysis was conducted in the current research to observe the crystalline and phase analysis of inorganic content in polymeric scaffolds, as shown in The XPS spectra collected from the as-synthesized BGH-3 sample is shown in The surface and porous morphologies are significant characters of any biomaterial for biomedical applications, especially bone tissue engineering. The rough surface and interconnected porosity are essential to proliferate, differentiate, and adhere to bone tissue support. These samples exhibit rough surface morphology, and it is also observed that increasing HAp amount caused interconnected porosity with regulated pore size, as shown in The presence of adequate porosity in the scaffold is essential to regulate critical processes such as nutrient delivery to cells, metabolite dispersion, pH stability, and cell signalling. In the early stages, the size of the pores is a significant factor for evaluating the closeness of cells. The later stages of tissue growth allow for three-dimensional cell-cell communication and space for cells to organize themselves in three dimensions. Because cell seeding in the scaffold’s centre and feeding the scaffolds’ inner surfaces are limited, smaller pores have their constraints. On the other hand, larger pores affect the scaffold’s stability and offer physical support for the cell seeding. Pores of sufficient size allow cells to migrate or adhere to a material’s surface . Table 1Porous scaffold must mimic the native extracellular matrix (ECM) with desirable mechanical behavior and porosity, an essential requirement for bone tissue regeneration. The mechanical properties of the scaffolds should be similar to those of native bone tissue. The mechanical strength can be determined by the impact resistance of polymeric scaffolds to retain structural integrity during implantation . MultiplThe polymeric scaffolds’ machinal properties were conducted via stress-strain curves, as shown in Wetting of material surfaces is a crucial feature for tissue engineering, and it has an impact on cell-material interactions. Wetting of polymeric material is a vital property to achieve a suitable biological response. Wetting measurements are an essential part of the scientific analysis of biomaterial features. Contact angle measurements are one of the most common methods for determining the wetting behavior of polymeric biomaterial surfaces . The hydThe ability of biomaterials to swell is a crucial feature that facilitates the interaction of the biological system with biomaterials. It also regulates the sustained release of therapeutic agents. Swelling is a characteristic of polymeric materials that occurs when a solvent penetrates the void space of the polymeric chain network. The swelling causes the polymeric materials to expand. In some cases, external triggers  can influence the swelling behavior of polymeric materials . The polScaffold swelling behavior and structural integrity are critical for tissue engineering clinical use. In biological fluids, mostly biopolymers swell quickly. Initial swelling is desirable in the in vitro culture studies because the resulting increase in pore size enhances 3D cell adherence and proliferation. On the other hand, persistent swelling would result in a loss of mechanical integrity that develops compressive stress in the surrounding tissue . The impThe essential characteristics of polymeric composites for biomedical applications are degradability and biocompatibility. Polymeric composites are generally biocompatible and low in toxicity. As a result, they do not cause any unwanted biological responses at the molecular, cellular, or organ level. They contain more water, and the hydrophilic functional groups in the polymeric network are more numerous . Because3, –CH3 and –COOH, etc.) of GO, HAp, and the polymeric matrix. This multifunctionality encourages cell adherence that supports cell viability and proliferation due to hydrogen bonding [The cell adherence and morphology of the polymeric scaffold were evaluated against MC3T3-E1 cell lines, as shown in  bonding . It was  bonding . The rouThe biological activities of polymeric were performed against MC3T3-E1 cell lines by in vitro analyses to determine cell viability a assay aExcellent translational biomaterials demonstrate a ton of potential performance in the lab but have limitations in commercialization due to cast and other research aspects. Here we reported the synthesis of polymeric composite materials via the free radical method to fabricate polymeric scaffolds using the freeze-drying technique. The results demonstrate that the fabricated scaffolds have porous morphology, adjustable wetting, swelling, degradation and mechanical testing. In vitro bioactivities were performed against MC3T3-E1 at different times and concentrations. It was found that increasing ceramic (nHAp) amount enhances physicochemical and biomechanical characteristics. BGH3 was the best polymeric scaffold due to high mechanical (Young’s modulus and compression) with appropriate porosity and surface morphology due to maximum HAp amount with fixed GO quantity. The increasing HAp amount enhances the cytocompatibility and biocompatibility of cell adherence, leading to cell viability and proliferation due to the highly interrelated porous scaffold structure. It was found from the results that these polymeric scaffolds have desirable structural, morphological and biocompatible properties with different load-bearing mechanical behaviors. These polymeric scaffolds would be promising biomaterials to regenerate fractured bone for bone tissue engineering applications.


Plasmodium. Out of 171 studies in Homo sapiens, Macaca mulatta, and Mus musculus, we identified 63 potential studies containing host and parasite data. While 16 studies  explicitly performed dual RNA-Seq, 47  originally focused on one organism. We found 915 experimental replicates from 20 blood studies to be suitable for coexpression analysis and used orthologs for meta-analysis across different host-parasite systems. Centrality metrics from the derived gene expression networks correlated with gene essentiality in the parasites. We found indications of host immune response to elements of the Plasmodium protein degradation system, an antimalarial drug target. We identified well-studied immune responses in the host with our coexpression networks, as our approach recovers known broad processes interlinked between hosts and parasites in addition to individual host and parasite protein associations. The set of core interactions represents commonalities between human malaria and its model systems for prioritization in laboratory experiments. Our approach might also allow insights into the transferability of model systems for different pathways in malaria studies.Dual RNA sequencing (RNA-Seq) is the simultaneous transcriptomic analysis of interacting symbionts, for example, in malaria. Potential cross-species interactions identified by correlated gene expression might highlight interlinked signaling, metabolic, or gene regulatory pathways in addition to physically interacting proteins. Often, malaria studies address one of the interacting organisms—host or parasite—rendering the other “contamination.” Here we perform a meta-analysis using such studies for cross-species expression analysis. We screened experiments for gene expression from host and IMPORTANCE Malaria still causes about 400,000 deaths a year and is one of the most studied infectious diseases. The disease is studied in mice and monkeys as lab models to derive potential therapeutic intervention in human malaria. Interactions between Plasmodium spp. and its hosts are either conserved across different host-parasite systems or idiosyncratic to those systems. Here we use correlation of gene expression from different RNA-Seq studies to infer common host-parasite interactions across human, mouse, and monkey studies. First, we find a set of very conserved interactors, worth further scrutiny in focused laboratory experiments. Second, this work might help assess to which extent experiments and knowledge on different pathways can be transferred from models to humans for potential therapy. Transcriptomes are often analyzed in a first attempt to understand molecular, cellular, and organismic events. A comprehensive profile of RNA expression can be obtained using high-throughput sequencing of cDNA from reverse transcribed expressed RNA. Such RNA sequencing (RNA-Seq) provides high technical accuracy at a reasonable cost, making it the current method of choice for transcriptomics , 2.In an infection experiment, RNA-Seq can assess host and pathogen transcriptomes simultaneously if RNA from both organisms is contained in a sample. On the one hand, it has been proposed to analyze transcriptomes of both organisms involved in an infection for a more complete understanding of the disease –5, such –Plasmodium spp. and the host are eukaryotic organisms with similar transcriptomes  for development and asexual expansion. While the nuclear machinery from both host and parasite cells produces mRNA in the liver, RBCs are enucleated and transcriptionally inactive in the mammalian host. In the blood stage of the infection, leukocytes or white blood cells (WBCs) provide most of the transcriptomic response and are, thus, the source of host mRNA. Correlation of mRNA expression can be indicative of different types of biological interactions: protein products could be directly involved in the formation of complexes and might therefore be produced at quantities varying similarly under altered conditions. Alternatively, involvement in the same biological pathways can result in coregulated gene expression without physical interactions. This broad concept of interaction has long been exploited in single organisms  , Plasmodium yoelii, Plasmodium chabaudi, and P. berghei in mouse studies and Plasmodium cynomolgi and Plasmodium coatneyi in macaque studies (in vivo) or hepatoma cultures (in vitro).We found 63 potentially suitable studies  of the data sets, we selected a total of 15 sub-data sets maximizing overlap between individual study networks. We concatenated them to construct the “overall” data set: four studies without thresholds , seven at intermediate , and four at stringent (70% “str”) thresholds  outnumber random expectations and are highly significantly , indicating a biological signal shared between data sets , possibly indicating differences in sampling from patients. Alternatively, this might hint toward idiosyncrasies in human blood studies using native samples with high parasitemia. The potential difficulty to apply RNA-Seq meta-analysis to these human samples highlights the need to transfer such data across model systems while controlling for concordance with the human system.Correlating expression on the “overall” data set resulted in a larger network  than on 13 out of 15 individual study data sets—two macaque data sets were exceptions . A totalRP004598 ) in whic10.1128/mSystems.00182-21.1FIG S1FIG S1, DOCX file, 0.08 MB.Histogram of correlation coefficients for gene expression correlation coefficients. (A) Histogram for the edges in the overall network. (B) Edges extracted to make the core network are represented. The correlation coefficients of these edges as computed for the overall network are shown. To make the core network, the overall network was used as a scaffold. Download Copyright © 2021 Mukherjee et al.2021Mukherjee et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The “overall” network is a highly connected graph with a total of ∼3.64 million edges . Even th10.1128/mSystems.00182-21.3TABLE S2Table S2, TXT file, 35.3 MB.Host-parasite interaction edges in the overall network. Download Copyright © 2021 Mukherjee et al.2021Mukherjee et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00182-21.4TABLE S3Table S3, XLSX file, 0.2 MB.Core networks. Twenty interactions found in common in seven individual data sets. Download Copyright © 2021 Mukherjee et al.2021Mukherjee et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00182-21.5TABLE S4Table S4, XLSX file, 0.4 MB.Enriched GO terms for the studies in Copyright © 2021 Mukherjee et al.2021Mukherjee et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Plasmodium.A node is important in a network if it is central, i.e., highly connected to other nodes in the network. The metrics node degree (DG), betweenness  and eigenvector centrality (EC) are network centrality measures that can quantify this. Similarly, an essential gene is classically described as a gene performing a function necessary for the viability of a cell or organism. We thus hypothesized central nodes in our parasite networks would tend to be more essential genes of P. berghei in mice, for which one study  quantifies essentiality of a gene.In quantitative terms, more essential genes are more important for cell growth, and thus, the disruption of those genes leads to larger growth defects. Such data are available for ne study  reportedne study  reportedFirst, we tested the different network metrics for predicting gene essentiality (RGR and MIS) as response variables in beta-regression models. EC generally resulted in the best models, and combination of the centrality measures did not improve the models in most cases . This mi10.1128/mSystems.00182-21.6TABLE S5Table S5, XLSX file, 0.01 MB.Beta regression models of centrality properties from gene coexpression networks to explain growth phenotypes. Download Copyright © 2021 Mukherjee et al.2021Mukherjee et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. falciparum studies did significantly predict P. falciparum MIS and P. berghei RGR. EC from P. berghei networks was also a significant predictor for both P. falciparum MIS and P. berghei RGR. Surprisingly, P. berghei network centrality was a better predictor of MIS in P. falciparum than the centrality in P. falciparum expression networks. EC from both the “overall” network and the “core” network was a significant predictor for gene essentiality. Considering effect sizes, P values, and Akaike information criterion (AIC), we conclude that centrality in the P. berghei network best explains both RGR and MIS, followed by the metric from the “core” network. Given the much smaller size of the “core” network, the predictive power of centrality within this network is striking.We then compared how well EC from different networks explained gene essentiality : EC in tWe can conclude from this analysis, that (i) our coexpression networks capture biological characteristics independently measured for the parasite and (ii) that parasite genes with a central position in the “core” network are important for the parasite’s growth and survival. Highly connected genes in the intraparasitic “core” network, however, might control the reaction to host signals without necessarily directly interacting with host genes.Plasmodium infection causes systemic endothelial activation of host blood vessels when the infected RBCs (iRBCs) sequester on the lining of blood vessels. This is accompanied with an increased interaction of endothelial cells with WBCs, as cell adhesion molecules on the vessel lining direct WBC trafficking to infected RBC sequestration sites  host and parasite GO terms based on the interactors of the genes in these enriched GO terms. From the “core” network, we identified a GO network of 617 host GO terms and 464 parasite GO terms. This analysis suggests that besides the detailed coexpression of gene products, broader enriched host and parasite processes or pathways are likely interacting in malaria infection  play roles in complex signaling pathways and are important for apicomplexan parasite virulence , T cells and B cells (adaptive immunity), and myeloid dendritic cells and NK cells (innate and adaptive). We found  in our “overall” network. Specific markers from monocytes were not significant but overrepresented . This likely indicates phagocytosis and killing of Plasmodium via respiratory burst in which neutrophils and monocytes have a central role . Marker genes for all other cell types were underrepresented . In the “core” network, marker genes were not significantly enriched, but those specific for monocytes, neutrophils, and NK cells were tentatively overrepresented. We then used the presence of these specific cell type markers to annotate network clusters with the cell type indicated , connected to 11 host genes. Dynamins are mechanochemical enzymes with a GTPase domain and one of the functions of dynamin-related proteins (Drp) is to traffic vesicles  are indicative of correlated gene expression originating from specific immune cells, we used immune cell marker genes established in reference We found  that speral role , 64. Spendicated . This refe cycle . WLL-vs fe cycle . In an efe cycle . Thus, ivesicles . DrpC is fission .10.1128/mSystems.00182-21.8TABLE S7Table S7, XLSX file, 0.4 MB.Immune cell gene marker identification in core and overall networks. Download Copyright © 2021 Mukherjee et al.2021Mukherjee et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. berghei but not in P. falciparum  was reported to be a pathogenesis-related protein in an in silico module-based subnetwork alignment approach using protein-protein interactions of Escherichia coli as references (Another module of three host genes included a NK cell marker correlated with two parasite genes—a 26S proteasome subunit (PBANKA_1206600) and ubiquitin fusion degradation protein UFD1 (PBANKA_1024700). The parasite 26S protease subunit 7072 was relciparum , 52. In ferences .One module had genes specific to three different immune cells—monocytes, myeloid dendritic cells (DC), and NK cells—the highest number of distinct immune cell types represented in a “core” network cluster. Among the 46 coexpressed parasite genes in this module were genes related to apicoplast biogenesis, mitochondrial fission, transcription, vacuolar transport from Golgi apparatus to the endoplasmic reticulum, and Fe-S cluster assembly proteins.Overall, these results  give an Plasmodium with laminin subunit beta-2 (LAMB2) in the host and (ii) negatively correlated (Pearson’s rho = −0.33) parasite 26S protease subunit and host LAMB2, both recovered only in mouse and monkey studies. LAMB2 is an extracellular high-affinity receptor and is associated with GO term “substrate adhesion-dependent cell spreading” to which infected erythrocytes (IE) have been reported to bind, along with other adhesion molecules such as ICAM-1 and vascular cell adhesion molecule (VECAM). Burgmann et al.  but were not recovered by any of the human-lciparum , 52. TLAium spp. . Along wium spp. . These cium spp. . Host ODium spp.  and in pium spp. .Plasmodium survival in the liver stages but not in the blood stages  and parasite with protein kinase (PBANKA_1016200). In the host, biotinidase makes biotin available from dietary sources. Host biotin was found to be essential for d stages . In gened stages . PutativWe propose that interactions between these proteins  or interlinkage of associated pathways may be worth further scrutiny in mechanistic investigations.Our analysis recovers both very broad and well-known processes involved in host-parasite interactions as well as strong interaction signals for narrower processes, including specific interacting gene pairs. This indicates that our analysis of interaction networks might uncover novel links between host and parasite processes helping to focus further research into detailed mechanisms of host-parasite interactions. Further analyses building on the methodology and concepts conceived here will involve networks derived from liver studies curated here, in which host and parasite cells interact more directly than in the blood. Methodologically, we show that analyses of expression from different host-parasite systems as multilayer networks can link host and parasite network modules. Meta-analysis of coexpression networks can thus assess whether host-parasite interactions are shared among different studies and treatments, different tissues, and different host-parasite systems. This allows the identification of processes at different scales of molecular pathway organization as depicted above.Moreover, the sharing of pathways among coexpression networks from different host-parasite systems might indicate how easily results on those pathways can be translated between different host-parasite systems—here presented in a “core” network. Our results can thus provide insights into how easily and for which pathways observations made in malaria models can be translated to human malaria.Plasmodium reads for our meta-analysis. We first selected studies with library_strategy “RNA-Seq” and with “Plasmodium” in the study title, abstract, or sample attributes fields using the function dbGetQuery. Then we used the getSRA function with the query “ AND RNA-Seq.” This function searches all fields. We manually curated the combined results and added studies based on a literature review using the terms described for the getSRA function in PubMed and Google Scholar. We disregarded studies on vectors and nontarget hosts and studies derived from cell culture conditions devoid of transcriptionally active host cells. In these databases, all experiments submitted under a single accession number are given a single “study accession number” and are collectively referred to as a “study” here onwards. We used prefetch and fastq-dump functions from SRAtoolkit v2.8.2 to download all replicate samples  of the selected studies. The curation and the download of the studies were performed on 21 January 2019 and updated on 24 July 2020.Sequence data generated in biological experiments is submitted to one of the three mirroring databases of the International Nucleotide Sequence Database Collaboration (INSDC): NCBI Sequence Read Archive (SRA) , EBI EurPlasmodium sp. genome assemblies and gene annotation files were downloaded from Ensembl version 43. Simultaneous mapping against both genomes should avoid nonspecific mapping of reads in regions conserved between host and parasites. We quantified the sequencing reads mapped to exons using the countOverlaps function of the GenomicRanges package v3.7  and not by chance  . As computational costs for these permutations are too high, we used uncorrected P values to rank genes as initially proposed by Reid and Berriman  as evidence of coregulation.Reid and Berriman  recommeng genes) , 99. To The construction of host-parasite gene coexpression networks require RNA-Seq runs to have both host and parasite transcript expression. To fulfil these criteria, we implemented thresholds based on host and parasite transcript expression on the selection of runs. If a study had at least five runs with 50% detectable transcriptome expression from the host and from the parasite, we defined two thresholds for runs: (i) “intermediate” (int) with at least 50% detectable host and parasite transcriptome expression and (ii) “stringent” (str) with 70% detectable host and parasite transcriptome expression. If a single study had less than five runs in the intermediate threshold, these runs were pooled with runs from other such studies.To compare sub-data sets with runs selected at different thresholds and including all runs without thresholds, we calculated the Jaccard index for every pairwise combination of these data sets.The Jaccard index is defined as ∩B||A∪B| , where AOrthologs are genes in different species that have been derived from a single gene in the last common ancestor of those species by vertical descent. This means that orthologous genes from different species, in general, are likely to perform the same function .Plasmodium from PlasmoDB release 37. Orthologs were identified using OrthoFinder v2.2.7   and mutaP values for variables in question.The centrality properties measured for our networks were node degree (DG), eigenvector centrality (EC), and betweenness (BW) using their dedicated functions in igraph package in R: “degree,” “betweenness,” and “eigen_centrality.” RGR and MIS (as response variables) were modeled first with a single centrality measure as a predictor and then with combinations of two centrality measures: DG with EC and DG with BW. “Nested” models were compared based on likelihood ratio tests and the more complex model was accepted when delta likelihood exceeded 2. In addition to this, the Akaike information criterion (AIC) was computed for each model to compare models, and again models were considered differing in explanatory power at a delta-AIC of 2. Models on different data sets (and different response variables) were compared without explicit statistical testing discussing differences in In general, R (v3.4.3 - 4.0.0)  was usedhttps://doi.org/10.5281/zenodo.4535898 (as used in this article) and GitHub repository https://github.com/parnika91/CompBio-Dual-RNAseq-Malaria (under further development).All code is available at