P < 0.001, P = 0.034). Choroidal thickness was positively correlated with scleral thickness (P = 0.031). A thick sclera along with a thick choroid were demonstrated in CSC eyes using AS OCT. Scleral characteristics might be involved in the pathogenesis of CSC by affecting outflow resistance of venous drainage in choroidal circulation.The aim of this study is to compare the scleral thickness of central serous chorioretinopathy (CSC) eyes with controls using anterior segment optical coherence tomography (AS OCT). This prospective case control study included 15 patients (15 eyes) with CSC and 15 age and gender matched healthy subjects. All subjects underwent spectral domain OCT with enhanced depth imaging and swept source AS OCT of temporal sclera. We investigated difference in scleral thickness between the two groups and relationship between choroidal and scleral thickness. Among the 15 eyes in the study group, 1 eye had acute CSC, 4 had recurrent CSC, 7 had inactive CSC, and 3 had chronic CSC. There was no significant difference in terms of age, gender, axial length and spherical equivalent between the two groups. The choroidal and scleral thickness of the study group were significantly greater than those of the control group ( While the underlying pathophysiology of CSC remains unclear, choroidal vascular disturbance demonstrated on indocyanine green angiography (ICGA)—for instance, increased permeability of the choriocapillaris, vascular congestion, and venous dilation—is thought to be a principal mechanism behind the detachment of the neurosensory retina and RPE3. Recent studies using enhanced-depth imaging (EDI) optical coherence tomography (OCT) provided additional evidence supporting the role of the choroid in CSC4. Compared with healthy eyes, CSC eyes have increased subfoveal choroidal thickness (SFCT)  and dilated choroidal vessels 5.Central serous chorioretinopathy (CSC) is a disease characterized by a localized exudative detachment of the neurosensory retina and retinal pigment epithelium (RPE)7. The pathogenesis of UES has been explained as outflow obstruction due to decreased transscleral outflow and compression of the vortex vein by an abnormal sclera 8. Although the severity of choroidal congestion and exudative retinal detachment in CSC differs from that in UES, both are common findings in these conditions9. However, little information is available regarding the characteristics of sclera in CSC eyes. In this study, we aimed to investigate the scleral thickness of CSC eyes using anterior segment (AS) OCT and compare the results with those of normal eyes.As the choroid of the eye is primarily a vascular structure lacking in autoregulation, impairment in drainage of choroidal flow could cause congestion of the choroidal venous system, leading to pachychoroid and choroidal hyperpermeability, as seen in uveal effusion syndrome (UES)P = 0.361, P = 0.651, P = 0.125, P = 0.878, respectively) (Table P = 0.143).We enrolled 15 eyes from 15 patients diagnosed as CSC as the study group, and 15 eyes from 15 age- and gender-matched subjects as the control group. Among the 15 eyes in the study group, 1 eye had acute CSC, 4 had recurrent CSC, 7 had inactive CSC, and 3 had chronic CSC. At the time of enrollment, 6 out of 15 eyes in the study had no prior treatment, while 9 eyes had received different treatments . Correlation analysis showed a moderate positive linear relationship between SFCT and the sub-LR scleral thickness  . Figure The median SFCT in the study and control groups was 443.0 µm  and 294.0 µm , respectively , it was difficult to compare the thickness of the sclera at any particular point. Moreover, we only measured the temporal quadrant because it was difficult to expose the sclera for accurate measurements in other quadrants. However, we cannot rule out the possibility that confounding factors specific to temporal area may have affected our results. Nevertheless, the findings by Imanaga et al. suggest that these effects are minimalOCT Fig. . The vorIn conclusion, in eyes with CSC, both the sclera and the choroid were thicker than those in normal eyes. Scleral characteristics, by affecting outflow resistance, may be another contributing factor in the development of CSC. Therefore, the balance between inflow and outflow of choroidal circulation should be considered and novel treatment strategies aimed at relieving outflow obstruction are indicated in the management of CSC.1. We excluded patients with (1) a choroidal thickness < 300 µm; (2) an axial length > 25 mm; (3) any other retinal abnormalities, including diabetic retinopathy, retinal vein occlusion, choroidal neovascularization, and posterior uveitis; (4) previous vitreoretinal surgery and trauma; (5) a history of taking medications that could induce CSC-like retinopathy, such as mitogen-activated protein kinase inhibitors32; (6) poor-quality images that could not be evaluated because of low resolution.We performed this prospective, cross-sectional, case–control study at Kangwon National University Hospital, Chuncheon, Korea between October 2019 and November 2019. The institutional review board of Kangwon National University Hospital approved the study, which adhered to the tenets of the Declaration of Helsinki. All patients gave their written informed consent prior to their enrollment in the study. We consecutively enrolled patients diagnosed with CSC as the study group, and formed the control group from age- and gender-matched healthy subjects. We based diagnosis of CSC on ocular examination including spectral-domain (SD) OCT and fluorescein angiography (FA) results. CSC is classified in accordance with the suggestion of Daruich et al., and all subtypes of CSC were included in this studyAll participants underwent a comprehensive ophthalmologic examination, including measurement of best-corrected visual acuity, intraocular pressure, slit-lamp biomicroscopy, and dilated fundus examination. We measured axial length (AL) with a partial coherence laser interferometer , and determined choroidal thickness from images obtained using the EDI mode of a Spectralis OCT device , analyzing them with the associated software (Version 6.0). We obtained the horizontal and vertical sections, comprising 100 averaged scans passing directly through the center of the fovea, in a 5° × 30° rectangle centered on the macula. We measured the SFCT from the outer portion of the hyperreflective line corresponding to the RPE to the inner surface of the choroid/scleral boundary. Two independent graders measured the SFCT and we used the average measurements in statistical analysis.We imaged the eyes of all participants with a swept source AS OCT . We captured images of temporal sclera during 45° nasal gaze position using ‘bleb wide’ mode  or median (interquartile range), and we used the Student t test or Mann–Whitney U test as statistical methods of comparing variables, as determined based on the above results. We performed Spearman’s correlation analysis to examine the relationship between choroidal thickness and scleral thickness. We expressed the inter-observer reliability of the scleral thickness measured by the two observers as the ICC.We used SPSS version 22.0 software  to perform the statistical analysis, and considered


Fossilized invertebrate embryonic and later developmental stages are rare and restricted largely to the Ediacaran-Cambrian, providing direct insight into development during the emergence of animal bodyplans. Here we report a new assemblage of eggs, embryos and bilaterian post-embryonic developmental stages from the early Cambrian Salanygol Formation of Dzhabkan Microcontinent of Mongolia. The post-embryonic developmental stages of the bilaterian are preserved with cellular fidelity, possessing a series of bilaterally arranged ridges that compare to co-occurring camenellan sclerites in which the initial growth stages retain the cellular morphology of modified juveniles. In this work we identify these fossils as early post-embryonic developmental stages of camenellans, an early clade of stem-brachiopods, known previously only from isolated sclerites. This interpretation corroborates previous reconstructions of camenellan scleritomes with sclerites arranged in medial and peripheral concentric zones. It further supports the conjecture that molluscs and brachiopods are descended from an ancestral vermiform and slug-like bodyplan. The Cambrian is known as a period of rapid animal diversification, but the development of these animals is not well characterized. Here, Steiner et al. describe a new assemblage of Cambrian eggs, embryos and early postembryonic stages from Mongolia that provides insight into ancient bilaterian development and evolution. Animal bodyplans emerged as a consequence of evolutionary changes in the genetic regulation of embryological development. Insights into developmental evolution are usually sought in comparative developmental biology but, surprisingly, the fossil record has begun to provide more direct insights, manifest as fossilized eggs, embryos, and larvae of marine invertebrates3. Existing reports include almost complete cnidarian life cycles5, a possible ctenophore embryo6, scalidophoran embryos, and larvae10, as well as possible mollusc embryos4 which have impacted on macroevolutionary theories of developmental model, life history strategy, as well as the evolution of bodyplan symmetry3. The influence of this dimension of the fossil record is strictly limited by a paucity of sites where they are preserved. So far, early developmental stages of invertebrates in Orsten-type preservation have been reported from the Ediacaran to Ordovician of South China, Australia, Laurentia, and Siberia15.The origin and early evolution of animals is among the most formative episodes in Earth history, establishing the basis of modern animal biodiversity and, through their ecological expansion, precipitating a fundamental change to the Earth systemMarkuelia and, in particular, microscopic bilaterian postembryonic developmental stages preserved to an almost unparalleled cellular fidelity. The juveniles may represent early stages in the life cycle of co-occurring tommotiids, such as Camenella mongolica and Camenella parilobata, in which case they would appear to corroborate the hypothesis of slug-like bodyplan for stem-brachiopods23 and, possibly, ancestral trochozoans.Here, we report a new assemblage from the Cambrian Stage 3 of the Mongolian Dzhabkan Microcontinent, that includes fossil eggs, embryos attributable to the early scalidophoran 25. This outcrop of the Terreneuvian Bayan Gol Formation exposes a >1140 m sequence of thick siliciclastics interlayered with thinner limestones or calcareous bioherms, overlain by massive limestones of the Salanygol Formation (ca. 360 m thick). The upper 160 m of the Salanygol Formation commonly contains redeposited archaeocyathans, while the lower part is a monotonous algal limestone devoid of a fossil fauna. Three thin horizons within the topmost 5 m of the Salanygol Formation contain phosphatized eggs, embryos, and postembryonic articulated life stages . This fossil deposit is exceptional for preserving eggs, embryos, and cuticularized articulated organisms  located at the Dzhabkan Microcontinent  six large saddle-shaped central sclerites  and a peripheral zone with strongly asymmetric sclerites Fig. . As suchtes Fig. , (b) onetes Fig. , as welltes Fig. . The thr39. They are implicated in debates over the nature of tommotiid bodyplans, the evolutionary assembly of the brachiopod bodyplan, and  utilized as an interpretative model for resolving the affinity of halkieriids. Following Bengtson16, the bodyplan of camenellans has invariably been inferred as slug-like, with a multimembrate dorsal scleritome. Bengtson16 reconstructed the scleritome as composed of serial transverse rows comprised of a single medial approximately symmetrical mitral sclerite flanked by a pair of asymmetric sellate sclerites. This model was followed in reconstructing the scleritomes of other camanellans  and adapted for other tommotiids23. Based solely on isolated sclerites, it is astonishing that these reconstructed scleritomes bear any resemblance to reality, let alone the very precise correspondence of the bilaterian postembryonic developmental stages to the original reconstruction of Bengtson16. Based on the consistent occurrence of ridges and protuberances in the juveniles, we hypothesize that adult camenellans developed a dorsal scleritome with organophosphatic assymetric sclerites arranged in the peripheral zone of a slug-like body and more symmetrical sclerites in the central medial zone, and with some morphological variation in the anterior position . Figures were assembled using the computer software Adobe Photoshop CS6, Adobe Illustrator CS6 and Microsoft Ecxel 14.2 for Mac, while texts were handled by Microsoft Word 14.2 for Mac.The phosphatized embryos, postembryonic developmental stages, and adult sclerites were extracted with 10% buffered acetic acid from carbonates, followed by sieving (>64 and >125 µm) and hand-picking of the acid-resistant residue (fraction >125 µm <2 mm) under a binocular microscope. Scanning electron microscopy (SEM) was carried out at the Department of Earth Sciences, Freie Universität Berlin with a ZEISS Supra 40 VP Ultra . Specimens selected for investigation in SEM were rinsed in pure ethanol, mounted on aluminum sample stubs with adhesive carbon film, and sputtered for 6 min with gold in a BioRad elemental SEM coating system. Synchrotron Radiation X-ray Tomographic Microscopy (srXTM) was conducted at the X02DA TOMCAT beamline of the Swiss Light Source, Paul Scherrer Institut, Villigen, Switzerland. Measurements were obtained with an operating voltage of 15 KeV, exposure of 150 ms, using a 20 µm LuAg:Ce scintillator, and a 20× objective, yielding reconstructed tomographic data with voxel dimensions of 0.325 µm. A total of 1501 projections were taken equiangularly through 180Further information on research design is available in the Supplementary InformationReporting Summary


IntroductionComputer vision syndrome (CVS) is defined as a group of vision-related symptoms that result from the continuous use of devices with digital displays, such as computers, tablets, and smartphones. Students nowadays can find resources and books online on their smartphones easily, hence, reducing the use of paper-based reading materials. This might lead to a number of ocular symptoms. In this study, we aim to assess the prevalence and determine the risk factors of CVS among students at King Saud Bin Abdulaziz University for Health Sciences (KSAUHS) in Jeddah.Materials and methodsThis is an observational descriptive cross-sectional study design. Students of Colleges of Medicine, Applied Medical Sciences, and Science and Health Professions at KSAUHS were asked to fill an electronic self-administered survey. The survey instrument included questions on demographic information, digital devices using habits, frequency of eye symptoms, and ergonomic practices. ResultsThe sample size was 334 students, 55% of whom were males. The most used device was the mobile phone (78%), and the most common reason for using an electronic device was for entertainment (80%). The frequency of reported eye symptoms was as follows: headache (68%), feeling of an affected eyesight (short- or long-sightedness (65%)), eye itchiness (63%), burning sensation (62%), excessive tearing (58%), unclear vision (52%), redness (51%), dryness (48.3%), photophobia (47%), painful eye (44%), foreign body sensation (40%), excessive blinking (40%), difficulty in focusing on near objects (31%), halos around objects (28%), double vision (21%), and difficulty moving eyelids (9%). The most commonly applied ergonomic practice was adjusting display brightness based on the surrounding light brightness (82%). The rest of the ergonomic practices were less applied as follows: taking breaks while using the device (66%), sitting with the screen on face level (59%), sitting while the top of the screen on eye level (43%), sitting with the screen more than 50 cm away (32%), using antiglare filter (16%). The number of eye symptoms reported was significantly greater in female students (using Mann-Whitney U test) , students who wear glasses , and students who observe glare on their screens .ConclusionCVS symptoms are commonly reported among health sciences students who use different electronic devices. The occurrence of CVS symptoms was significantly higher among female students, those who observe glare on screens, and those who wear eyeglasses. However, long duration of device use was not significantly associated with increased CVS symptoms. Ergonomic practices are not usually applied by most of the students, which necessitates more efforts to increase their awareness of the correct way of using devices. Computer vision syndrome (CVS), also referred to as digital eye strain, is defined as a group of vision-related and muscular symptoms that result from continuous use of devices with digital displays, such as computers, televisions, tablets, and smartphones. This syndrome started to arise after the usage of computers in the mid-twentieth century . With thStudy population and samplingThe study was conducted at KSAUHS, Saudi Arabia. 334 students from the College of Medicine, Applied Medical Sciences, and Science and Health Professions at KSAUHS were asked to fill electronic self-administered questionnaires. The sample size was calculated using the prevalence of previous studies (70%). The study was conducted from March 2018 to July 2019 with the data collection conducted during April 2018. Verbal consent was acquired from all of the participants after explaining the confidentiality measures and the objectives of the research. Convenient sampling technique was used to collect the data, and all students who were willing to participate were included. Ethical approval was obtained from the ethics review committee of King Abdullah International Medical Research Center (KAIMRC), Saudi Arabia. Confidentiality of data was maintained thoroughly as no personal information was obtained and all questionnaires were kept anonymous. Study instrumentsAn electronic self-administered questionnaire was used to collect demographic data, symptoms of CVS, and data about computer/electronic device usage. Demographic data collected include age, gender, the college that the participant attends, the electronic device used the most, and the main purpose of using the said device. Symptoms of CVS included the presence of pain in and around the eyes, burning sensation in the eye, sore/irritated eyes, sensation of foreign body, excessive tearing, twitching of eyelids, difficulty moving the eyelids, eye dryness, headaches, blurred near vision, blurred distant vision, red eyes, double vision, and changes in visualizing colors. The participants were also asked to report the frequency by marking one of the following - never, rarely, often, or most of the time. Moreover, the data collected on computer/electronic device usage were daily computer usage in hours, whether or not rests in between were taken and the length of that rest, the position of the device, usage of glasses or contact lenses, usage of anti-glare filters, and any chronic condition which affects them. The aforementioned questions were used to assess the risk factors based on the literature review done on CVS. The data collection sheet was provided to participants in Arabic but then was translated into English for data entry and analysis.Statistical methods and variables Data were entered using Microsoft Excel 2017 and analyzed by Statistical Package for the Social Sciences (SPSS) version 22. For the analysis, descriptive statistics and Mann-Whitney U test were performed with a confidence level of 95%. The main outcome was CVS which is defined as a combination of eye and vision problems associated with the continuous use of computers, tablets, e-readers and cell phones . A p-valA total of 334 students were included in the study. The median age of the participants is 20 years of age (interquartile range= 2). 55.4% 185) of the sample were males. The most commonly used device was the mobile phone (78%) with the main goal of using that device was entertainment (80%). 97.3% (325) of the study participants had at least one symptom of CVS. The symptoms most experienced were headache (68%), feeling of affected eyesight (i.e. temporary long or short-sightedness) (65%), and itchy eyes (63%); whereas, the symptom least experienced was difficulty moving the eyelids (9%)  reported at least one symptom. Compared to previous studies that found a prevalence of 67.4% among computer office workers in Sri-Lanka, 72% in the United Arab Emirates (UAE), among computer using university students, 80.3% in the Indian city of Chennai among medical and engineering university students, and 89.9% of five Malaysian universities students experience at least one of the CVS symptoms ,11. NeveMultiple studies have reported that participants experienced more pronounced symptoms after using the device for more than six hours or spending more than seven hours a day on the computer which was a significant predictor of CVS ,12,13. HCVS symptoms are commonly reported among health sciences students who use electronic devices. The frequency of CVS symptoms was significantly higher among female students, those who observe glare on the screens, and those who wear eyeglasses. On the other hand, increased duration of device use was not significantly associated with a higher frequency of CVS symptoms. It was also found that ergonomic practices are not usually applied by most of the students. Therefore, more efforts should be directed toward educating the students on the correct way of using electronic devices.


Pregnancy among adolescents, whether intended or not, is a public health concern as it is generally considered high risk for both mothers and their newborns. In Zambia, where many women engage in early sexual behaviour or marry at a young age, 28.5% of girls aged 15–19 years were pregnant with their first child in the year 2013–2014. This study sought to explore associations between maternal age and neonatal outcomes among pregnant women in Lusaka, Zambia.P < 0.05.This was a secondary analysis of data nested within a larger population-based prospective cohort study which was implemented in three government health facilities-two first level hospitals and one clinic in Lusaka, Zambia. Women presenting to the study sites for antenatal care were enrolled into the study and followed up for collection of maternal and neonatal outcomes at 7, 28 and 42 days postpartum. The study’s primary outcomes were the incidence of maternal and newborn complications and factors associated with adverse neonatal outcomes. Statistical significance was evaluated at a significance level of The study included 11,501 women, 15.6% of whom were adolescents aged 10–19 years. Generally, adolescence did not have statistically significant associations with poor maternal health outcomes. However, the risk of experiencing obstructed labour, premature rupture of membranes and postpartum hemorrhage was higher among adolescents than women aged 20–24 years while the risk of severe infection was lower and non-significant. Adolescents also had 1.36 times the odds of having a low birthweight baby  and were at risk of preterm birth . Their newborns were in need of bag and mask resuscitation at birth . Advanced maternal age was significantly associated with increased odds of hypertension/ pre-eclampsia  and preterm labour .Adolescence is a risk factor for selected pregnancy outcomes in urban health facilities in Lusaka, Zambia. Health care workers should intensify the provision of targeted services to improve neonatal health outcomes.NCT03923023 (Retrospectively registered). Clinical trial registration date: April 22, 2019.Clinical trial number and URL:  Over the years, there has been a dramatic shift in childbearing age, globally . WhereasAdvanced maternal age at birth, defined as 35 years and older –9. PregnThe World Health Organisation (WHO) reports that an estimated 18 million girls aged 13–19 years give birth annually, translating into approximately 1 in 4 girls bearing children by the age of 19 . ApproxiStudies suggest that pregnancy complications are more often the result of socioeconomic factors rather than the biological age of the mother , 19. BesAlthough there has been a steady but uniform decrease in adolescent pregnancies from 137 to 96 per 1,000 adolescents in LMICs, 96 to 40 per 1,000 in middle income countries, and 46 to 13 per 1,000 in high income countries, the prevalence in sub-Saharan Africa (SSA) still remains high , 23. ConThis was a secondary analysis of data nested within a larger population-based prospective cohort - the Preterm Resources, Education, and Effective Management for Infants (PREEMI) study - which was implemented using a step-wedge model. Between May 2015 and September 2017, data were collected for births recorded in three public health facilities – Chawama and Chipata First Level Hospitals and George Health Centre – and surrounding catchment areas in Lusaka, Zambia. Study sites were selected on the basis of their high number of births one year prior to initiation of activities. During the implementation period, trained study staff – Quality Improvement Nurses (QI Nurses) and Community Liaison Officers (CLOs) – registered all pregnant women residing within the defined catchment areas and followed them until 42 days postpartum.The PREEMI program activities were implemented as part of routine obstetric care and aimed to reduce neonatal mortality through the introduction of proven cost-effective clinical interventions such as Helping Babies Breathe (HBB) and Kangaroo Mother Care (KMC). Additionally, the study sought to enhance obstetric services and reduce gaps between health facility and home births by providing medical supplies, onsite clinical mentorship, and strengthening linkages between health facilities and community structures. Maternity service users were not engaged in the development of this study.Health facilities were open 24 hours a day, 7 days a week, provided antenatal care (ANC), labour and birth and postnatal services with at least one nurse-midwife on duty throughout the day. As part of the national scale-up plan of Essential Newborn Care (ENC) services, nurse-midwives were trained to identify obstetric complications, accurately administer antibiotics for treatment of neonatal sepsis, antenatal corticosteroids for preterm labour, chlorhexidine gel (7.1 percent) for cord care and provide neonatal resuscitation with bag and mask. At community level, activities were implemented by Safe Motherhood Action Groups (SMAGs), community-based volunteer groups that are established to serve as a critical link between communities and health facilities thus supporting the Ministry of Health’s (MOH) efforts to reduce critical delays associated with individuals' decision-making about seeking health care, including life-saving maternal and newborn services.Members of respective SMAGs were trained in KMC, early identification of danger signs at any time during pregnancy or labour and birth and immediate referral to higher levels of healthcare for further management. They were responsible for household follow ups during the postnatal period, at seven, 28 and 42 days, and were supervised by the CLOs. During follow-ups, data were collected on home births, outcomes , and early (7-day) and late (28-day) neonatal mortality as well as baseline clinical characteristics of both mothers and their newborns.Since the PREEMI program used a health system strengthening approach to reduce the risk of severe morbidity and mortality due to preterm birth complications in the selected health facilities and their wider catchment areas, we provided targeted services to all pregnant women attending ANC services at the three implementation sites. Given that two of the three implementation sites were first level referral hospitals, they provided services to women living within and outside of the health facilities’ catchment areas. However, while all women received the same quality of care, only women who lived within the catchment area and met the study’s inclusion criteria were eligible for enrolment into the parent study. Women residing outside the catchment areas but were seeking a one-off ANC service at one of the study sites were excluded from the study.To be eligible for inclusion in this analysis, pregnant women should have registered for their first ANC visit or given birth at any of the study sites between May 2015 and September 2017. In 2015, the three implementation sites recorded a combined total of 14,350 births, including patients seeking services from outside the facility catchment areas . Given tUsing study-specific data collection tools, study staff collected data from enrolled participants at health facilities during ANC, labour and delivery and postnatal visits or in the community during outreach activities. At community level, data on miscarriages, stillbirth, neonatal and maternal outcomes were collected by the CLOs through existing community structures  and SMAGs) and submitted to the QI Nurse at each study site. To facilitate data collection in the communities, the CLOs had weekly meetings with community volunteers, SMAGs, and NHCs.The QI Nurses were responsible for collecting data at the health facility level. For quality control and quality assurance purposes, the QI Nurses and CLOs reviewed all the data before submitting them to the data team for entry. On a monthly basis, trained data associates performed consistency and completeness checks before and during data entry and at the time of producing reports. All study staff were trained in Good Clinical Practice (GCP), the study protocol, data collection and data collection tools. As per guidelines, GCP refresher trainings were held every three years, while refresher trainings pertaining the study protocol and data collection were conducted on an annual basis.To determine the association of maternal age with adverse neonatal outcomes, this study used the 13 Core Outcome Sets (COS) relevant to women’s and newborn health and shortlisted in the CROWN database . The priThe primary neonatal outcomes of interest included the occurrence of low birthweight (< 2500 grams), preterm birth (infant born before 37 weeks’ gestation), the need for resuscitation at birth, and neonatal mortality during the first 7 and 28 days after birth. Additional outcomes of interest included miscarriage and stillbirth. We also examined a combined category of outcomes indicative of the occurrence of at least one poor maternal health outcome, and similarly a combined category of outcomes for the occurrence of at least one poor neonatal health outcome. All outcomes were self-reported by the mothers and ascertained by cross referencing data in the health facility registers.The association between maternal age and adverse maternal and neonatal health outcomes was examined using logistic regression, yielding odds ratios (ORs) as our effect measure of interest. Consistent with WHO definitions, adolescent pregnancy was defined as pregnancy among mothers 10–19 years of age . The disa priori for inclusion as potential confounders based on direct acyclic graph (DAG) based analysis and existing literature on predictors of maternal and neonatal outcomes, namely: maternal education, attendance to antenatal care, and parity. All analyses were conducted in STATA 15  and statistical significance was evaluated at an alpha significance level of P < 0.05.In addition, the analysis examined unadjusted univariate associations between maternal age and adverse maternal and neonatal outcomes using chi-squared tests and fisher’s exact tests, where appropriate. Separate logistic regression models were constructed to examine unadjusted and adjusted associations between maternal age and each outcome of interest. Adjusted models included covariates specified A total of 11,535 adolescents and women were enrolled into the study. However, this analysis was restricted to 11,501 mothers after the exclusion of 34 mothers with missing values for maternal age. Of those enrolled, 1,788 (15.6%) were aged 10–19 years old, 3,980 (34.6%) aged 20–24 years old, 4,651 (40.4%) aged 25–34 years old and 1,082 (9.4%) aged ≥ 35 years old. Characteristics of the study population by maternal age are presented in Table In the overall population, as well as within each maternal age group, over 90% of the sample had a vaginal birth assisted by a nurse or nurse-midwife. Less than 5% of adolescent mothers, as well as mothers in the reference age group of 20–24, gave birth at home. Chawama and Chipata first-level hospitals were the most prevalent locations of birth enrolled in this study . Mean parity at enrolment increased with maternal age. Unsurprisingly, adolescent mothers had lower parity (mean = 0.1), as they were enrolled in this study at their first pregnancy that resulted in a viable birth.p < 0.001).The incidence of adverse maternal and neonatal health outcomes stratified by maternal age is presented in Table In this study, we found significant associations between adolescence and neonatal outcomes of low birthweight, preterm birth, and the need for resuscitation at birth in unadjusted and adjusted analysis Table . AdolescMaternal adolescence was protectively associated with the need for babies to be resuscitated at birth . Babies born to mothers of advanced maternal age similarly had a lower odds of needing resuscitation at birth . Advanced maternal age was also significantly associated with odds of having a stillbirth . Using combined outcome categories, adolescence was not associated with odds of experiencing at least one adverse maternal outcome or at least one adverse neonatal outcome  at first ANC enrolment.The evaluation of obstetric complications in a cohort of pregnant mothers facilitated the identification of the most common risks among adolescents. There are no marked differences between maternal and neonatal outcomes of adolescents and older mothers. In fact, there were more significant outcomes in the women older than 35 years. While the Zambian government continues to adapt and support the implementation of evidence-based interventions, health care workers must adhere to national guidelines for providing quality healthcare for mothers and their newborns during the continuum of care to ensure improved health outcomes must provide evidence-based.


Each unit had a retention time of 1.12 days and a hydraulic load rate of 0.2 m/day. To evaluate the efficiency of the treatment, physical, chemical and biological parameters were monitored for three consecutive months. Water samples in the influent and effluent of each experimental wetland were analyzed. At the end of the monitoring, Cyperus Papyrus had a better removal of total phosphorus (50%), ammoniacal nitrogen (69.69%), biochemical oxygen demand (69.87%), chemical oxygen demand (80.69%), total coliforms (98.08%) and fecal coliforms (95.61%). Meanwhile, Phragmites Australis eliminated total solids better (62.85%). These data may be useful for comparative purposes on domestic wastewater treatment using Phragmites Australis and Cyperus Papyrus planted in VSSFCW.The data of this document highlights a comparative analysis between the Phragmites Australis and Cyperus Papyrus planted in vertical subsurface flow constructed wetland (VSSFCW) independently implemented at pilot scale for the treatment of domestic wastewater. At the exit of the primary lagoon a pipe was installed to feed a constant flow of 0.6 m Specifications table•The data presented are used to analyze the treatment capacity of domestic wastewater using two species of vegetation  in vertical subsurface flow constructed wetland (VSSFCW).•The physical-chemical and microbiological parameters data serve to provide a clear picture of the water quality that could be obtained when using Phragmites Australis and Cyperus Papyrus in VSSFCW, therefore, these data could be useful for communities or cities that have domestic wastewater with characteristics similar to this research.•To make known that unconventional treatments in small population centers or in rural areas, is possible using VSSFCW.•To run new experiments using these data as initial information, researchers can recognize the efficiency of Phragmites Australis and Cyperus Papyrus to develop a new set of experiments using other vegetation species and compare them with these.•These data allow choosing a suitable vegetation for a VSSFCW that you want to implement in the treatment of domestic wastewater.13—N), nitrate (NO3—N), total phosphorus (TP), biochemical oxygen demand (BOD5), chemical oxygen demand (COD), total coliforms (TC) and fecal coliforms (FC). The data obtained for the physicochemical and microbiological parameters of the wastewater (influent) and treated water in the VSSFCW (effluent) are shown in 3—N, TP, FC The data presented in this article refers to the quality of the treated water in two vertical subsurface flow constructed wetland (VSSFCW). Phragmites Australis was planted in a first wetland and Cyperus Papyrus was planted in a second wetland. The data obtained during the tests included parameters such as: pH, total suspended solids (TSS), electrical conductivity (CE), alkalinity (Alk), ammoniacal nitrogen .Data presented in 22.13 day−1.The VSSFCWs were implemented together with the Wastewater Treatment Plant in the city of Santa Isabel, Ecuador (Longitude 79.313732 °W and Latitude 3.298460 °S). This plant has a primary treatment through a lagoon. The effluent from this lagoon was conducted to the two VSSFCW, one for each species, Phragmites Australis and Cyperus Papyrus. Each wetland operated independently but with a continuous water flow of 0.6 m2.25 concentration of 100 mg L−1 (influent) and the desired BOD5 concentration of 10 mg L−1 (effluent) was considered; a water residence time of 1.12 days was determined for the system to operate continuously To determine the size of the VSSFWCs, the methodology cited by García 52, necessary for the reduction of BOD5. In −1. The granulometry of the material recommended by A substrate depth of 0.7 m was considered, the deeper the substrate, the greater the load that the system can process, but if the substrate is too deep, the bottom conditions become anaerobic and can reduce the removal of BOD2.32  to avoid infiltrations. The treated water drainage and conduction system were built with a 50 mm PVC pipelines and fittings. For the continuous supply of water (influent) to the VSSFWCs, 16 mm diameter polyethylene hoses were used. These hoses were drilled lengthwise to regulate flow at each point in the wetland, forming a closed-loop that allowed the influent to be distributed equally over the entire surface of the wetland. Six vertical aeration pipes connected to the drainage collection system were installed 2 .Fig. 3VS2.4−1. During two months the development of the plants and the purified effluent were observed. After four months of planting, the plants reached a suitable height. The experiment started in January and ended in July. The sampling was carried out from April to July.After the Phragmites Australis and Cyperus Papyrus were planted, the wetlands were filled for a week. From the second week on, the wetland was fed water at a flow rate of 0.6 m day3—N), nitrate (NO3—N), total phosphorus (TP), biochemical oxygen demand (BOD5), chemical oxygen demand (COD), total coliforms (TC) and fecal coliforms (FC) according to the standard methods for the examination of water and wastewater.The performance efficiency of the Phragmites Australis and Cyperus Papyrus in VSSFCW was evaluated, for which water samples were collected biweekly for three months to measure: pH, total suspended solids (TSS), electrical conductivity (EC), alkalinity (Alk), ammoniacal nitrogen (NHThe data presented in this article are related to the research article entitled “Performance of Phragmites Australis and Cyperus Papyrus in the treatment of municipal wastewater by vertical flow subsurface constructed wetlands” 5, COD, total coliforms, fecal coliforms, ammonia nitrogen and phosphates was 80.69, 69.87, 98.08, 95.61, 69.69 and 50.0% for Cyperus Papyrus and 75.39, 64.78, 96.02, 93.74, 70.70 and 49.38% for Phragmites Australis respectively. These data allowed to determine that Cyperus Papyrus was a little more efficient compared to Phragmites Australis, therefore, Cyperus Papyrus could be a species of macrophyte ideal for wetlands built on a large scale, due to its high effect of eliminating pollutants present in wastewater domestic. These data allow choosing a suitable vegetation for a VSSFCW that you want to implement in the treatment of domestic wastewater.As it can be observed in The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Arsenic trioxide has shown a strong anti-tumor effect with little toxicity when used in the treatment of acute promyelocytic leukemia (APL). An effect on glioma has also been shown. Its mechanisms include regulation of apoptosis and autophagy; promotion of the intracellular production of reactive oxygen species, causing oxidative damage; and inhibition of tumor stem cells. However, glioma cells and tissues from other sources show different responses to arsenic trioxide. Researchers are working to enhance its efficacy in anti-glioma treatments and reducing any adverse reactions. Here, we review recent research on the efficacy and mechanisms of action of arsenic trioxide in the treatment of gliomas to provide guidance for future studies. Glioma is the most common primary malignant tumor of the central nervous system, accounting for about 50–60% of intracranial tumors. Its invasion and recurrence rates are significantly higher than those of other intracranial tumors . Due to Elemental arsenic is insoluble in water and acid. It has almost no toxicity, but when exposed to air, it readily oxidizes to highly toxic arsenic trioxide. Traditional Chinese medicine has applied arsenic and arsenic-containing medicines to treat a variety of intractable diseases since ancient times , but theIn glioma, arsenic trioxide exerts its anti-cancer effects via regulation of apoptosis and autophagy; its impact on the cell cycle; promotion of the production of intracellular reactive oxygen species (ROS), which results in oxidative damage; inhibition of tumor stem cells; and enhancement of the effects of radiotherapy and chemotherapy. However, glioma cells and tissues from various sources have different responses to arsenic trioxide, and it has some limitations and side effects in tumor therapy –10. ReseThere are two forms of programmed cell death, apoptosis and autophagy , 12. ApoAutophagy is characterized by the formation of acidic vesicular organelles in the cytoplasm . This caSimilar stimuli can induce apoptosis, autophagy or both, possibly because apoptosis and autophagy have common effector proteins  and upstream pathways  . Therefo. showed that treatment with a low concentration of arsenic trioxide (2 μM) caused glioma cell death but could not induce apoptosis. U373 cells showed the loss of microvilli and the presence of a large number of autophagic vacuoles. The same researchers studied the occurrence of autophagy and found that arsenic trioxide upregulated mitochondrial cell death protein BNIP3, thereby opening mitochondrial permeability transition pores and destroying the integrity of the mitochondrial membrane [In experiments with six malignant glioma cell lines , Kanzaw et almembrane .An inhibmembrane . TherefoSimilar studies have also shown that inhibiting cathepsin L (CatL) activity partially reduces lysosomal activity, thus affecting autophagy. This can also cause the transition from autophagy to apoptosis in U87MG cells that were treated with a low concentration of arsenic trioxide (2 μM).The drug effect is more obvious in multicellular spheroids than in cell monolayers .The effects of arsenic trioxide on apoptosis and autophagy in glioma may be affected by the drug concentration and the p53 gene type. As arsenic trioxide-induced apoptosis in glioma requires complete p53 protein function, autophagy may become the dominant form of programmed death when apoptosis is inhibited . In a wiFurther study showed that the induction of autophagy in glioma cells may also be related to the heat shock response. After treatment of U118 with arsenic trioxide, the expression of the heat shock protein HSP70 increased. In addition, the HSP70-family protein HSC73 bound to soluble proteins and initiated their transport to lysosomes or lysosomal compartments, resulting in complete protein degradation .. found that arsenic trioxide had both apoptotic and autophagic effects on U118 cells, related to the inhibition of survivin [. studied the expression profiles of apoptosis- and cell cycle-related genes in two glioma cell lines (U87MG and T98G) of different p53 types treated with arsenic trioxide (2 μM). The results suggested that arsenic trioxide inhibited the growth of U87MG cells expressing wild-type p53 mainly by affecting the expression of genes involved in cell cycle arrest, stress and toxicity. Furthermore, it inhibited the growth of p53-mutant T98G cells mainly by upregulating the apoptosis pathway mediated by Bcl-2 and tumor necrosis factor. They also found that arsenic trioxide was more likely to cause apoptosis of p53-mutant cells [However,other reports have been inconsistent with the above-mentioned results Table . For exasurvivin . Zhao etnt cells . A Separnt cells .Table 1SIn conclusion, further study is needed to determine whether apoptosis or autophagy of glioma cells is induced by arsenic trioxide. However, regardless of the p53 type of gliomas, low-dose arsenic trioxide (1–2 μM) can induce programmed cell death in vitro, and it is effective as a single or combined therapy for glioma .There are other mechanisms by which arsenic trioxide regulates programmed cell death in glioma cells. Some studies have found that 5 μM arsenic trioxide can upregulate the expression of the death receptor DR5 and facilitate its binding to tumor necrosis factor-related apoptotic ligand (TRAIL). This leads to both the cleavage of pro-caspase-3 (the precursor of caspase-3) and apoptosis via extrinsic pathways. The upregulation of DR5 induced by arsenic trioxide may be mediated by CCAAT/enhancer-binding protein homologous protein (CHOP) . CHOP isApoptosis is also thought to be related to glycosylation. The sugar chain structure of cell surface proteins has an important role in regulating cell survival –35. ArseIn cells treated with arsenic trioxide (6 μM) for 48 h, about 40% of the water-soluble arsenic present was found to exist in the metallothionein (MT) protein component. MTs can bind to metal elements, thereby protecting cells from arsenic toxicity. Further studies of the interactions between arsenic trioxide and MT subtypes, with different doses or durations of treatment, indicated that the expression of the MT gene in each subtype increased or decreased to a varying degree. The mRNA expression levels of MT1X, MT1F and MT2A increased the most (up to 13 times), whereas those of MT1A, MT1E and MT3 decreased or remained unchanged. The increase in expression of MT1X, MT1F and MT2A may be one of the mechanisms by which glioma cells resist the toxicity caused by arsenic trioxide therapy. On the other hand, MT3 has low metal inducibility and is capable of rapid binding and release of metals. Its isoform is mainly expressed in the brain and it is necessary for the normal lysosomal function of astrocytes. In astrocytes with MT3 deletion, the activities of several lysosomal enzymes decreased significantly, and the death of glioma cells induced by oxidative stress was substantially weakened . After tCompared with other organs, the brain seems to be particularly sensitive to ROS stress. Although it accounts for only 2% of bodyweight, it consumes up to 20% of the body’s oxygen supply. Large amounts of ROS are produced in the brain tissue during oxidative phosphorylation . Since tThe mechanisms by which arsenic trioxide induces apoptosis include stimulate on of ROS production, mitochondrial aggregation, Bax oligomerization, dissipation of membrane potential, and mitochondrial membrane collapse, followed by the release of apoptotic factors, the caspase cascade, and, finally, cell death. Increased ROS levels were detected in both A172 and T98G cell lines treated with 50 μM arsenic trioxide. However, there was no mitochondrial aggregation, membrane potential dissipation or Bax oligomerization in these T98G cells. The mitochondria of T98G cells may be less sensitive and responsive to oxidative stress, and thus resistant to arsenic trioxide–induced mitochondrially mediated apoptosis .A study using rat C6 and 9L cell lines showed that 5 μM arsenic trioxide could strongly inhibit cell viability and induce apoptosis by downregulating the expression of Bcl-2 and upregulating the expression of Bax. This change is related to the mitochondrial damage caused by the production of intracellular ROS. However, the inhibition rate for normal rat glial cells was less than 10% of that for rat glioma cells .While arsenic trioxide increased the production of ROS in glioma cells, it also increased the expression of heme oxygenase-1 (HO-1) and its upstream effector Nrf2. HO-1 can protect against oxidative stress and antagonize the effects of ROS. Therefore, inhibiting the expression of the gene encoding HO-1 or Nrf2 can enhance the oxidative damage caused by arsenic trioxide .Iron plays an important part in the production of ROS by mitochondria . MitoferOther studies have shown that arsenic trioxide could induce the expression of XPC  in U87 cells. XPC may be involved in the regulation of the intracellular redox dynamic balance. When it was silenced, U87 cells were more sensitive to arsenic trioxide .Telomeres are a special DNA structure located at the ends of chromosomes, which are gradually shortened with each cell division. It enables cells to avoid proliferative disorder by stabilizing the telomeres, especially through overexpression of its catalytic subunit hTERT, which is found in 85% of cancers, including glioblastoma . ArsenicIn most solid tumor cells, arsenic trioxide induces G1 or G2/M phase arrest of the cell cycle . Cell cyThe anti-cancer effects of arsenic trioxide may be related at least in part to epigenetic regulation of miRNAs. It may alter the expression levels of a large number of miRNAs with important roles in cancer–related signaling pathways by affecting DNA demethylation . Many of. treated U87 cells with 4 μM arsenic trioxide for 48 h and applied gene chip technology to analyze the changes inexpression of 88 cancer-related miRNAs. They found that 60 miRNAs showed increased expression and 28 showed decreased expression [Shidfar et alpression . Nine mi. treated U251 cells with arsenic trioxide and showed that it directly inhibited potassium hERG channels at a post-transcriptional level by upregulating miR-133b, thereby causing cell apoptosis [In a separate study, Wang et alpoptosis .To some extent, the recurrence of glioblastoma is mediated by glioblastoma stem cells (GSCs). Arsenic trioxide can reduce the expression of stem cell markers  of GSCs and inhibit the Hedgehog and Notch pathways, thus reducing their sphere forming ability, inhibiting their DNA repair ability, and reducing drug resistance . A combi. compared the effects of temozolomide combined either with arsenic trioxide or with the Hedgehog pathway inhibitor vismodegib. Both combinations increased the anti-glioma effect of temozolomide, with similar effects, and inhibited the Hedgehog pathway in glioblastoma [Bureta et alblastoma .When U87MG, U251MG and U373MG GSC cells were treated with arsenic trioxide, the protein levels of Notch1 and Hes1 decreased significantly, indicating that it inhibited the survival and proliferation of tumor stem cells by blocking the activity of the Notch signaling pathway . A furthCurrently, promyelocytic leukemia (PML) protein is the main target in the treatment of APL, which is the most common use of arsenic trioxide , 60. In GBM can be divided into four molecular subtypes: proneuronal, neural, classical and mesenchymal. GSCs are key promoters of the occurrence and evolution of GBM and lead to drug resistance and recurrence of tumors. The mesenchymal subtype is more invasive and has a poorer prognosis than the proneuronal subtype . Bell etCombined treatment strategies have become the most common approach to overcoming malignant tumor resistance to anti-cancer drugs. Arsenic trioxide has potential in combined strategies but its toxicity and side effects are an issue.A combination of arsenic trioxide and silibinin was shown to synergistically inhibit U87 metabolic activity, cell proliferation, and gelatinase A and B activities; increase cell apoptosis; decrease the mRNA levels of cathepsin B, urokinase type plasminogen activator, matrix metallopeptidase 2 (MMP-2), MMP-9, survivin, Bcl2 and CA9; and upregulate caspase-3 mRNA expression . The mecGSH is the most important intracellular antioxidant. Its depletion can increase the toxicity of arsenic in various cell types via mechanisms including increased oxidative stress and free active arsenic concentration, loss of arsenic-bound mercaptan, and reduction in the detoxification by GSH–dependent methylation and the binding of arsenic to GSH. Silibinin sensitization did not affect GSH levels but doubled the accumulation of arsenic. Accumulation of arsenic may be related to the inhibition of the ATP-binding cassette transporter and multidrug resistance-associated protein, which would slow down the excretion of arsenic and increase its accumulation . It was Lin et al. found that berberine could enhance the arsenic trioxide-mediated inhibition of the migration and invasiveness of glioma by reducing the activation of protein kinase C signaling and MMP-2 in the extracellular matrix .Mesbahi et al. found that the most common genetic change in malignant glioma was the amplification of epidermal growth factor receptors . Arsenic trioxide treatment alone could induce the activation of the EGFR signaling pathway in U87MG and A172 cells. Over activation of this pathway may be directly related to resistance to arsenic trioxide as a drug. When erlotinib, an inhibitor of EGFR, was used in combination with arsenic trioxide, ROS levels increased and there were significant synergistic effects on survival, proliferation and migration as well as on the cell cycle . The actMany researchers have also studied the combined effects of arsenic trioxide and radiotherapy. Ning et al. treated U87MG and SNB75 cells with a combination of radiotherapy and arsenic trioxide in vivo and in vitro. The results showed that arsenic trioxide sensitized glioma to radiation, with the greatest effect when it was administered 4 h after radiotherapy. However, the effects of arsenic trioxide alone or before radiotherapy were limited and this form of radio sensitization was time and sequence dependent , 71. ThiA study of the influence of 2 μM arsenic trioxide and 4 Gy irradiation on U118-MG cells found that the proportion of mitotic cells increased significantly. Arsenic trioxide may inhibit the activation of G2-phase DNA damage checkpoints, thereby allowing cells with DNA damage to enter mitosis from the G2 phase. Entering mitosis in the presence of DNA damage leads to centro some inactivation and obstruction of chromosome separation, delaying the mitotic process and inducing mitotic abnormalities. This could lead to mitotic arrest and subsequent autophagy in malignant glioma cells. This combination could also induce autophagy of U118–MG cells by inhibiting PI3K/Akt and activating ERK1/2 signaling pathways .Before 2000, clinical trials of arsenic trioxide focused on the treatment of APL. After treatment with a dose of 0.15 mg/kg/day for 12 to 39 days, most patients with APL achieved complete remission . After iPhase I and II clinical trials of glioma therapies were conducted at Northwestern University in Chicago to select the best combination dosage of arsenic trioxide, temozolomide and radiotherapy. When arsenic trioxide (0.2 mg/kg) was added to the standard glioma treatment regimen of temozolomide combined with chemotherapy, the survival rate of patients with anaplastic astrocytoma increased, but there was no significant improvement in patients with malignant glioblastoma , 85. ThiIn the in vitro BBB model constructed by Tao et al., the transport ratio of arsenic trioxide was 2.21 ± 0.19%. In vivo, the elimination half-life of arsenic trioxide at the end of treatment was 4.41 ± 0.87 h, and the area under the drug-time curve was 0.18 ± 0.03 μg h/ml. After 24 h of intravenous injection, the arsenic trioxide uptake rate in glioma was 0.8 ± 0.3% ID/g, and the mean residence time was 6.17 ± 1.29 h. In this study, polyacrylic acid (PAA) was grafted onto mesoporous silica nanoparticle (MSN) cores to form MSN lipid capsule nanoparticles modified by self-assembled angiopep-2 (amino acid residues TFFYGGSRGKRNNFKTEEY). These could be loaded with arsenic trioxide (ANG-LP-PAA-MSN@As2O3).Owing to the specific recognition and binding of the BBB by angiopep-2, and the high expression of low-density lipoprotein related receptor 1 in glioma cells, the ability of arsenic trioxide to cross the BBB and target glioma increased, and pH-sensitive release . The samAlthough arsenic trioxide exerts anti-glioma effects through a variety of mechanisms, multidrug resistance and the existence of the BBB render glioma insensitive to it. In addition, serious adverse reactions to high dose arsenic trioxide limit its clinical applications. Therefore, it is necessary to further study the efficacy on glioma of a combination of drugs with arsenic trioxide as well as the efficacy of drugs encapsulated with arsenic trioxide carriers.In addition, prior to preclinical trials and clinical application, precision medicine principles should be used to guide experimental research. It is essential that we search for tissue markers of glioma that are sensitive to arsenic trioxide. Finally, most studies to date have been conducted in vitro. Therefore, more preclinical and clinical studies should be conducted to elucidate the anti-cancer effects of arsenic trioxide in the treatment of glioma.


Bladder and bowel dysfunction (BBD) is a common yet underdiagnosed paediatric entity that describes lower urinary tract symptoms (LUTS) accompanied by abnormal bowel patterns manifested as constipation and/or encopresis. LUTS usually manifest as urgency, urinary frequency, incontinence, and urinary tract infections (UTI). Despite increasing recognition of BBD as a risk factor for long-term urinary tract problems including recurrent UTI, vesicoureteral reflux, and renal scarring, the mechanisms underlying BBD have been unclear, and treatment remains empirical. We investigated how constipation affects the lower urinary tract function using a juvenile murine model of functional constipation. Following four days of functional constipation, animals developed LUTS including urinary frequency and detrusor overactivity evaluated by awake cystometry. Physiological examination of detrusor function in vitro using isolated bladder strips, demonstrated a significant increase in spontaneous contractions without affecting contractile force in response to electrical field stimulation, carbachol, and KCl. A significant upregulation of serotonin receptors, Htr2a and Htr2c, was observed in the bladders from mice with constipation, paralleled with augmented spontaneous contractions after pre-incubation of the bladder strips with 0.5 µM of serotonin. These results suggest that constipation induced detrusor overactivity and increased excitatory serotonin receptor activation in the urinary bladder, which contributes to the development of BBD. The clinical spectrum of paediatric lower urinary tract dysfunction (LUTD) is wide, including over- or under-active bladder, frequent- or infrequent voiding, voiding postponement, urgency, enuresis and nocturia. LUTD may be associated with other conditions including neuropsychiatric problems and gastrointestinal dysfunctions, constipation being the commonest comorbidity of LUTD. Constipation is typically defined as infrequent bowel evacuations (less than three bowel movements per week), abnormally large stools, and difficult or painful defecation. Functional constipation is a common problem in childhood with the prevalence of up to one-third of children2. Constipated children were reported to be 6.8 times more likely to have LUTD compared to those with normal bowel function, and up to 84% of functionally constipated children suffer from encopresis which is also often associated with LUTD5. With increasing recognition of the prevailing comorbidity of LUTD and bowel disturbances in children, the condition has been labelled with a standardized term, bladder and bowel dysfunction (BBD) by the International Children's Continence Society in 20146. Due to a diverse spectrum, BBD is often not recognized by child, family or the referring professional, instead, the secondary symptoms of urinary and/or faecal incontinence or urinary tract infection (UTI), prompt the child to be evaluated by specialists9. Hence the diagnosis of BBD is often delayed, and the treatment mainly being symptomatic management, which increase a risk of prolonged and more serious conditions such as recurrent UTI, faecal impaction, rectal prolapse, vesicoureteral reflux, renal failure, and irritable bowel syndrome13. In addition, clinical studies found that adults with LUTD and/or bowel symptoms often experienced BBD symptoms in childhood, underscoring the importance of recognizing BBD early on and to address both, LUT and gastrointestinal dysfunctions, simultaneously16.Functional urologic and bowel problems are common in childhood, account for up to 40% of consultations in paediatric urology, and can cause both physical and psychosocial distress for children and families17. Physical pressure onto the LUT decreases the functional capacity of the urinary bladder, constricts the urethra, thereby resulting in urgency, dysfunctional voiding, urinary retention and urinary incontinence18. This explains the improvement of lower urinary tract symptoms (LUTS) following the medical relief of constipation in the majority of cases with BBD symptoms19. However, other studies found no improvement or persistent LUTS with release of intra-rectal pressure in children with BBD22.The aetiology of BBD seems to be multifactorial, and the mechanical compression of large stool in the colon or rectum during constipation is considered to be a principal cause for LUTD development24. Other possibility includes histological and/or molecular changes in the tissues including inflammation, fibrosis, alterations in intercellular connections, signalling molecules, receptors and ion channels28. As such, the pathophysiological mechanisms underlying BBD are complex, yet not understood very well. In the current study, we examined the effects of functional constipation on the LUT early in life using a murine model of constipation at young age.Normal function of the urinary bladder and gastrointestinal tract depends on their components including the nerves, muscle, connective tissues and mucosa, and the coordinated interactions among them. Prolonged BBD causes muscle instability in both bladder and colon, suggesting that BBD affects the muscles and nerves that control normal bowel and bladder function. One proposed mechanism is the development of bladder-bowel cross-organ sensitization via overlapping neural pathways at both peripheral and central level.29. Briefly, a lubricated 2.0 mm diameter polyethylene tubing was inserted into the anus of mice (1 cm) under anaesthesia. After injection of 2% lidocaine into the skin next to anal opening, a purse-string suture  was snugly placed in the external sphincter region around the tubing, and then the tubing was removed. To create functional constipation model (N = 42), the suture remained in place for four days. Sham operated animals underwent the same procedure except for the immediate removal of the suture after its placement . Mice were maintained in the animal facility (14-h light: 10-h dark cycle) with access to water and chow ad libitum. Rectal sutures and perianal area were checked to ensure the integrity of the model. The colorectum and urinary bladders were harvested for examination at four days after the surgery. Urine samples were collected from the bladders (N = 12 per group) and tested with urinalysis strips . All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Colorado Denver. All experiments were performed in accordance with relevant guidelines and regulations, as well in compliance with the ARRIVE guidelines 2.0 (https://arriveguidelines.org/arrive-guidelines).Male C57BL/6J mice (4-week-old) were divided into two groups: constipation model, and sham group. Constipation was induced as previously described by Heredia et al30. Each mouse had free access to water but no food during the 3 h test period. All VSA experiments were started at 10 am. The urine spots on the filter paper were imaged using ultraviolet light on a transilluminator, and the number and area of the spots were analysed using Adobe Photoshop CS6 . Faecal pellets were collected and air dried at least 16 h following VSA (N = 8 per group), and the number and the weight of pellets were recorded.In order to evaluate voiding patterns, void spot assays (VSA) were conducted at four days after the surgery, as previously described (N = 22 per group)15 immediately before undergoing the constipation or sham surgery. Cystometry was performed in unanesthetized unrestrained mice 4 days after the surgery (N = 4 per group) as described previously15. The tip of the exteriorized bladder catheter located at the base of the mouse neck was connected to a pressure transducer and an infusion pump of the cystometry station . Room temperature saline was infused into the bladder at the rate of 10 μl/min. Each animal was observed for minimum four voiding cycles of reproducible micturition patterns. Urodynamic values recorded continuously during testing, and four parameters; maximum intravesical pressure at micturition, functional bladder capacity, voided volume, and the number of non-void contractions (NVC) per voiding cycle were analysed using Cystometry Analysis Software . The NVC were defined as intravesical pressure rises greater than one-third of average maximal voiding pressure in each animal without triggering micturition.For urodynamic evaluation, mice underwent surgical catheter implantation in the bladder as previously described30. Spontaneous contractions in each bladder strip were collected for 2 min after initial equilibration for 30 min in Tyrode’s solution. Detrusor contractility was examined in the responses to a series of electrical field stimulation , the acetylcholine receptor agonist carbachol , high KCl (125 mM replaced NaCl in Tyrode’s solution), a mixture of purinoceptor agonists  (n = 14–15 per group). Contractile responses to EFS were also recorded after 20 min of incubation with the following substances: (1) ADP/ATP to desensitize purinoceptors, and (2) atropine  (n = 10–12 per group). Peak force of the contractile response was calculated in grams of tension per weight of individual bladder strip. Spontaneous contractions in the bladder strips were collected for 2 min after 30 min of (1) initial equilibration in Tyrode’s solution, (2) incubation with 5-hydroxytryptamine (5-HT) at 0.5 µM, and (3) incubation with a mixture of 5-HT at 0.5 µM and a selective Htr2 receptor antagonist, ketanserin (+)-tartrate31  at 200 nM (n = 16 per group). All data analyses were performed using PowerLab Lab-Chart version 8.1.9 . Calcium imaging of denuded detrusor sections from each group of mice  was performed with 10 µM Cal-520 AM  in Tyrode’s buffer according to the manufacturer’s protocol. The relative amplitude of Ca2+ transients was expressed as F/F0, where F represents the total fluorescence in an event of fluorescent increase, and F0 represent the basal fluorescence. Images were taken every 0.3 s for 3 min.In vitro physiological evaluation of detrusor contractility and the baseline spontaneous activity was conducted using freshly isolated bladder strips from mice in each group as described previously32. Area measurement of the distal colon sections was performed for the 4 tissue layers, mucosa, muscularis mucosa, submucosa, and muscularis externa, as well as the oval shaped non-staining area in the mucosa which was considered as mucus vesicles. All measurements were conducted in a blind fashion to avoid biased interpretation. For immunostaining, sections were subjected to heat-induced antigen retrieval . All antibodies used in this study are listed in Table Paraformaldehyde-fixed paraffin sections (5 µm thickness) of the urinary bladders and the distal colon from each group were stained with Masson’s trichrome staining15. Expression levels of each gene were calculated as fold changes based on ∆∆Ct values. Data were normalized to the mean of three housekeeping genes: β-actin (Actb), Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) and TATA box-binding protein (Tbp). Western blot analysis was performed to assess the protein expression level as previously described (N = 4 per group)15. The antibodies used in this study are listed in Table Total RNA isolated from the urinary bladders (N = 5 per group) was transcribed into cDNA and used in real-time quantitative PCR (qRT-PCR) as previously describedBlood serum samples were collected from each group of mice (N = 15 per group) with BD Microtainer serum separator tubes  and flash frozen. Serotonin ELISA Kit  was used to quantify serotonin concentration in mice serum samples. All ELISA measurements were made in duplicate.https://www.graphpad.com/quickcalcs/Grubbs1.cfm) was used to detect outliers, which were excluded from the analysis in detrusor physiology test in vitro. A probability value of p < 0.05 was regarded as significant. Results are expressed as means ± standard error of the mean (SE).All data were analysed using two-tailed unpaired t-test using GraphPad Prism 8.4.3  between two groups. GraphPad outlier calculator  .Bladder strips showed comparable contractile responses to EFS, CCh, ADP/ATP and KCl between the two groups. Inhibition of purinergic or muscarinic receptors suppressed EFS-evoked contractile response at similar level in bladder strips from both groups Fig. A,B. Howe33. Three genes, 5-HT receptor subtype 2a and 2c (Htr2a and Htr2c) and Transient receptor potential vanilloid 2 (Trpv2) were significantly upregulated in the constipation group in comparison to the control group  at the protein level in the bladder after four days of functional constipation  . The changes of spontaneous contractions between with and without ketanserin were statistically significant in both the frequency  and the amplitude  in the constipation groups, but not in the control group .To investigate how the Htr2 upregulation in the bladder induced by functional constipation impacts detrusor function, the responses of the bladder strips to 5-HT were evaluated. The 5-HT caused a small contractile response that was less than half of the response to EFS at 2 Hz, with a trend of higher force produced in the constipation group especially at lower concentrations (0.1 and 0.5 µM) compared to the control group Fig. A. On the36. Spontaneous voiding behaviour tests demonstrated that constipation led to urinary frequency with decreased volumes per-void in mice. It is possible that large stool in the colorectum takes up abdominal space and prevents the bladder to expand fully, therefore contributing to the decreased functional bladder capacity. Cystometry, an independent in vivo bladder function test, confirmed urinary frequency accompanied with a decreased functional bladder capacity in the constipation group. Additionally, increased NVC suggested bladder overactivity. These results suggested that constipation at young age can induce LUTS and contribute to the development of BBD. During urodynamic tests in awake mice, the control animals usually moved to a corner of the cage immediately before they started voiding as a part of voiding behaviour in mice as described previously37. However, the same behaviour was observed only in about half of the micturition cycles recorded in each mouse with constipation. We consider that this behavioural phenotype in mice with constipation was a manifestation of urinary urgency and/or incontinence, another LUTS common in BBD cases.In the present study, we investigated the impact of functional constipation onto the urinary bladder at histological, physiological, and molecular levels. The murine model employed in this study recapitulated clinical and pathological features of functional constipation in children including faecal impaction, faecal incontinence, colorectal distension, and growth retardation38. The urinalysis results showed normal parameters for both groups, indicating that UTI, and bladder lesions were not responsible for LUTS observed in the constipation group. Cystometry showed that the voided volume was approximately equal to the volume infused in each micturition cycle, and no intermittent void was detected in both groups of mice. Altogether, these results suggest that neither bladder outlet obstruction nor detrusor-sphincter dyssynergia developed in the constipated animals tested in urodynamic study. Histological examination also confirmed the absence of detrusor hypertrophy or fibrosis in the bladder, suggesting no bladder outlet obstruction developed along constipation in this study32. Based on the current results, we propose three potential explanations for constipation-induced bladder overactivity observed during awake cystometry. First, the bladder experienced an additional external pressure from the overdistended colorectum, which contributed to the rises in intravesical pressure without causing micturition (NVCs). Second, the convergent neurons that receive afferent inputs from both the colon and the bladder were likely continuously stimulated during constipation and sent aberrant signals to the bladder causing an increase in sensation and overactive symptoms. Studies has been shown that colorectal distension or inflammation alters bladder sensations and detrusor activities, and vice versa in both humans and experimental animals39, suggesting the existence of "cross-organ sensitization" between the colon and bladder. This might be one of the reasons why successful treatment of constipation leads to an improvement of LUTS in many patients as previously reported19. However, not all children with BBD improve LUTS after successful resolution of constipation22. This prompted us to hypothesize the third possibility that constipation, especially in chronic condition, may trigger alterations in the bladder physiology itself . Our physiological studies in vitro revealed a prominent augmentation of spontaneous contractions of the bladder strips alongside enhanced spontaneous excitation of the detrusor muscle cells in mice with constipation. These results provided evidence that constipation negatively impacted the detrusor itself, and likely altered its physiology independent from the neural inputs. On the other hand, the detrusor contractile responses evoked by EFS, carbachol and KCl as well as the proportion of purinergic and muscarinic contribution to EFS-triggered contractility did not altered following constipation. This result suggested that constipation had no significant impact on the contractile apparatus, the balance between excitatory and inhibitory neurotransmitter release as a whole, or their receptors in the bladder.Constipation is also associated with increased occurrence of UTI in children, and UTI can cause symptoms similar to those of overactive bladder40. The effect of selective serotonin uptake inhibitors (SSRIs) as well as agents acts on 5-HT receptors on LUT function has been extensively studied in both clinical and experimental settings, however, demonstrated contradictory results43. The discrepancies among studies may be due to differences in the pharmacological properties, affinities to different receptors of each agent, availability of receptors, and age and gender of study cohorts44. Htr2 receptors couple to Gq/11 protein which activates inositol 1,4,5-triphosphate pathway leading to intracellular Ca2+ increase33. Our in vitro experiments demonstrated 5-HT at 0.5 µM further augmented overactivity in the bladder strips from the constipation group, which was reversed by the Htr2 antagonist. These results suggest that the increase in the functional Htr2 receptors expressed in the bladder contribute to the constipation-induced detrusor overactivity and related LUTS at peripheral level (in situ). This notion agrees with the association between bladder hyperactivity and Htr2 upregulation in the bladder described previously44. Both groups of animals had a normal level of serum 5-HT and the comparable expression of 5-HT transporter, Sert, in the bladder, suggesting that 5-HT bioavailability was unaffected by constipation. Therefore, the detrusor cells could be activated and contract spontaneously at the normal level of 5-HT through Htr2 following a period of constipation. Other 5-HT receptor subtypes  did not show difference in the expression level in the bladder following constipation, suggesting their contribution to 5-HT induced detrusor hyperactivity may be minor34.Molecular studies revealed comparable expression levels of many genes involved in regulation of detrusor contractile function including muscle contractile apparatus, ion channels, neurotransmitter receptors as well as a cholinergic neuron marker in the bladder, corresponding to the data from physiological evaluation of the bladder. Yet, two 5-HT receptors, Htr2a and Htr2c, were significantly upregulated in the bladder following four days of constipation. Accumulating evidence suggests that 5-HT contributes to LUT function by modulating the activity of the detrusor, external sphincter, urothelium, and neural pathways at both central and peripheral levels46, consequently, an elevated activation of Htr3 pathway facilitates bladder overactivity. Our data showed an equal level of Htr3a receptor expression in the bladder from both groups, suggesting the involvement of Htr3 pathway in bladder overactivity observed in the constipation group to be minor. However, it is possible that constipation induced changes in afferent activity at the level of dorsal root ganglia or spinal cord, and therefore contributes to bladder overactivity observed in the constipation group. We did not address this hypothesis in this study, and it needs to be evaluated in future studies.The comparable intensity and distribution of Uchl1 IR in the bladders between the two groups indicate that constipation did not alter bladder innervation at appreciable level. The bladder strips from both groups generated equal contractile responses to EFS alongside the equal level of Chat gene expression, suggesting that the proportion of motor and sensory neurons in the bladder was not affected following constipation. Lines of evidence demonstrated that Htr3 plays a predominant role in bladder afferent firing and micturition reflexes at spinal and spinobulbospinal levels2+ permeability, and is expressed in the bladder along with several other TRP channels including Trpv1, Trpv4, Trpa1, and Trpm828. TRP channels are expressed in nerve fibres and urothelium in the bladder wall, and suggested to act as sensors of stretch and/or chemical irritation28. Yet, the functional significance of Trpv2 in bladder sensation is considered to be minor as the receptor was shown to be not essential for heat or mechanical nociception or hypersensitivity in the adult mouse. On the other hand, several lines of evidence indicate that Trpv2 plays a role in Ca2+ influx in different types of myocytes, and there is an association between an upregulation of Trpv2 and abnormal or leaky Ca2+ influx in pathologic skeletal muscle47. Since Trpv2 is expressed in detrusor cells in addition to nerves and urothelium28, we speculated that the upregulation of this receptor may contribute to the elevated spontaneous Ca2+ transients in detrusor cells and associated unstable baseline detrusor excitability detected in the constipation group. This conjecture requires to be tested in future studies.An ion channel, Trpv2 also showed a significant elevation in the bladder following four days of constipation. Tprv2 is a member of transient receptor potential (TRP) cation channel family with high Ca2+ regulation in the detrusor myocytes, and be associate with the persistence of LUTS even after successful treatments of constipation in BBD22. The data obtained in this study suggest that local administration of selective antagonists of Htr2 and Trpv2 may offer therapeutic options for BBD patients. Future studies focused on long-term effects of functional constipation on LUT function, as well as a possible prevention and reversibility/progression, will allow for deeper understanding of the mechanisms underlying BBD.The present study provides initial evidence of association between functional constipation and LUTD, including the changes in detrusor responses to different stimuli and symptoms of bladder overactivity. The elevated expression of Htr2 and Trpv2 in the urinary bladder might account for impaired intracellular CaSupplementary Information.


Additionally, it has high diffusivity, low viscosity and much lower dielectric constant. For instance, whereas dielectric constant of subcritical water is 80 at 298 K, it is 2 at 673 K. The point of this research paper is to assess the impacts of different reaction parameters on cellulose conversion as the principle segment of lignocellulosic biomasses for the production of value-added chemicals, particularly levulinic acid. Hazelnut shell waste was chosen as model biomass since hazelnut is a standout amongst the most cultivated agricultural crops in Turkey. Besides, Turkey provide 70% of the world's total hazelnut production. It was found that as reaction temperature increases, a considerable improvement on the amount of formed levulinic acid and conversion of hazelnut shell was observed. For instance, when the reaction temperature, time and acid concentration were 280°C, 120 min and 50 mM, respectively, levulinic acid yield and conversion of hazelnut shell were found as 13.05 and 65.40%, respectively. Addition of H2SO4 enhanced the production of levulinic acid from waste hazelnut shell. Another method which is hybrid process could be used to produce value-added chemicals from lignocellulosic biomass. Hybrid process basically combines hydrolysis and electrolysis in subcritical water. Subcritical water has much lower dielectric constant than liquid water at ambient temperature. So, it was claimed that if constant current was applied to the reaction medium through specially designed electrodes in subcritical water environment, electrolysis could alter the hydrolysis reaction of cellulose in a way of protonation of intra-and inter-molecular hydrogen bonding around anode and as a result electrolysis in subcritical water could decrease necessary thermal energy to hydrolyze the β(1–4) glycosidic linkage. Therefore, we developed a green hybrid process by combining hydrolysis and electrolysis in subcritical water without using any toxic, organic solvents and catalyst. Effects of especially applied current and temperature on the product distribution and conversions of cellulose were revealed and hydrothermal electrolysis reaction pathway of cellulose was proposed. The significance of the interaction indicated that, applied voltage had major impact on cellulose hydrolysis. Maximum cellulose conversion (82%) was achieved at 230°C and 180 min of reaction time in 25 mM of H2SO4. Application of 8.0 V of applied voltage to the reaction medium at reaction temperature of 230°C increased the TOC conversion (50.3%) with acid concentration of 25 mM in comparison with current-free experiments. Thus, the idea of electrochemically generated acid layer due to the dissociation of water around anode is supported. As future perspective, the output of the study gave an idea about converting cellulose and various biomass wastes, which may have high cellulose, content and led the way in obtaining valuable chemicals from no utilized real biomass sources such as hazelnut shell waste. The studies with other biomasses are undergoing.Recently, liquefaction of biomass in subcritical water to convert it into value-added substances has been broadly attracting attention. However, there is a gap in literature about the levulinic acid, which is a high worth substance, production from biomass using subcritical water. As a green chemistry approach, decomposition of biomass could be obtained using subcritical water effectively. In this case, water uses as a solvent so that it gives a possibility to take place a reaction for the decomposition of biomass. Subcritical water, which liquid water and its temperature is higher than the normal boiling point of water, has higher ion product as well as higher concentrations of H Biomass is a renewable energy source and the interest for biomass increases day by day. Since 50% of total biomass consists of plant-derived or lignocellulosic biomass and its content made-up from cellulose (38–50%), which is one of the most prevalent organic compounds in the World, hemicelluloses (23–32%), and lignin (15–25%) 2 and RbCO3 catalysts. Bio-oil yield enhanced by the addition of catalysts.In literature, there are several researches about the natural biomass decomposition in subcritical water. Chan et al.  studied Electrochemical methods such as electrochemical degradation of lignin  in which cylindrical type titanium electrode was used as anode and reactor wall was behaved as cathode. In this research activity, effects of different parameters which are direct current, temperature, time and proton donor (sulfuric acid) concentration on the conversion of cellulose were investigated. The conversion of TOC and yields of products  were determined.2SO4 was used as an electrolyte and also be a proton source. Hydrothermal electrolysis products of cellulose were investigated. After series of experiments, levulinic acid, 5-HMF and furfural was determined as the main decomposition products of cellulose by hydrothermal electrolysis. Detailed ionic and radical mechanisms of cellulose under constant voltage were revealed.After obtaining the optimum conditions, it was proposed when constant voltage is applied, the degradation mechanism of cellulose and hydrolysis product distribution can selectively change. Constant voltage ranges from 4.0 to 8.0 V was applied between anode and cathode. 25 mM of HAll chemicals used in experiments were at analytical grade and all solutions were prepared using de-ionized water. Microcrystalline cellulose (MCC) was purchased from Sigma Aldrich. The standards of reagents used in HPLC analysis are fructose (≥99%), levulinic acid (98%), lactic acid (98%), glycerolaldehyde (99%), glycolaldehyde (99%), 5-HMF (99%), glycolic acid (99%), and pyruvic acid (98%) and they were purchased from Merck. Sulfuric acid (96–98%) was also obtained from Merck. Phosphoric acid (85–90%) were purchased from Fluka. Biomass feedstock, hazelnut shell, was supplied from Ordu, Turkey. Hazelnut shells were dried in an oven at 60°C and then, they ground into small pieces (~1 mm) using a laboratory type grinder.Elemental analysis of hazelnut shell was performed via CHNS-932, Leco, USA branded elemental analyzer. Moisture and ash contents of hazelnut shell (biomass) were determined by Thermal Gravimetric Analysis . Hazelnut shell contains cellulose, hemicellulose and lignin and their amount were determined by Van Soest Method  was used for detection of aldehydes and organic acids. TOC conversions in the liquid products were determined by TOC analyzer (Shimadzu TOC-VCPH).The concentration of liquid products was determined via High Performance Liquid Chromatography (HPLC) and Gas Chromatography with Mass Spectroscopy  were used to identify the liquid products which were unidentified in HPLC analyses. In HPLC analysis, sugar column  was used to separate products clearly and the temperature of column was 40°C. 3.75 mM HExperiments related to liquefaction of cellulose and hazelnut shell waste were performed in a Parr Instrument Company, USA branded 316 stainless steel autoclave (300 mL). The reactor equipped with gas inlet and outlet valves, liquid sampling valve, pressure gage, safety rupture disc, magnetic-driven impeller, internal thermocouple, and cooling loop  with specially designed anode (titanium electrode) and it is illustrated in o: 76 mm, L: 165 mm) was acted as cathode. The experiments were carried out to understand the reaction pathway by using H2SO4 solution (25 mM) at 200°C and applying constant voltage  for 180 min. Liquid samples were collected in every 30 min. after the desired reaction temperature was reached. Collected liquid samples were filtered and analyzed immediately without any further treatment  passing through the electrodes. Specially designed cylindrical type titanium electrode  was used as anode and cylindrical reactor wall  and (3), respectively.Cellulose has glycosic bonds so that is show high chemical resistance. In order to break these bonds, subcritical water can be used. Hence, usage of subcritical water provides a favorable environment and reaction medium for degradation of cellulose to desired product (levulinic acid)  yield at different reaction times was investigated and the results are given in 2SO4 concentration  on the product distribution was also investigated. In that case, yield of levulinic acid enhanced with respect to the increasing H2SO4 concentration. While the lower yield of levulinic acid was observed as 4.8% using 5 mM H2SO4), the highest yield was observed as 38% with the addition of 125 mM H2SO4. Similar trend was observed in the yield of formic acid. The yield of formic acid was observed as 2.6 and 21% using 5 and 125 mM H2SO4, respectively  was used excessively and long reaction time (≥60 min). Two main reaction pathways were possible for hemicellulose. When hydrothermal reaction was performed at low H2SO4 concentration (0–5 mM) at 200°C, acetic acid formation was favored by the cleavage of acetyl , which is illustrated in o: 76 mm, L: 165 mm) was used instead of cathode. To purpose the reaction pathway of degradation of cellulose when current was applied, the reaction conditions were as follows: 25 mM H2SO4 solution at 200°C with application of constant voltages  for 180 min.Experiments of hydrothermal electrolysis were performed at a constant current which are 0 and 2 A passing through the electrodes. In this set-up, anode was specially designed cylindrical type titanium electrode , cylindrical reactor wall (D−11 of ionic product concentration (200–300°C range) and dielectric constant is ≈10  glycosidic bond of cellulose.The hypothesis of this is that application of direct current into reaction medium under hydrothermal condition would create activated species in terms of ionic and radical molecules due the redox reactions of water and sulfuric acid. Formation of these molecules can alter the decomposition of cellulose as in the postulated reaction mechanism . Applica2SO4 was used and 1 A current was applied to the reaction medium, the required reaction temperature for the maximum cellulose conversion of 82% decreased from 230 to 200°C , so that TOC conversion enhanced. As current values increased, concentration of radicals in the reaction medium increased. However, TOC conversion decreased when higher current (2 A) was applied. Additionally, the application of direct current (1–2 A) affected the formation yields of 5-HMF and levulinic acid. While the yield of 5-HMF enhanced by direct current, levulinic acid concentration decreased. During the reaction, variable electrode potential was observed because of the application of constant current to reaction medium. Hence, the change in electrode potentials leads to formation of various activated species within the reaction medium. Davis et al. . Selectivity values of cellulose on formation of furfural, 5-HMF and levulinic acid were determined as described in material and methods section. The control reaction was carried out by applying voltage under hydrothermal conditions (200°C) in the absence of acid catalyst. Results are listed on Effect of applied voltage (4.0 and 8.0 V) on product selectivity and TOC conversion in hydrothermal reaction medium was investigated in the presence and absence of acid catalyst (H•−) could enhance under hydrothermal conditions by applying voltage. Hence, in the light of literature, it is possible to say that higher TOC conversions could be achieved by higher voltage. If concentration of radical species increases, decomposition products could form via radical based mechanism. The coupled effect sulphuric acid (25 mM) and applied voltage can be explained by the oxidation of sulphuric acid to form the sulfate radical (SO•4−) near anode , TOC was recorded as 13.8% . WhereasThe conversion of fructose to furfural under hydrothermal conditions could be proposed by two reaction pathways  was applied, various activated radical species such as SOnd 5-HMF . Additiond 5-HMF .Hydrolysis of cellulose in sub-critical water conditions has been widely searched because of the versatile properties  of hot compressed water as reaction medium. Thus, high ionic product concentration and its high diffusivity to intra and inter molecular hydrogen bonds enhanced the protonation of β(1–4) glycosidic linkage of cellulose that yield degradation products which are glucose, fructose, 5-HMF, furfural, and levulinic acid.2SO4) concentration was investigated. The main products of hydrothermal acid-treatment were clarified as levulinic acid, furfural and acetic acid by HPLC analysis. As reaction temperature increases, a considerable improvement on the amount of formed levulinic acid and conversion of hazelnut shell was observed. For instance, when the reaction temperature, time and acid concentration were 280°C, 120 min and 50 Mm, respectively, levulinic acid yield and conversion of hazelnut shell were found as 13.05 and 65.40%, respectively. Addition of H2SO4 enhanced the production of levulinic acid from waste hazelnut shell.As model biomass, hazelnut shell waste was converted to value-added chemicals under hot compressed water. In the context of experimental study, the effect of reaction temperature, reaction time and acid  in comparison to current free experiments. The enhancement in TOC was conducted for the formation of an acid layer around anode due to the self-dissociation of water that hydrolyses cellulose. It was proposed that application of constant voltage could selectively change the degradation mechanism of cellulose and hydrolysis product distribution. The detailed product distribution of decomposition of microcrystalline cellulose under subcritical water condition by application of direct current was investigated and reaction pathway for hydrothermal electrolysis of cellulose was proposed.All datasets generated for this study are included in the article/supplementary material.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.