Organogenesis and regeneration are fundamental for developmental progress and are associated with morphogenesis, size control and functional properties for whole-body homeostasis. The liver plays an essential role in maintaining homeostasis of the entire body through various functions, including metabolic functions, detoxification, and production of bile, via the three-dimensional spatial arrangement of hepatic lobules and has high regenerative capacity. The regeneration occurs as hypertrophy, which strictly controls the size and lobule structure. In this study, we established a three-dimensional sinusoidal network analysis method and determined valuable parameters after partial hepatectomy by comparison to the static phase of the liver. We found that mechanical homeostasis, which is crucial for organ morphogenesis and functions in various phenomena, plays essential roles in liver regeneration for both initiation and termination of liver regeneration, which is regulated by cytokine networks. Mechanical homeostasis plays critical roles in the initiation and termination of organogenesis, tissue repair and organ regeneration in coordination with cytokine networks. Ishikawa, Takeo et al. present a 3D liver sinusoidal network analysis method and identify important parameters after partial hepatectomy. They find that in coordination with cytokine networks, mechanical homeostatic signalling, including shear stress and tension, plays critical roles in the initiation and termination of liver organ regeneration. After birth, as well as after disease and injury, organs are maintained by cell turnover, tissue repair, and organ regeneration under homeostatic conditions. Regenerative phenomena, such as whole-body regeneration in planarians and limb and tail regeneration in urodele amphibians, are widely distributed in nature, although the regenerative capacity varies among species3. During regeneration, undifferentiated cells, including stem cell-derived and/or progenitor-derived cells, traditionally referred to as blastema cells, accumulate at the amputation area, and growth and differentiation of these undifferentiated cells leads to restoration of the missing part. In mammals, the regeneration capacity is very restricted, and only tissue repair occurs in most organs following disease and injury. These differences in regenerative capacity among species are known to depend on the distribution of the wide variety of stem cells with different potentials, comprising pluripotent, organ-inductive and tissue stem cells4. Although the molecular mechanisms, such as gene expression networks and cytokine cascades, have been elucidated in both organogenesis and regeneration in each species, it remains unknown how the events, including physical and molecular mechanisms, are induced and completed to maintain the balance of homeostasis.Organogenesis occurs only during embryonic development through complex processes involving the formation of organ germ, which arises from organ-inductive potential stem cells, and subsequent morphogenesis, resulting in the unique morphology and function of individual organs6. In contrast to other organs with specific shapes and morphologies, the mass/volume of the hepatic lobule is essential for liver function but not organ morphology. The liver is also well known to have a high regenerative capacity among mammalian organs7. Mammalian liver organ regeneration is known to be a form of compensative hypertrophy, in which the volume of an organ increases due to enlargement (hypertrophy) and proliferation (hyperplasia) of its component cells8. The cellular and molecular mechanisms underlying compensative hypertrophy, which is also observed in the kidney9, and regeneration in amphibians are clearly different because the liver is a functional organ unit that receives blood from the portal vein and hepatic artery and passes blood to the suprahepatic inferior vena cava (SHIVC). Past studies have revealed that liver regeneration, i.e., compensatory hypertrophy, is complexed process involving multiple types of cells, such as endothelial cells as well as bone marrow cells, and tightly regulated by the cytokine network, including hepatocyte growth factor (HGF), epidermal growth factor (EGF), Tumour necrosis factor (TNF), vascular endothelial growth factor (VEGF), Hedgehog, Wnt/β-catenin, and transforming growth factor-beta (TGFβ)13. Despite multiple studies regarding the molecular mechanism of liver regeneration, in which large haemodynamic alterations occur during liver regeneration, how the liver senses its situation and the triggers for the initiation and termination of regeneration remain largely unknown. Knowledge of these aspects is expected to elucidate the sensing mechanisms associated with successive molecular networks.The liver plays an essential role in maintaining homeostasis of the entire body through its functions, including the storage and metabolism of substances, detoxification, and production of bile, through the three-dimensional spatial arrangement of hepatic lobules, which are cord-like structures consisting of hepatocytes and sinusoids15. Organ morphology and function, which are controlled by the traction force balance in cells, are essential for maintaining organ homeostasis16. Cells sense physical stimuli, including frictional force (shear stress) and tension traction force, from the surrounding environment via morphological changes in cell shapes, including changes in the cytoskeleton and membrane, and respond to mechanical-biological stimuli through mechanotransducers17. In the vascular structure, the various physical forces generated by blood flow, including shear stress and traction force on the surface of endothelial cells, are known to play important roles in the specific morphological and physical properties of blood vessels18. The extension of vascular scaffolds under high blood pressure is known to play an important roles in the specific morphological and physical properties of blood vessel structures19. Endothelial cells have several receptors, including P2X4 and Piezo1, as physical force sensors for vascular development and diacylglycerol-sensitive canonical transient receptor potential (TRPC) subunits for sensing membrane stretching, along with G protein-coupled receptors (GPCRs) for sensing these mechanical stresses22. Based on these observations, it is hypothesized that mechanical sensors of blood vessels in the liver would have key roles as the triggering and termination signals associated with the successive cytokine networks during liver regeneration.Mechanical homeostasis, including gravity, shear stress, osmotic pressure, and tension, is crucial for organ and tissue development, including the establishment of morphological and functional propertiesIn this study, we identified the fluctuating parameters of the liver sinusoidal network, which were examined by a three-dimensional structural analysis of the network, under liver regeneration by using partial hepatectomy. The operation models related to these physical fluctuations in sinusoidal networks included a bypass vessel to inhibit the sinusoidal blood flow increment after partial hepatectomy, and small-liver transplantation to a large animal to increase blood flow to the transplanted liver; these models indicated that mechanical homeostasis, including tension and shear stress, is essential for the triggering signals for liver regeneration. Erk phosphorylation in both human umbilical cord endothelial cells (HUVECs) and endothelial cells isolated from the liver was observed after shear stress loading and was inhibited by a GPCR inhibitor, suramin. The production of TGFβ1, which is known to inhibit hepatocyte proliferation, was also regulated by haemodynamics in the early liver regeneration process. These results indicated that mechanical homeostatic signalling, including shear stress and tension, to sinusoidal endothelial cells prior to HGF production plays essential roles in the initiation and termination of liver organ regeneration.The liver is divided into five lobes, i.e., the left lobe (LL), left median lobe (LML), right median lobe (RML), right lobe (RL), and caudate lobe (CL). These lobes are mainly composed of hepatocytes and blood vessels Fig. . In eachint Fig. . We founint Fig. . The sinint Fig. . To analint Fig. . We founint Fig. . Next, tint Fig. . We alsoint Fig. . We defiint Fig. . These fQ) and volume (V) of the entire liver are expressed as the sum of each lobe  is increased after PH. After regeneration, the total liver volume is recovered, and the blood flow efficiency in each lobe is defined as below using the regenerated volumes of the RL (V′4) and CL (V′5).4 cell per mm3 to 9 × 104 cell per mm3 by 1 day after PH , which results in removal of the RML, LML, and LL, and the liver remnant is composed of the RL and CL Fig. . The mecobe Fig. . We also PH Fig. . During  PH Fig. . At the mentclass2pt{minim PH Fig. . As expe PH Fig. . ImportaWe further examined the causal relationship between the increase in sinusoidal blood flow velocity and liver regeneration. To this end, we developed a surgical rat model that can suppress the increased blood flow velocity in sinusoids by redirecting about 70% of blood flow of portal vein into the inferior vena cava by delegating the ligation of the portal vein to reduce adjusted, so that the cross-sectional area isto 1/3 of the original Fig. . In cont21. Therefore, we analysed how vascular endothelial cells are involved in the initiation of liver regeneration.Past studies have demonstrated that vascular endothelial cells, which are distributed throughout the liver, can detect the change in shear stress in blood vessels and shift the entire liver to the regeneration phase23. We found that the intracellular calcium concentration increased immediately after shear stress loading at 15 dyne/cm2, and this response was neutralized by applying suramin, an inhibitor of GPCRs  Fig. . When enCRs Fig. . We furtECs Fig. . Next, wECs Fig. . In cleaECs Fig. . We furt24  model in which a small liver isolated from a young rat was orthotopically transplanted into an aged large rat, and assumed that the transplanted liver regenerates similar to the PH model due to an increase in blood flow Fig. . We orth24 Fig. . Consist24 Fig. . In the 24 Fig. . The num24 Fig. . In bothel Figs.  and 5k. ion Fig. . These rion Fig. . Taken tIn our current study, mechanical homeostasis indicated by fluctuation parameters, including tension and shear stress on liver sinusoidal networks, plays an essential roles in the initiation of liver regeneration. The physical fluctuations are converted to chemical signalling prior to cytokine production. It is suggested that mechanical homeostasis is involved in maintaining liver homeostasis, including the pathology and functions of both steady-state and regeneration of the liver in vivo. These findings contribute to the understanding of organ homeostasis and functions through tension homeostasis, and they indicate that mechanical homeostasis could be essential for the initiation and termination of organ regeneration and growth.8. Hepatic lobule structure improves metabolic efficiency, and the mass/volume of hepatic lobules is essential for total liver functions in vivo25. Previous studies, including a liver regeneration model in silico, indicated that the hepatic lobule structure ensures optimal exchange of metabolites in sinusoids27. During liver regeneration by partial hepatectomy, rapid change of hepatocyte membrane potential occurs immediately after partial hepatectomy possibly mediated by satellite cells29. Simultaneously, haemodynamic changes, such as changes in the portal pressure and physical force caused by blood flow, might be affected by sinusoidal structural parameters, such as the vascular diameter or volume, depending on the physical vasodilation force from inside the sinusoid31. Moreover, previous studies suggested the relationship between change in blood flow rate and resulting shear stress and liver regeneration34. However, the relationships between the effect on sinusoidal structure and liver regeneration remain unclear36. In the current study, we revealed the detailed structural changes in the sinusoids, including quantitative parameters, by accurately measuring the spatial arrangement of the sinusoids and found that the sinusoidal volume and the surface area drastically changed at only 6 h after the partial hepatectomy, similar to changes in parameters associated with liver volume prior to the biological reactions for liver regeneration, such as HGF expression, as previously reported37. Furthermore, our developed bypass model of partial hepatectomy, which suppresses the increase in blood flow and changes in sinusoidal structure, revealed that the increase in these haemodynamic changes is essential for the early stage of liver regeneration. These observations suggest that alterations in mechano-homeostasis, which is maintained by stable blood flow in sinusoids, would be the first triggers/initiators of liver regeneration.Liver organ regeneration is known to be a form of compensative hypertrophy, occurring through the enlargement (hypertrophy) of parenchymal cells, proliferation (hyperplasia) of its component cells and vascular remodelling38. The traction force balance in cells also plays an important role in maintaining organ homeostasis by controlling organ morphology and function16. The balance among extracellular forces exerted on neighbouring cells and the traction forces generated by cells themselves is termed tensional homeostasis39. Among many cells and tissues, endothelial cells in vascular networks are well known to be constantly exposed to mechanical stress, including shear and tension stress on their cell membrane, caused by blood flow and/or pressure41. Previous studies reported several essential molecules related to these mechanical forces on endothelial cells42. The mechanoreceptor P2X4, which is a type of Ca2+-ion channel on the surface of endothelial cells, followed by proteins such as calmodulin and downstream protein kinases in intracellular cascades, regulates cellular behaviour, including increased production of nitric oxide and prostacyclin, which cause vasodilatation43. The TRP ion channel is also known to regulate cellular functions, including endothelial permeability in actin-stress fibre formation, and is coordinated with G-protein-coupled receptors (GPCRs) as a sensor for membrane stretching42. It is also known that several GPCRs, including angiotensin receptor type-1 and bradykinin B2 receptor, are activated by changes in the traction force balance of endothelial cells and regulate endothelial cell behaviour through conformational changes by partial rotation of the receptor unit without ligands46. In previous studies, liver regeneration was shown to be sensitive to suramin, which is an inhibitor of GPCRs47, suggesting that mechanical homeostasis through suramin-sensitive GPCRs has an important roles in the response of liver regeneration50. In this study, we observed a delay in liver regeneration in bypass surgery to decrease the blood flow of the portal vein increment after partial hepatectomy. We also observed an early-stage proliferation of parenchymal cells in the liver and downregulation of serum TGFβ1, which is known to be an inhibitor of parenchymal cell growth. We also found that Ca2+ influx, the downregulation of TGFβ1 and Erk phosphorylation in both HUVECs and primary isolated LSECs occurred in the shear stress model in vitro and were sensitive to suramin treatment. We could not evaluate whether a similar mechanism exist in vivo situation because of the experimental limitations, such as absence of reliable method to monitor Ca2+ influx and phosphorylation of ERK in vivo. Nevertheless, these findings strongly suggest that the balance of suramin-sensitive mechanical homeostasis in endothelial cells in the sinusoidal network in the liver plays an essential roles in liver regeneration, i.e., compensative hypertrophy prior to cytokine networks.Environmental mechanical stresses, including shear stress, osmotic pressure, and tension, are known to play an essential roles in the morphological and physical properties of tissues and organs during both embryogenesis and homeostasis after birth38. During organogenesis, tissue repair, and organ regeneration, mechanical homeostasis is considered as one of the fundamental and primitive triggers for cellular proliferation and tissue maturation prior to cytokine networks and is also essential for the termination of tissue and organ morphogenesis38. The mechanisms by which organs sense the timing to start and terminate organogenesis and regeneration are fundamental, but remain unanswered questions in developmental and medical biology53. Mechanical stress is thought to be a fundamental and primitive trigger not only for the initiation of cell proliferation and tissue maturation but also for the termination of regeneration to prevent over-regeneration55. In organogenesis, the cardiac loop is formed by unbalanced forces in left–right looping directionality in cardiac morphogenesis, and several mechanoreceptors mediate vascular network formation56. After birth, muscle or bone component cells receive mechanical force and regulate the balance between the proliferation and differentiation phases57. Endothelial cells also sense high blood pressure and enter vascular remodelling to reduce the pressure20. During liver regeneration, Hippo signalling defines an organ size through gene expression controlled via nuclear localization of the mechanical stress receptors YAP/TAZ61. These findings suggest that the balance of mechanical homeostasis, including traction force, shear stress, and other mechanical stresses, plays an essential roles in organogenesis, tissue repair, and organ regeneration. It is also well known that several cytokines, such as HGF, EGF, and inflammatory signals, mediate the liver regeneration process64. In this study, the change in haemodynamics following partial hepatectomy resulted in the disturbance of mechanical homeostasis, which is sensed through GPCRs and drives subsequent cytokine cascades as a trigger in the initiation phase. Furthermore, in our experiments, including the 3D-sinusoidal analysis, s-OLT model, and endothelial cell assay in vitro, these mechanical stress parameters continued to reach the equilibrium rates of those in the normal static phase and were involved in the termination process of liver regeneration for a return to equilibrium. Although further confirmation in vivo situation is necessary, these results strongly suggest that physical sensing of mechanical homeostasis is an essential system for both the primitive trigger in the initiation phase and sensing of liver size in the termination phase of liver regeneration, which is coordinated with cytokine networks.During embryogenesis and organ and/or organ system regeneration, the regulation of organ size and morphology is thought to play essential roles in proper organogenesis and regeneration of the organ and/or organ systemIn conclusion, we demonstrated that mechanical homeostasis in liver sinusoidal networks plays an essential roles in both the initiation and termination of liver regeneration. Our results show the impact of mechanical homeostasis in linking cytokine cascades in a wide variety of phenomena, such as organogenesis, tissue repair, and organ regeneration. Further studies on the detailed molecular mechanisms of mechanical homeostasis, including traction force balance and shear stress, which are sensed by GPCRs and other sensing molecules on endothelial cells as mechanical sensors, would provide a valuable information for understanding the roles of both systems of mechanical homeostasis and cytokine networks during organogenesis, morphogenesis, and regeneration in vivo.5–15-weeks-old male Wistar rats and 5–8-weeks-old male C57/BL6 mice were purchased from SLC Inc. (Japan). All handling and care procedures for the rats conformed to the National Institutes of Health (NIH) guidelines for animal research, and all experimental protocols involving animals were approved by the Riken Animal Care and Use Committee (Permit Number: A2014-02-12). All efforts were made to minimize suffering.x, y, and z coordinate system.To visualise the sinusoidal structure, we prepared a solution of fluorescein 5-isothiocyanate  conjugated to gelatine (Sigma-Aldrich Japan), and dissolved in 1 mL dimethyl sulfoxide  at pH 11. The FITC solution and 20% (w/v) gelatine solution were mixed for conjugation at 37 °C for overnight. The unconjugated FITC was removed using a NAP-25 column . We perfused this FITC-conjugated gelatine from the left ventricle, and livers were harvested and fixed with Zamboni solution at freezing temperature. From these samples, frozen sections (300 μm thick) were prepared after fixation using 8% formalin containing Hoechst 33342 dye . The fixed sections were washed with PBS (−) and examined using laser confocal microscopy . These three-dimensional imaging data were analysed using Measurement, FilamentTracer, and Surface in Imaris software (Bitplane) with an The livers were flushed with saline and fixed with 10% formalin . Tissue sections (5 μm thick) were taken after paraffin embedding and staining with haematoxylin and eosin (HE). For Brd-U staining, sections were processed under 2 N HCl for 15 min at room temperature and washed three times using PBS. After blocking, sections were first stained with anti-Brd-U antibody  and then with FITC-conjugated goat anti-rat IgG antibody .2PO4/2H2O , Na2HPO4/12H2O , HEPES , glucose , ethylene glycol tetra-acetic acid , NaHCO3 , and phenol red . A secondary perfusion was performed for 8–15 min using collagenase solution , which contained NaCl , KCl , NaH2PO4/2H2O , Na2HPO4/12H2O , CaCl2 , HEPES , glucose , NaHCO3 , phenol red , and soybean trypsin inhibitor . After the secondary perfusion, the liver was split into multiple tissues, and the liver digestion was corrected through mesh filtration  in 2% FBS MEM. To obtain the hepatocytes, we washed the digestion using MEM and corrected the pack with two centrifugations at 50×g. For purification of LSECs, we used the supernatant that was removed hepatocytes and performed MACS using anti-CD45 microbeads , anti-PE microbeads , and anti-CD146 PE-conjugated antibody . We used the fraction of CD45− and CD146+ as LSECs.The preparation of liver cells was performed by using the collagenase perfusion method. Abdominal incisions were performed on the rats under anaesthesia, and 18G catheters  were inserted into the PV and inferior vena cava. The initial perfusions  were performed using the perfusion solution,which included NaCl , KCl , NaHLSECs and HUVECs were cultured with the endothelial cell growth medium BulletKit for maintenance . For the shear stress loading experiment, these cells were seeded onto a microslide I 0.4 Luer  coated with collagen 1A. We perfused endothelial cell basal medium  supplemented with 0.5% BSA and an antibiotic-antimycotic mixed stock solution  to load shear stress using a peristaltic pump. For inhibition of GPCRs, we precultured the cells with ECM supplemented with 0.25 μg/mL suramin sodium  an hour before shear stress loading.Animals were incised at the abdomen to expose the surface of the liver and placed on a plate made of thin glass to observe the liver surface at a wavelength of 488 nm under 1.5% isoflurane inhalation anaesthesia. We obtained time-lapse images to observe the erythrocytes moving at the liver surface using a high-speed imaging system constructed of an EM-CCD camera ImagEM X2 and high-speed recording software . We tracked erythrocytes in every frame and measured the moving distance over ten frames using Imaris software.The medium was replaced with ECM supplemented with 10 μM Indo-1 AM  and 0.02% Pluronic F-127  for 50 min to incorporate the dye and ECM for 20 min to de-esterize the intracellular dye. We obtained time-lapse images using a two-photon laser microscope  by perfusing HBSS without calcium  under a 20-cm hydraulic head distance to obtain a non-pulsatile flow of 11 mL/min. In the suramin group, we used these media supplemented with 0.25 μg/mL suramin sodium.Rats were incised at the abdomen transversely, and the infrahepatic inferior vena cava was half-clamped to anastomose the vessel graft that was obtained from the other rat portal vein. After anastomosing, we clamped the portal vein and anastomosed it to the other end of the graft. Before opening the clamp, we stenosed the portal vein at the hepatic hilar section using a plastic tubular band made from 20G Surflo  and opened the clamp. Continuously, we resected the left and median lobes of the liver for 2/3 partial hepatectomy according to the Higgins and Anderson method.Donor rats at 5 weeks of age were incised at the abdomen transversely, and the resected ligament was connected to the liver and ligated to the left diaphragmatic vein, portal branchings, right renal vein, and right adrenal vein under 2% isoflurane. The hepatic artery from the common hepatic artery to the proper hepatic artery was preserved, and branches such as the gastric artery and splenic artery were ligated. After a tail vein injection of 100 U heparin sodium , a 22G catheter  was inserted into the bile duct as a stent. We clamped the thoracic aorta and flushed out the blood by perfusing saline from the abdominal aorta before liver donation. A plastic cuff of 20G catheter  for the portal vein and 12G catheter  for the infrahepatic inferior vena cava (IHIVC) was attached to each donor’s vessel carefully and with full dilation. To reconstruct the hepatic artery, we preserved the aortic wall around the branching to use it as a Currel’s patch. We anaesthetized the recipient rat at 15 weeks using 1.5–2% isoflurane and removed the liver after clamping the portal vein, IHIVC, and suprahepatic inferior vena cava (SHIVC) and ligating the proper hepatic artery. We anastomosed the SHIVC manually by reefing the recipient’s vascular end and inserted the portal cuff into the recipient portal vein. After reflowing to the transplanted liver, the IHIVC cuff was inserted into the recipient’s vessel for subsequent reconstruction of the hepatic artery by anastomosing to the abdominal aorta and allowing blood to flow again. The bile ducts were connected to each other by the stent, and several clots in the abdomen were washed out using warm saline before abdominal closure.RNA was isolated from hepatocytes and LSECs using the RNeasy Plus Mini Kit (Qiagen) and was reverse transcribed using SuperScript VILO  to obtain cDNA. Real-time PCR was performed on an Applied Biosystems QuantStudio 12K Flex (Thermo Fisher Scientific) using SYBR Premix Ex Taq II  or a TaqMan Gene Expression Assay system . The amount of expression of each target gene was normalized to the expression level of GAPDH. The primer pairs used for real-time PCR are listed in Supplementary Table The cells were washed with ice-cold PBS, and the proteins were extracted using RIPA buffer supplemented with protease and phosphatase inhibitor cocktails . The lysates were centrifuged at 13,000 rpm for 5 min, and supernatants were quantified using Bradford reagent. Western blotting was performed using p44/42 MAPK antibody , phospho-p44/42 MAPK antibody  and beta actin antibody , and western blot images were analysed with ImageQuant LAS 3000 .Culture medium and serum were quantified using an ELISA kit for rTGFβ1  according to the manufacturer’s protocol.t-test with two-tailed test. We repeated same experiments at least three times. Each sample size are indicated in the figure legend.Statistical significance was determined based on Student’s Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Data 1Reporting Summary


Total anomalous pulmonary venous connection (TAPVC) is a rare congenital cardiac malformation, and prenatal detection of TAPVC malformation remains a challenging. TAPVC can be easily missed or misdiagnosed in prenatal examinations. This study was aimed to use the modified vascular corrosion casting technique to prepare fetal cardiovascular casts with TAPVC and investigate the utility of cardiovascular casting for the demonstration of fetal TAPVC. The retrospective study enrolled twenty fetuses  with TAPVC diagnosed by prenatal echocardiography and casting technique from May 2015 to May 2020. Pre- and postnatal medical records, including results obtained by prenatal ultrasound, postpartum computed tomography angiography, as well as anatomic and cardiovascular casting findings were carefully reviewed and analyzed. In twenty cases, 80% (16/20) had intra- or extracardiac malformations. The TAPVC types were supracardiac (n = 8), cardiac (n = 6), infracardiac (n = 4), and mixed (n = 2). The diagnosis of 1 case each of supracardiac and cardiac TAPVC was modified to partial anomalous pulmonary venous connection; additionally, 4 malformations were missed and 2 were misdiagnosed, including an anomalous left brachiocephalic vein in supracardiac TAPVC, abnormal inflow of the hepatic vein and a double inferior vena cava in infracardiac TAPVC; and bilateral ductus arteriosus in infracardiac TAPVC; a tetralogy of Fallot in cardiac TAPVC that was corrected to right ventricular double outlet; and an absence of ductus arteriosus that was misdiagnosed as slim ductus arteriosus. Comparing with ultrasound, casting technique has its own superiority in exhibiting TAPVC abnormalities, especially in certain types such as course, origin and absence abnormalities of ductus. Postpartum cardiovascular casts can accurately depict the branch structure of the heart’s larger vessels, and may be used as a clinical assessment and teaching method in complex cardiac malformations. The main feature of this condition is that all 4 pulmonary veins (PVs) fail to form a direct connection with the left atrium, instead draining into the right side of the heart via different routes of systemic venous return4. Examination of the fetal venous system has become an increasingly important aspect of prenatal monitoring, because isolated venous abnormalities are not only potentially fatal, but may also reflect more extensive heart malformations or genetic syndromes6. TAPVC is the only condition among cyanotic heart diseases that involves venous malformation, and it is easily misdiagnosed7. Early diagnosis of TAPVC—especially prenatally—is critical because after birth, cyanosis often occurs through the mixing of blood in the systemic and pulmonary circulations, which may be accompanied by PV reflux obstruction8 and lead to a poor outcome without timely intervention. Early diagnosis of abnormal venous drainage is also an important consideration for surgery12. Recognizing TAPVC in utero remains clinically challenging for obstetricians; in a retrospective analysis of birth records from 1998 to 2004, only 1.9% of TAPVC cases (8 in 424) were prenatally diagnosed13.Total anomalous pulmonary venous connection (TAPVC) is a rare condition that accounts for 0.7%–1.5% of cases of congenital heart disease14.Because of its noninvasiveness and convenience and real-time monitoring capabilities, prenatal ultrasound is becoming the preferred method for diagnosing TAPVC, especially with the development of new scanning technologies such as color flow Doppler imaging and spatiotemporal image correlation, which has improved the diagnosis rate. However, given the lack of awareness of this rare malformation and the fact that it is often accompanied by complex cardiac and intracardiac malformations, TAPVC can be easily overlooked or misdiagnosedIn the present study, we retrospectively analyzed the clinical data of 20 cases of fetal TAPVC diagnosed by prenatal ultrasound, and compared the results of antenatal echocardiography with postpartum echocardiography, postpartum computed tomography angiography (CTA), and anatomic findings to establish the characteristics of different types of TAPVC and their venous return. In order to clearly display the 3D structure of fetal vessels, modified cardiovascular casts of some specimens were also used for postpartum diagnosis.The study was approved by the ethics committee of Xiangyang No. 1 People’s Hospital Affiliated with Hubei University of Medicine. All pregnant women enrolled in our study provided written, informed consent. We retrospectively analyzed 20 cases of TAPVC diagnosed by ultrasound before delivery between May 2015 and May 2020. The gestational age ranged from 22 + 4 to 29 + 6 weeks , and maternal age was between 19 and 36 years .Prenatal ultrasound was performed according to guidelines of the International Society of Ultrasound in Obstetrics and Gynecology and American Society of Echocardiography using a Voluson E8 or 730 ultrasound system  equipped with RM6C, C4-8, and C4-8D transducers (4–8 MHz). The number, location, and specific PV drainage routes were visualized in a short axis section of the heart.Postpartum diagnosis was based on ultrasound, CTA, surgery, anatomic examination, and cardiovascular casts. For pregnant women who chose natural delivery, postpartum diagnosis was made according to the results of the operation or postpartum ultrasound. Some pregnant women chose to have labor induced because of complex intra- or extracardiac malformations in the fetus, and donated the specimens to our maternal and fetal medical center. In these cases, the postpartum diagnosis was based on CTA, anatomy, and cardiovascular casts. Postpartum ultrasound was performed using a GE Vivid 7 ultrasound system  with a probe frequency of 2.5–12 MHz. Reflux into the left atrium and cardiac malformations were visualized by analyzing Cardiac segments and aortic cross sections of the superior sternal fossa.For cardiovascular casting, the specific steps are as follow: (1) Fill in the fetal basic information record form and documentary; (2) The fetal weight, body length, head circumference, chest circumference, abdominal circumference, eye distance, upper limb length and lower limb length were measured and recorded; (3) Take photos before casting; (4) Incision of abdominal wall, separation of umbilical vein, umbilical vein intubation; (5) The left umbilical artery was cut off; (6) Heparin (5–10 ml) and acetone (20–50 ml) were injected through umbilical vein catheterization to wash blood and blood clots in cardiovascular system. The injection amount of acetone was adjusted according to the situation of blood washing until no blood flowed out. The mixture of self-coagulating denture powder and denture water was perfused slowly with pressure of 30 ~ 60 ml, and the injection time was controlled at about 30 min. Finally, the mixture of self-setting denture powder and denture water was infused with a pressure syringe for about 10 h; (7) After 24 h, the fetal specimens perfused with the mixed reagent of self-setting denture powder and denture water were immersed in 30% hydrochloric acid solution; (8) After about 2 weeks, the perfused fetal specimens were taken out from hydrochloric acid solution, and the corroded soft tissues were washed carefully with running water. The soft tissues that were not washed clean were washed carefully with syringe until there was no soft tissue on the surface of cardiovascular cast; (9) The fetal cardiovascular cast specimens were registered and photographed. The ratio of materials used for perfusion was as follows: (1) Denture powder: Denture water: dibutyl phthalate: barium sulfate = 20:40:6:6; (2) The total amount of perfusion reagent used for perfusion: 30 ~ 60 ml; (3) Speed of filling: 0.5 ml/s; (4) The temperature of perfusion environment: room temperature 20–25 ℃; (5) Humidity of perfusion environment: 50% ~ 70%; (6) Concentration of hydrochloric acid in acid tank: 30%.Informed consent was drawn from all patients. All methods were carried out in accordance with relevant guidelines and regulations. The study was approved by the ethics committee of Xiangyang No. 1 People’s Hospital Affiliated with Hubei University of Medicine with reference number XYFH20150124, and date of approval January 24, 2015.A total of 20 fetuses with a prenatal diagnosis of TAPVC were included in the analysis. The median age of the pregnant women was 27.6 years , and the median gestational age at diagnosis was 25 weeks . Of the 20 cases, 18 were confirmed as TAPVC by postpartum diagnosis, and the other 2 (cases 4 and 15) were modified to partial anomalous pulmonary venous connection. The different types of TAPVC were supracardiac, 44% (8/18); cardiac, 22% (4/18); infracardiac, 22% (4/18); and mixed, 11% (2/18). Intra- or extracardiac malformations were observed in 78% (14/18) of cases, and 22% (4/18) were confirmed as isolated TAPVC. Among TAPVC fetuses with intra- or extracardiac malformations, 78% (11/14) showed atrial isomerism, 43% (6/14) had pulmonary artery stenosis, 28% (4/14) had a single ventricle, 21% (3/14) had an endocardial cushion defect, 14% (2/14) had a single atrium, and 21% (3/14) had a right aortic arch. Four malformations were misdiagnosed, including an anomalous left brachiocephalic vein in supracardiac TAPVC (case 3), abnormal inflow of the hepatic vein and a double inferior vena cava in infracardiac TAPVC (case 9); and bilateral ductus arteriosus in infracardiac TAPVC (case 14). Two malformations were misdiagnosed, including a tetralogy of Fallot in cardiac TAPVC that was corrected to right ventricular double outlet (case 11); and an absence of ductus arteriosus that was misdiagnosed as slim ductus arteriosus (case 13)  were complicated by intra- or extracardiac malformations, and 61% (11/18) were complicated by heterotaxia syndrome; as the latter can lead to atrial heterogeneity and a change in the position of the atrium, it is critical for clinicians to identify the true left and right atria, which may be a key step in the diagnosis of TAPVC. Following pre- and postnatal diagnoses, fetal echocardiography showed no direct connection between the PV and left atrium but revealed the convergence of PVs at the back of the atrium to form a common vein stem; an increased distance between the posterior wall of the atrium and descending aorta; and a visible ascending or descending vertical vein. These features not only exist in a fetus with complex intra- or extracardiac malformations, but also in isolated TAPVC.20.Our modified cardiovascular cast is another method for postmortem evaluation of fetal TAPVC. In China, our team was the first to improve and patent the modified vascular casting technology. We previously demonstrated that the cardiovascular cast can accurately show various complex fetal cardiac malformations, which can facilitate prenatal diagnosis and reduce the risk of overlooking or misdiagnosing the Complex cardiac malformations23.Several studies have evaluated the clinical utility of postmortem magnetic resonance imaging (MRI) and micro-CTHowever, casting may better reveal some rare malformations, especially smaller branching anomalies that are difficult to detect by other imaging modalities. In the future, this new approach of casting combined with micro-CT or MRI may be used to develop 3D digital models for clinical assessment and teaching.The results of our study demonstrate that prenatal ultrasound can be used to accurately diagnose most cases of fetal TAPVC. By analyzing the echocardiography findings, the specific type of fetal TAPVC can be identified. Identifying the PV drainage route is critical for diagnosing different types of TAPVC. Finally, a postpartum cardiovascular cast can accurately depict the branch structure of the heart’s major vessels, which can guide prenatal diagnosis of TAPVC for timely management that can lead to an improved outcome.


Background: The members of the Chromobox (CBX) family are important epigenetic regulatory molecules with critical biological roles in many tumors. However, no study has analyzed or verified their role in lung adenocarcinoma (LUAD).Methods: UALCAN and Oncomine databases were used to analyze CBX expression in LUAD, and the cBioPortal database was used to analyze CBX genetic variations. The Kaplan-Meier plotter and UALCAN databases were used to identify molecules with prognostic value. Gene Ontology pathway, receiver operating characteristic curves, and tumor-infiltrating immune cell analyses were used to clarify the biological function of the CBX hub molecules. Paired tumor samples and lung adenocarcinoma cell lines were collected for molecular functional assays to validate the results of the bioinformatics analysis.Results: CBX3/5 may have a cancer-promoting effect and its expression is associated with a poor patient prognosis, while CBX7 shows an opposite trend. CBX3/5/7 can regulate signaling pathways, regulate tumor immune cell infiltration, and has diagnostic value. Molecular biology experiments show that CBX3/5 is highly expressed in LUAD patients; in vitro it promotes the proliferation and migration of the LUAD cell line and can regulate the expression of the corresponding cytokines. CBX7 has opposite effects.Conclusion: Our bioinformatics analysis and subsequent experimental verification confirmed the CBX family members acted as hub signaling molecules in LUAD. The results provide new potential targets for the diagnosis and treatment of this cancer. Lung adenocarcinoma (LUAD) is one of the deadliest cancers worldwide, and the incidence is increasing annually . LUAD acCBXs are transcriptional repressors found in normal human cells that regulate cell senescence. Their expression is downregulated in colon cancer, gastric cancer, and hepatocellular carcinoma. As a kind of epigenetic regulatory complex, CBXs repress target gene transcription by modifying chromatin . The eigin vitro studies to demonstrate that high expression of CBX3/5 in LUAD tissues promoted tumor cell proliferation and migration of tumor cells and promoted the infiltration of cytotoxic cells, CD8+ T cells, and other immune cells. CBX7 has opposite biological functions. Our results provide new potential therapeutic targets for the diagnosis and treatment of LUAD.Bioinformatics analysis is increasingly performed to stimulate and identify new research directions. This approach was used to elucidate the relationship between LIM domain kinase 1 and immune cell infiltration in LUAD . Other rhttp://ualcan.path.uab.edu/), Gene Expression Omnibus Database , and the expression of CBX mRNA in multiple types of cancer was analyzed in the Oncomine database (https://www.oncomine.org/resource/login.html) (http://www.proteinatlas.org/). The association analysis between members of the CBX family and the prognosis of the LUAD patient was explored using the UALCAN and Kaplan-Meier (KM) plotter databases   , which palysis/) . The twohttp://www.cbioportal.org/) derives from the Cancer Genome Atlas (TCGA) database in the United States, which allows a comprehensive analysis of genomic data from various types of tumors from multiple dimensions +Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, respectively. Data were visualized using the ggplot2 R package and presented as a bubble graph . Log-ranThe STAT R package was used to analyze genes co-expressed with CBX3/5/7. The screening threshold was a Pearson’s correlation coefficient >0.5. These data were visualized using the ggplot2 R package and are presented as a heatmap.p-values were obtained by the Wilcoxon rank sum test.Patients were divided into two groups according to the median expression values of CBX3/5/7 and then the GSVA R package and a single-sample gene set enrichment analysis (ssGSEA) immune infiltration algorithm was used to compare immune cell infiltration between two groups. The data are presented as box plots. The Human LUAD A549, SPCA1, H1299, and H1975 cell lines were cultured in Dulbecco’s minimum essential medium supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin solution at 37°C in a cell incubator.Thirty-six LUAD and paired normal tissue samples were obtained from patients who underwent surgery at the Third Affiliated Hospital of Soochow University. None of the included patients had undergone radiation, chemotherapy, or other preoperative treatment. This study complied with the Ethics Code of the World Medical Association set out in the Declaration of Helsinki. Studies involving human participants were reviewed and approved by the Ethics Committee of the Third Affiliated Hospital of Soochow University (IRB number: (2021) 005). All patients provided their written informed consent to participate in this study.Small interfering RNA (siRNA) targeting CBX3, CBX5, and Negative control (NC) were purchased from GenePharma , and were transfected into LUAD cell lines using Lipofectamine 3000  according to the manufacturer’s instructions. CBX7 cDNA was cloned into the pcDNA3.1+ vector purchased from Genebay Biotech  and then transfected into LUAD cell lines using Lipofectamine 3000.−ΔΔCt method. interleukin (IL)-6, or interferon (IFN)-γ in culture supernatants of LUAD cell line were quantified using IL-6/IFN-γ Quantikine ELISA kit  according to the manufacturer’s instructions.Tissue (100 mg) or cells were ground to powder in liquid nitrogen, 1 ml of TRIzol was added to fully lyse the cells, and RNA was extracted. The PrimerScript Reverse Transcription Kit  was used for cDNA preparation and the TB Green™ Premix Ex Taq  was used for qPCR. Relative gene expression levels were analyzed using the 23 cells/well) and 10 μl of CCK8 reagent  was added to each well at five time points . After 1 h of incubation in a cell incubator at 37°C, the optical density of the sample (OD) at 450 nm was detected using a microplate reader.The proliferative ability of the cell lines was examined using the Cell Counting Kit-9 (CCK8) assays. Cells were seeded in a 96-well plate  was added to the upper chamber and 800 μl of medium containing 20% FBS was added to the lower chamber. After incubation for 24 h, cells that had not migrated in the upper chamber were removed. After fixation and crystal violet staining, photographs were taken under a microscope.Transwell inserts were inserted into a 24-well plate and 200 μl of the cell suspension  was used to perform a one-way analysis of variance analysis and paired Student’s t-tests to analyze differences between multiple groups or two groups, respectively.We used the UALCAN database to explore the expression of members of the CBX family in LUAD and found significant differences between them . To ensuGenetic variants are also important for tumorigenesis, so CBX gene variants were analyzed using the cBioPortal database . We founConsidering the importance of molecular prognostic markers, we used the KM plotter database to explore the predictive utility of CBXs in LUAD. CBX1, CBX3, and CBX5 were associated with poor prognosis fig4, whiTo clarify the mutual regulatory relationships, we analyzed genes co-expressed with these three CBX molecules. By mining and analyzing the transcriptome data of TCGA-LUAD, we identified 437 positively and 72 negatively correlated genes for CBX3 , 65 posiTo verify the predictive prognostic efficacy of CBX3, CBX5, and CBX7, we used TCGA datasets and performed LASSO regression to reduce dimensionality and build a prognostic model based on CBX3, CBX5, and CBX7 expression  × CBX3 + (0.2588) × CBX5 + (−0.2601) × CBX7). Patients were divided into low- and high-risk groups according to the median risk score. The heatmap of the expression profiles of prognostic genes in low- and high-risk groups is shown in + T cells, natural killer (NK) cells, neutrophils, and macrophages  showed a reduction in the numbers of infiltrating B cells, cytotoxic cells, CD8rophages . Patient17 cells . The gro17 cells . In tumorophages . Accordiin vitro studies. We initially selected five lung adenocarcinoma cell lines to detect the expression of CBX3/5/7 by qPCR. Considering the transfection efficiency and gene expression profiles, we used the A549 and H1299 cell lines for the subsequent experiments  from the GEO database. The results showed that the expression trend of other molecules was consistent with those from the TCGA except for CBX2. If a molecule has a consistent expression trend in various types of tumors, it indicates that it has pan-cancer characteristics and may play a strong role in the regulation of tumorigenesis. Therefore, we also used the Oncomine database to analyze the expression of members of the CBX family in different types of cancer. The results showed that CBX1/2/3/5/7 are dysregulated in lung cancer and show a unified trend in cervical cancer and colorectal cancer. Unfortunately, the Oncomine database does not separate lung cancer according to pathological type, i.e., lung adenocarcinoma and lung squamous cell carcinoma. This also led us to collect clinical samples from our own patients for verification. In general, CBX1/2/3/5/7 have the greatest potential and merit study among the members of the CBX family. We also investigated modifications in the CBX family members at the genomic level. Interestingly, CBX7 was not associated with any deletions and mutations, despite its mRNA level being lower in LUAD tumor tissues. A possible reason may be that of histone modification, which is responsible for regulating the degree of chromatin compaction, which thereby interferes with the binding of transcription factors . There mWe evaluated the expression of members of the CBX family and their associations with the prognosis of patients with LUAD, since molecules with prognostic utility may be more worthy of further study. Because the underlying algorithms used in different datasets may differ slightly, verifying our findings across multiple databases yields more reliable conclusions. We used the UALCAN and KM plotter databases to analyze CBX family members and the prognosis of patients with LUAD, to increase the accuracy of our study. It should also be kept in mind that in survival analysis, numerical variables are generally used, although we should also use both categorical and continuous parameters. Without considering the dual aspects for analysis, the results obtained may be subjected to statistical bias. The two databases we selected applied two different methods for prognostic analysis. Finally, our results showed that CBX3/5/7 levels correlated with prognosis in LUAD patients, albeit in different ways. The expression of CBX3/5 was associated with poor prognosis, while CBX7 showed the opposite trend. What is remarkable about our results is that CBX8 was highly expressed in LUAD, but the higher the expression, the better the prognosis. We speculated that the high expression of CBX8 in lung adenocarcinoma patients may increase the tumors sensitivity to radiotherapy and chemotherapy. Furthermore, compared to normal tissues, the number of lymphocytes infiltrating tumors was increased, a phenomenon which has been confirmed to be associated with increased sensitivity of tumors to immunotherapy and beneficial effects on prognosis of patients . WhetherNext, we analyzed the genes co-expressed with CBX3/5/7 and performed GO and KEGG Pathway analyses. The results showed that CBX3 was related to the cell cycle and p53 signaling pathways, both of which are closely related to tumorigenesis . CBX3 ha+ T cells. Conversely, patients with higher levels of CBX7 expression had significantly increased infiltration of all three cell types. For this reason, we performed additional in vitro experiments. To date, many studies have reported that several tumor factors influence the secretion of cytokines in tumor cells, which may have an impact on the infiltration of immune cells in the tumor microenvironment. LINC01116 can promote tumor proliferation and neutrophil recruitment through DDX5-mediated regulation of IL-1β in glioma cells; and melanoma-derived factors may alter the maturation and activation of differentiated tissue-resident dendritic cells (Many recent studies have also reported prognostic signatures constructed by some genes or noncoding RNA in LUAD, glioma, and clear cell renal cell carcinoma . We alsoic cells . Previouic cells . Lung caic cells , which iic cells . Recent ic cells . These rTo confirm our findings, we collected primary tissue samples from LUAD patients at our institution. qPCR evaluation of these samples were performed to verify the expression trends of CBX3/5/7. Furthermore, knocking down CBX3/5 expression in A549 cells and overexpressing CBX7 in H1299 cells, we demonstrated that CBX3/5/7 inhibited the proliferation and migration of LUAD cell lines, respectively. CBX3/5 promoted the expression of some cytokines, while CBX7 exerted inhibitory effects. These cytokines have also been shown to play a regulatory role in immune cell recruitment within tumors .Our findings should be considered in the context of the several limitations of the study. We did not study the biological functions of other members of the CBX family, there was no in-depth study of the mechanisms, and no animal experiments were carried out. Our bioinformatics analysis was not sufficiently exhaustive. We did not find any immune-related items among the results of the TOP 10 functional pathways in the enrichment analysis, and thus, we added additional experimental data to investigate immune activity in LUAD. The results obtained by different algorithms also differed, which also prompts the need for additional experimental verification, which will be addressed in future studies investigating pathway enrichment. Further, we did not explore the reasons for the contrasting biological functions among CBX3, CBX5, and CBX7, which will be addressed in future studies.In summary, we investigated the impact of CBX3/5/7 expression on LUAD including their prognostic value and ability to regulate immune cell infiltration and cell cycle transition. Our results suggest that CBX3/5/7 may be key molecules in LUAD and that these could be used for diagnostic purposes or may provide a rationale to develop new therapies.


Tenebrio molitor L.) is a classic edible insect with high nutritional value for substituting meats from vertebrates. While interest in mealworms has increased, the determination of carbohydrate constituents of mealworms has been overlooked. Thus, the aim of the present study was to investigate the carbohydrate content and composition of mealworms. In addition, the characteristics of mealworm chitin were determined as these were the major components of mealworm carbohydrate. The crude carbohydrate content of mealworms was 11.5%, but the total soluble sugar content was only 30% of the total carbohydrate content, and fructose was identified as the most abundant free sugar in mealworms. Chitin derivatives were the key components of mealworm carbohydrate with a yield of 4.7%. In the scanning electron microscopy images, a lamellar structure with α-chitin configuration was observed, and mealworm chitosan showed multiple pores on its surface. The overall physical characteristics of mealworm chitin and chitosan were similar to those of the commercial products derived from crustaceans. However, mealworm chitin showed a significantly softer texture than crustacean chitin with superior anti-inflammatory effects. Hence, mealworm chitin and chitosan could be employed as novel resources with unique advantages in industries.Mealworm ( The global population is still increasing and, consequently, likely food shortcomings need to be addressed. Furthermore, the increasing consumption of meat in developing countries affects the sustainability issue because livestock-rearing requires excessive energy inputs . Meat prTenebrio molitor L.) is one of the most popular species as a food and feed resource, and its extensive utilization has resulted in the advancement and establishment of commercial large-scale rearing systems 1/2.The color values  of the chitin powders were measured using a colorimeter . The whiteness index (WI) of mealworm chitin was calculated using an equation described in a previous study : WI = 10The surface images of chitin and chitosan extracted from the mealworms were obtained using FESEM , and EDS was simultaneously performed using the same instrument. α-Chitin from shrimp  was also analyzed as the reference sample. The chitin and chitosan samples were fixed on a stub and coated with platinum . The images of the samples were obtained at two magnifications . The carbon and nitrogen contents of the samples were analyzed by EDS, and the ratio of carbon and nitrogen in the samples was used to calculate the degree of acetylation (DA) of chitin and the degree of deacetylation (DDA) of chitosan using the following equations ,27:DA(%)Proton (1H) NMR analysis was conducted using a 600 MHz AVANCE 600 NMR spectrometer . The chitin and chitosan samples were dissolved in 2% deuterated acetic acid in D2O at a concentration of 2 M, and 1H NMR spectra were obtained at 70 °C . Additio−1 and the resolution was 8 cm−1 [1655/A3450) × 115.FT-IR spectroscopy was performed using a Nicolet 6700 spectrometer  to determine the chemical structural characteristics of chitin and chitosan obtained from the mealworms. The samples were mixed with potassium bromide and placed in a ZnSe/diamond disc. The analyzed frequency range was 4000–650 cms 8 cm−1 . The DDAs 8 cm−1 : DDA (%)XRD analysis was performed using a D8 ADVANCE instrument with DAVINCI  according to the method described by Yen et al. . The Cu 2 at 37 °C. The cells were seeded at a concentration of 5 × 104 cells per well in 96-well plates and maintained for 24 h. After removing the culture media and rinsing with phosphate buffer saline (PBS), 50 μL of mealworm chitosan-containing media was added to each well. Fifty microliters of LPS-containing media (2 μg mL−1) was added after an hour, and incubated for 24 h in an incubator. Then, the supernatant of each well was mixed with the same volume of Griess reagent. After reaction for 20 min, the absorbance of the solution was measured at 550 nm using a microplate reader .The anti-inflammatory effect of mealworm chitosan was tested with the lipopolysaccharide (LPS)-induced murine macrophage cells (RAW 264.7). RAW 264.7 cells were obtained from American Type Culture Collection  and maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum and 1% penicillin/streptomycin solution. The cells were incubated in an incubator with 5% COt-test or one-way analysis of variance (ANOVA) was conducted using the IBM SPSS statistics v.25 software . Duncan’s multiple-range test was also performed for the post-hoc analysis of ANOVA (p < 0.05). The data are presented as mean ± standard deviation (SD) based on triplicate measurements. After normality and homogeneity tests for data, the Student’s While carbohydrates are the major components of most foods, animal foods have abundant proteins and lipids but low carbohydrate contents. Accordingly, the crude carbohydrate content in dried mealworms was 11.45 ± 0.38%, implying that the undried mealworms contained approximately 3.4% carbohydrate . Among tAdditionally, a considerable gap was observed between the contents of crude carbohydrates and total soluble sugars in the mealworms; therefore, mealworm bodies were predicted to consist of abundant insoluble carbohydrates. Chitin and its derivatives are typical insoluble carbohydrates that are found in the insect integument, and 4.84 ± 0.43% of glucosamine (the monomer of chitin) is present in dried mealworm powder. This allows the consumers to intake abundant chitins upon eating mealworms. Moreover, mealworm chitins can be applied for mass production in industries because these are easy to rear on a large scale. Therefore, chitins were extracted from the mealworms and their overall characteristics were examined to verify their availability.Common chitin products in industries constitute those originating from crustaceans such as shrimp and crab. Because of their tough textures and difficult digestion, their shells are dumped as waste; therefore, this waste is typically used for preparing chitins. Chitin and chitosan can be employed as therapeutic agents, pharmaceutical carriers, and packaging materials as natural biopolymers . When chThe surface microstructural images of mealworm chitin and chitosan samples were obtained using FESEM , and comNMR is a reliable method for verifying the DA and DDA of chitin derivatives, which can assist in the determination of their structural properties . Therefo−1), C–H stretching (2878 cm−1), C–H deformations (1420 cm−1), C=O stretching in secondary amide , and C–N stretching in secondary amide (1320 cm−1). Mealworm chitin shows a spectral pattern similar to that of shrimp α-chitin. The chitin fibers are commonly classified into three types  based on their crystal structures, and each type shows different physical properties. The chitin polymer is generally arranged in parallel, and α-chitin has an inverse configuration between the adjoined chitin chains, but β-chitin consists of equally aligned chitin fibers [−1 (corresponding to the hydrogen bond between C=O and NH groups) and 1625 cm−1 (corresponding to the hydrogen bond between C=O and HOCH2 groups) are not observed, and β-chitin shows a peak at 1430 cm−1 instead of 1416 cm−1 [−1 (C–H stretching) and low absorbance at 3444 cm−1 (hydroxyl stretching) compared to the shrimp α-chitin. These distinct features are attributed to dibutyrylchitin (DBC), where chitin contains bonds of butyryl groups at the C3 and C6 positions [−1) have been detected in the data for the chitin extracted from silkworm pupa, but it shows a significantly smaller peak than that observed in the present study [The FT-IR spectra of the samples are shown in n fibers . There a416 cm−1 ,55,56. Aretching 78 cm−1, ositions . DBC is ositions . While Dnt study . Hence, To verify the crystalline structural characteristics of the chitin samples, XRD analysis was conducted . The α-cThe anti-inflammatory and anti-degenerative arthritis activities of glucosamine and chitosan are well known, but the efficacy of mealworm chitosan has not been sufficiently proven ,65. To vThe aim of this study was to determine the carbohydrate content and composition of mealworms as well as examine the characteristics of mealworm chitin and chitosan because chitin derivatives are the major carbohydrate constituents in mealworms. The mealworms had a low carbohydrate content (11.5%), while the soluble sugar content was only 3.2%. Instead, chitins constituted almost half of the total carbohydrate content in the mealworms. The prepared mealworm chitin exhibited a white flake form, and its yield was 4.7 g per gram of dried mealworm. The prominent structural type of mealworm chitin was α-chitin, similar to that observed for other insects, and it developed lamellar structures in the matrices. The calculated DA of chitin and DDA of chitosan were 98.3% and 89.4%, respectively, based on NMR analysis, which were analogous to those of the commercial products. As the DA and DDA values are closely related to the physical properties of the chitin derivatives and those of mealworms showed similar values, mealworm chitin derivatives could replace the commercial chitins without considerable changes in the product quality. The crystallinity index of mealworm chitin was relatively lower than that of the crustacean-derived chitins, indicating that mealworm chitin could be applied to the products with soft textures. Considering the FT-IR and XRD patterns, it was assumed that DBC was compounded with α-chitin in the mealworms, and this could be highly beneficial because DBC showed superior properties such as high solubility, suitability for fabricating films, and superior biodegradability; however, it has been rarely found in natural sources. In addition, mealworm chitosan showed excellent anti-inflammatory effects in the LPS-induced murine macrophage cell line. Therefore, the physical characteristics of mealworm chitin and chitosan are expected to be suitable for use in industries, and afford additional advantages such as tender texture and potent anti-inflammatory effects.


It also achieved higher permeatequality in these conditions. The Young’s modulus and ultimatetensile strength of the best-performing membrane increased by 38 and271%, respectively, compared to the pure polymer membrane, while bothhad similar porosities of ∼91%.This work demonstrates very highremoval rates (below the detectionlimit of 0.045 ppb) of inorganic arsenic from water using electrospunpolyvinylidene difluoride (PVDF) membranes enhanced by the additionof functionalized graphene oxide in membrane distillation. This showspotential for applications in the many parts of the world sufferingfrom arsenic-contaminated groundwater. These membranes were enhancedby the addition of reduced graphene oxide functionalized with superhydrophobicpolyhedral oligomeric silsesquioxane molecules (POSS-rGO) into thespinning solutions. The flux of the best-performing rGO-enhanced membrane(containing 2 wt % POSS-rGO) was 21.5% higher than that of the purePVDF membrane and almost double that of a commercial polytetrafluoroethylene(PTFE) membrane after 24 h of testing, with rejection values exceeding99.9%. Furthermore, the flux of this membrane was stable over 5 days(∼28 L m Arsenic can exist in four valence states: −3,0, +3, and +5, with inorganic As3+ and As5+ beingthe most common and relevant to groundwater contamination. Long-termexposure to these forms of arsenic increase risks of skin, lung, bladder,and kidney cancer as well as hypertension and cardiovascular disease,among others. Ingesting large doses of inorganic arsenic results ingastrointestinal symptoms, disruptions to cardiovascular and nervoussystem functions, and eventually death.5 There are a variety of both natural and man-made sources of arsenicin the environment, and both must be monitored in order to mitigaterisk to human health.Arsenic contaminationin groundwater is a growing problem, threateningdrinking water supplies in many parts of the world. Locations suchas the Ganga River Basin, encompassing considerable parts of India,Bangladesh, Nepal, and Tibet;6 By utilizing nonwetted hydrophobic membranes to separate heatedfeed water from the permeate collection stream, membrane distillationallows the passage of vapor through the membrane while keeping alldissolved inorganic substances in the feed water.7 This system is able to treat highly concentrated waterbecause it operates on a gradient in vapor pressure rather than hydraulicpressure like reverse osmosis and is therefore able to treat brinestoward and even beyond saturation. This is an advantage in the caseof arsenic removal since it can enable the complete recovery of waterfor zero liquid discharge applications.9Various techniques for removing inorganicarsenic from groundwaterexist, including oxidation, coagulation-flocculation, adsorption,ion exchange, and membrane filtration. However, these can suffer fromlow separation efficiency or require complex multistep processes inorder to operate effectively over time. Membrane distillation (MD)is a simple and robust technology for achieving very high removalrates of dissolved inorganic substances.11 Designinghigh-performance membranes is one of the main ways of achieving this.Over the years, it seems that phase inversion has all but given wayto electrospinning as the most effective way to fabricate MD membranes. By producing a network of randomly alignedpolymer fibers, it is possible to obtain membranes with very highporosity and highly interconnected pores, which have been appliedto applications ranging from tissue engineering,12 energy storage,13 air filtration,14 and others.17 In order to furtherenhance these properties, it is possible to incorporate nanomaterialsinto the polymer solutions prior to spinning as has been shown previously.20 In this work, graphene oxide (GO) was functionalized with superhydrophobicpolyhedral oligomeric silsesquioxane (POSS) molecules, which havea cage-like structure composed of silicon and oxygen atoms and representthe smallest possible silica nanoparticle size. This organic–inorganichybrid molecule has been shown to improve the thermal stability andmechanical properties of polymer composites due to strong hydrophobicinteractions with the matrix21 and hasbeen covalently grafted onto graphene oxide to induce superhydrophobicproperties.22 While most publications ongraphene-based membranes utilize stacked sheets of GO to form a networkof nanosized pores,23 here, the high strengthand tailorable surface of GO have been exploited and utilized as ananoadditive. In order to maximize the performance in MD, membraneporosity and hydrophobicity must be maximized. However, as porosityincreases, mechanical strength decreases and a balance must be struck.By functionalizing GO with POSS, thermally reducing it, and blendingit with the PVDF polymer, the aim was to simultaneously improve themechanical properties of the electrospun membranes, thereby increasingthe durability, allowing for porosities exceeding 90%, increasingthe hydrophobicity, reducing the chance of pore wetting, and increasingthe quality of the MD permeate. The efficacy of this approach wastested by using the membranes to treat saline arsenic-contaminatedgroundwater, simulating that from the Tacna region in Peru, usingair gap membrane distillation – the most thermally efficientvariant of membrane distillation.24 Inorganicfouling studies were also conducted using highly concentrated calciumcarbonate and iron(III) sulfate heptahydrate solutions to test thestability of the process in these harsh conditions. Calcium carbonate,iron oxides, and calcium sulfate are well-known inorganic foulantsin MD.28Muchresearch effort has been put toward achieving higher productionrates and lower propensity for pore wetting in MD in order for itto compete with other technologies.Figure S1. The C1s spectrum of POSS-GOexhibits a doublet, which was broken down into four peaks. The peakswere assigned to the C–C, C–N, C–O, and C=O bondspresent in POSS-GO as a consequence of the siloxane grafting. Accordingto the molecular structure of the siloxane, C–Si bonds (observedat 284 eV30) are expected. However, this peak is notsuggested by the software in any case, possibly due to the presenceof a huge peak for C–C, which makes the other peaks next toit negligible.31 Besides, a new band correspondingto C–N is observed around 285.6 eV.34 In addition, the peaks correspondingto C–O and C=O are decreased in POSS-GO as a consequence ofthe reaction between the epoxy and carboxylic groups with the aminegroup in POSS.31 The C1s spectrum of POSS-rGOshows a decrease in C–O in comparison to POSS-GO, which confirmsthe reduction of the GO. The degree of reduction can also be observedin the increase of the C/O ratio .Functionalizationgraphene oxide was analyzed by X-ray photoelectron spectroscopy. POSS-GOand POSS-rGO surveys expectedly show the presence of silicon and nitrogenas a consequence of the GO functionalization. The C1s high-resolutionspectra of both graphene derivative materials were acquired in orderto investigate the chemical modifications in detail and are presentedin –1, which originate from alkoxy CO stretching vibrations, epoxy COstretching vibrations, CC stretching in the aromatic ring, and COcarboxyl stretching, respectively. Meanwhile, the broad band from2990 to 3600 cm–1 is attributed to OH stretching.35 The spectrum is modified considerably for POSS-rGO, which shows a pronounced peak at 1110cm–1 as a result of Si–O–Si stretching.In addition, a weaker band in the range of 2750–3000 cm–1, attributed to the isobutyl groups, which extendfrom the POSS molecule, and the peak at 1650 cm–1, corresponding to the C=O stretch of the amide (which is absentfrom POSS), show the successful grafting of this functional moleculeonto graphene oxide.22In addition, the chemical functionalizationof GO was investigated using ATR-FTIR, as presented in Table S2. The high optical absorbance of the POSS-rGO resulted indark black dope solutions, even at the lowest loading of 0.5 wt %.However, when the polymer solidified during the electrospinning process,the appearance altered from transparent to white, occluding the darkcolor of the POSS-rGO and resulting in a light gray shade. This canbe seen in the optical photographs shown as inset images in Figure S2) also show a pronounced Si peak forthe GP 2 membrane, corresponding to POSS-rGO. In the elemental map,there are purple (Si) clusters concentrated on the surface of grapheneflakes, as highlighted by red dashed circles. This is further evidenceof successful grafting of POSS molecules onto the graphene as wellas successful incorporation of POSS-rGO into the electrospun membranes.Membranes werefabricated with concentrations of GO-based fillers in the electrospinningsolution ranging from 0.5 to 3 wt %. Prepared membranes were labeledas GP followed by the wt % used, so for instance, GP 3 is a membraneprepared from a dope solution containing 3 wt % POSS-rGO. The componentsof each dope solution are summarized in Figure S3. Smallcrystals do appear to have precipitated on the fibers upon dryingof the membranes but are not large enough to block the pores completely.However, the image of the PTFE membrane for the electrospun membranes (excluding GP 3), there are significantdifferences in the number of nanofibers under 300 nm. For pure PVDF,this proportion was just 4% compared with 12, 28, and 12% for GP 0.5,GP 1, and GP 2, respectively. This higher number of very small fibersfor the POSS-rGO membranes can be observed when looking carefullyat the SEM images. This is possibly the result of the conductivityof POSS-rGO increasing the electrostatic repulsion experienced bythe fiber as it travels to the collector, resulting in thinner fibers.A deviation from this trend is again seen in GP 3 for which only 2%of fibers measured were below 300 nm in size. This is possibly dueto the increased solution viscosity and agglomeration of the graphene,as has been observed elsewhere.39Capillary flow porometry revealed the electrospunmembranes tohave surprisingly narrow pore size distributions, as shown by representativeplots in 40In addition to narrow pore size distributions,these electrospunmembranes have incredibly high porosities of around 90%. 45It istherefore very promising to be able to fabricate membraneswith significantly higher porosities while retaining sufficient mechanicalproperties to withstand handling and high-shear testing environments.The highest porosity value belonged to GP 2 with a value of 91.9 (±0.4%)after hot-pressing. This is higher than most nanofiber membranes foundin the literature, which typically suffer reductions in porosity tobelow 90% due to post-treatment.49 These changes are likely due to the attractive interactions betweenthe PVDF polymer chains and the branched alkyl groups extending fromthe silica core of the POSS molecule. In addition, the high surfacearea and high intrinsic strength of the graphene basal plane providestrong interfacial interaction with the polymer, increasing both thestrength and stiffness of the membranes.51 As seen from the SEM images  measurements.These are presented together in The liquid entry pressure values do notfollow the same trend asthe water contact angle values but do relate to the maximum pore sizevalues for the membranes. The membrane with the smallest LEP was GP2 with a value of 0.159 ± 0.007 bar. This membrane also had thelargest maximum pore size value of 10.56 ± 1.05 μm, morethan twice that of GP 0.5, which had the largest LEP value of 0.321± 0.013 bar. The remaining three membranes have very similarmaximum pore size values, and their LEP values lie within one standarddeviation of each other, indicating the link between maximum poresize and liquid entry pressure. Intuitively, the largest pore in amembrane is the one that requires the least amount of pressure toforce liquid through, all else being equal. It is important to notethat these LEP values are considerably lower than that of the commercialPTFE (3.683 ± 1.677 bar). This is due to the high intrinsic hydrophobicityof PTFE compared to PVDF but also the significantly smaller maximumpore size value of 0.40 (±0.09) μm. These low LEP levelsdid not seem to affect the ability of these membranes to achieve highsalt rejection in membrane distillation experiments, as the followingsection highlights.54 Most of the membranes, including the commercial PTFE, showa similar flux pattern over 24 h. A slight decline (<5%) in fluxis observed over the first 3 h for all membranes except GP 0.5 andGP 3, which showed declines of 9.1 and 15.1%, respectively. This gradualdecline continued over 24 h of testing except in the case of GP 2,whose flux was stable at a value of 27.94 ± 1.77 L m–2 h–1. This was 21.5% higher than the pure PVDFmembrane nearly double that of the commercial PTFE membrane afterthe same time period. This increased flux can be largely attributedto the increased hydrophobicity and larger mean pore size of thismembrane compared to others, as shown in 2 permeability for GP 2was 57% higher than the pure PVDF membrane despite their porositiesbeing almost identical. Larger pore sizes are known to reduce theresistance to mass transfer in MD but increase the risk of pore wetting.55 In this case, the high hydrophobicity of themembrane successfully prevented wetting despite its mean pore sizevalue of 9.80 ± 0.73 μm being 1–2 orders of magnitudelarger than is typical for MD membranes. It is possible that the membranethickness affected the flux performance, particularly with respectto the PTFE membrane, which was more than twice as thick as the electrospunmembranes. However, the difference in thickness between the electrospunmembranes is not particularly significant. Furthermore, previous workhas suggested that the membrane thickness plays a much less significantrole in increasing the mass transfer resistance than the air gap,which is orders of magnitude thicker.58 In order to furthertest the flux stability of this membrane (GP 2), a 5 day continuousMD experiment was conducted, yielding a final flux value of 28.30L m–2 h–1 and a correspondingpermeate conductivity value of 1.786 μS cm–1. This is evidence of the high stability of the separation processfor this type of feed solution. The more severe flux decline observedfor the GP 3 membrane may be the result of increased temperature polarizationdue to the high thermal conductivity of the POSS-rGO. While at thelower loadings, this is unlikely to contribute significantly to temperaturepolarization; at 3 wt %, this property of the graphene may becomedetrimental to MD performance.The flux and permeateconductivity values from the MD experiments are summarized in –1. This corresponds to very high salt rejection values of >99.9%.More importantly, the arsenic levels in the permeate for all membranes,including PTFE, were below the detection limit of ICP-MS (<0.045ppb). This means that all samples produced water of significantlyhigher quality than recommended by the WHO (<10 ppb). Though nottested using the electrospun membranes, the air gap membrane distillation(AGMD) system with the commercial PTFE was also effective in removingAs3+ from a 300 ppb sodium meta-arsenite solution. In thiscase again, the arsenic levels were below the detection limit. HighAs rejections using commercial MD membranes have been also reportedin the literature.59In general, all electrospun membranes produced very high qualitypermeates with conductivities of less than 2 μS cm–1 calciumcarbonate and 2 g L–1 iron sulfate heptahydratewere added to the feed solution to create near-saturation conditions.Upon dissolving, the ions will dissociate and recombine to form precipitatesonce the saturation limit has been reached. Two well-known inorganicfoulants in membrane distillation are calcium sulfate and calciumcarbonate as they become less soluble at higher temperatures. Previousstudies have shown that these crystals can form on the membrane surface,eventually leading to pore blocking and flux decline.60 The normalized flux values for the pure PVDF, GP 2, and commercialPTFE membrane were plotted as a function of time to assess the fluxstability in these harsh conditions.In order to test themembranes under fouling conditions, 10 mg L–1 compared with 40–60 μS cm–1). However,after 24 h of testing, the permeate conductivities increased for allmembranes to between 60 and 70 μS cm–1 forthe PVDF and PTFE membranes and 50–63 μS cm–1 for GP 2, owing to the very high solute concentration resultingin partial wetting, which in turn enabled transport of inorganic solutesacross the membrane. This lower permeate conductivity for the GP 2membrane suggests that it had slightly better wetting resistance thanthe other two membranes but in all cases, the permeate quality isstill very high and well within the range for safe drinking. Anotherdifference between the membranes is the variability in the normalizedflux values. For the PTFE membrane, the data points are highly scattered,showing rapid variation in flux values, whereas the PVDF values weremore narrowly distributed and the GP 2 values more still. This rapidvariability could be due to the presence of precipitates in the feedsolution causing temporary pore blocking and then being removed dueto the shear forces from the feed flow. It may be expected to affectthe PTFE membrane more because the pores are significantly smallerand so may be more easily blocked. Nevertheless, overall, the fluxwas pretty stable for the membranes. The normalized flux values wereaveraged (excluding outliers) for the beginning and end of each test.The percentage differences of these values were −10.4, −8.7,and −6.4% for the PVDF, GP 2, and PTFE membranes, respectively.In other words, the average flux decline was highest for the PVDFmembrane, then GP 2, and then the commercial PTFE membrane.As can be seen in 61 Despite this, at theend of the experiments, the membranes were removed and though thoroughlyrinsed in DI water and still had visible coloration from the feedwater, suggesting that some precipitation did indeed occur on themembrane surface. This is further evidenced by the observed increasesin permeate conductivity and the EDX data depicted in Figure S2. Pronounced peaks are observed forcarbon and fluorine from all three membranes, as expected given theirchemical makeup. In addition, the elements Fe and O are also prominentin the EDX spectra and overlap in the element maps. This is suggestiveof iron oxides precipitating on the membrane, which are responsiblefor the red/brown color of the feed solution. The spectrum for theGP 2 membrane shows the presence of silicon, which originates fromthe POSS functional group on the graphene. Trace amounts of silicondetected on the PTFE membrane are likely due to contamination, andsimilarly, trace amounts of copper present in the electrospun membranesmay be the result of contamination from the stainless steel collectorplate on which the membranes were formed. Small peaks correspondingto sulfur are present on the electrospun membranes, again suggestingsome precipitation of sulfate crystals on the membrane, although thispeak is missing for the PTFE membrane, suggesting that precipitationoccurred preferentially away from the membrane surface. Furthermore,the absence of calcium peaks from all spectra indicates that no CaCO3 crystals precipitated on the membrane but instead remainedin the vessel or tubing. Similarly, the absence of an arsenic peaksuggested that the arsenic was retained in the feed solution and didnot adsorb onto the membrane in detectable quantities. The presenceof platinum is a result of the membrane coating process during thesample preparation.During the periods where the permeate was externally collectedrather than recirculated, the feed water became more concentratedto the point where crystals were clearly visible in the water andon the surfaces of the feed vessel and tubing. This precipitationaway from the membrane surface may explain the relatively stable fluxvalues observed in these experiments. Once precipitation was initiatedin the vessel, subsequent precipitation and crystal growth were favoredthere rather than on the membrane surface. This phenomenon has beenreported before.In the case of the electrospun membranes,the crystals that didprecipitate on their surfaces did not grow sufficiently large to completelycover the pores, whereas the PTFE membrane has a much more denselycoated surface. This may be the reason why the normalized flux valueswere much less stable for the PTFE membrane and may be an advantageof the electrospun architecture in more severe cases of inorganicfouling. Future work will involve longer-term experiments with variousprocess conditions to see how best to control inorganic fouling toenable continuous operation.Finally, by the end of the experiments,the feed volume had decreasedfrom 1600 to approximately 600 mL – a reduction of 62.5%. Thiscould have been continued, but this was the limit imposed by the geometryof the feed vessel. This highly effective concentration is usefulin the context of arsenic removal where the discharge of brine intothe local environment is highly dangerous.–2 h–1 over 5days of continuous testing, while the pure PVDF membrane showed a10.9% flux decline over just 24 h. This best-performing membrane hadnearly twice the flux of a commercial PTFE membrane, but all membranesin all cases showed very high (>99.9%) rejection of salt, highlightingthe effectiveness of air gap membrane distillation in removing inorganiccontaminants. These membranes also performed well when treating ahighly concentrated solution containing calcium carbonate, iron sulfate,sodium chloride, and sodium arsenate dibasic heptahydrate. The POSS-rGOmembrane demonstrated more stable flux over 24 h of testing than thepure PVDF membrane and also higher rejection values. Fouling withiron oxides was evident from EDX measurements but seemed to be moreprominent on the commercial PTFE membrane due to the crystal sizebeing comparable to or greater than the pore size. The much largerpore sizes of the electrospun membranes meant that they were not blockedor covered by the foulant crystals. Minimal amounts of sulfur andno traces of calcium were found from the EDX measurements, which suggestthat AGMD may be a suitable technology for zero liquid discharge applications.In the case of treating arsenic-contaminated groundwater, this approachis necessary in order to prevent further environmental damage.Inconclusion, air gap membrane distillation experiments showedperfect rejection of arsenic from simulated groundwater of the Tacnaregion, Peru. High-performance electrospun PVDF membranes were enhancedin terms of mechanical properties, hydrophobicity, and membrane distillationperformance with the addition of hydrophobic POSS-functionalized graphene.The optimal loading was 2 wt % with respect to the polymer, whichresulted in a 280% increase in the ultimate tensile strength comparedto the pure PVDF membrane. This membrane demonstrated a stable fluxof ∼28 L mN,N′-dicyclohexylcarbodiimide(DCC) and tetrahydrofuran (THF) were purchased from Sigma Aldrich(Germany). Electrospinning solutions were prepared using polyvinylidenedifluoride  and N,N-dimethylformamide(DMF), both purchased from Sigma Aldrich, Germany, as well as acetone. Millipore deionized (DI) water (18 MΩcm resistivity) was used for the preparation of feed solutions alongwith sodium arsenate dibasic heptahydrate and sodium meta-arsenite,which were purchased from Sigma Aldrich. NaCl, CaCO3, andFeSO4·7H2O also used for the feed solutionswere purchased from Acros, Belgium. All reagents and materials wereused as received.Forthe functionalization reaction, GO (1wt % aqueous suspension) was purchased from William Blythe , aminopropyl isobutyl polyhedral oligomeric silsesquioxane (AM0265– referred to here as POSS) was purchased in powder form fromHybrid Plastics (USA), and 22 Briefly, 100 mgof GO was freeze-dried from an aqueous suspension using liquid nitrogen.The dried GO was then redispersed in 50 mL of THF in a sonicationbath  for 2 h. This wasthen decanted into a 250 mL round bottom flask along with 2 g of POSSand 100 mg of DCC. This mixture was sonicated for a further 10 minand then refluxed at 80 °C for 48 h. Following this, the remainingsolvent was evaporated and the powder was heat-treated at 120 °Cfor 8 h to partially reduce the GO. The powder was then redispersedin 50 mL of THF, poured into approximately 500 mL of methanol, andthen filtered using a homemade polyacrylonitrile filter (0.2 μmpore size). This last step was repeated three times to remove anyunreacted POSS, and the powder (POSS-rGO) was then placed in a vacuumoven at 80 °C and then stored for further use. To investigatethe chemical changes further, the whole procedure was repeated withoutthe heat treatment step removed. This yielded POSS-GO (note that thisis now nonreduced GO), which was characterized for comparison purposesbut not used for the preparation of membranes. The reaction schemefor the functionalization of GO with POSS can be found in Figure S4.Functionalizationof GO with POSS occurred via amide formation, following the same methoddescribed elsewhere.Table S2 in the Supporting Information. This was done by firstproducing a 20 mg mL–1 solution of POSS-rGO in DMFvia sonication followed by the addition of acetone and a final stepof stirring overnight at 40 °C until the polymer completely dissolved.The electrospinningpolymer solutions were prepared by dissolving 1.4 g of PVDF powderin 8.6 g of a DMF/acetone mixture with a ratio of 1:2, making solutionswith a total weight of 10 g in each case. This solvent mixture containedvarious quantities of POSS-rGO as described in 2 was connectedto the opposite terminal of the high voltage supply, again using acrocodile clip, and was placed 20 cm from the tip of the needle.Electrospun membraneswere prepared using a setup that consisted of a syringe pump (ColeParmer), a high voltage supply, and a stainless steel tray, whichwas used as a collector. Prior to spinning, the dope solutions wereindividually drawn into a 10 mL plastic syringe , whichwas left standing on end for a few minutes to allow any bubbles toescape. Then a 19G 1.1 × 50 mm needle  whosesharp end was flattened by abrading it with sand paper was fixed tothe syringe. This was then clamped onto the syringe pump, and theneedle was connected to the high voltage supply using a crocodileclip. The collector plate with an area of 552 cm–1, respectively.Once the syringe needle and collectors were connected, the programon the syringe pump was run, and the high voltage supply was switchedon. The voltage and dope solution flow rate were kept constant foreach dope solution at 18 kV and 5 mL h–2. This was then placed in an oven at 170 °C,just below the melting temperature of PVDF, for 1 h in order to compactthe fibers and increase the mechanical stability of the membrane.After this post-treatment, the membrane was removed and stored forfurther use.After the dope solution was deposited, the membrane was left todry overnight under a fume hood. The membrane was then carefully peeledoff the collector plate and placed flat on a 250 × 230 mm sheetof tempered glass. An identical piece of glass weighing 785.2 g wasplaced on top of the membrane, exerting a pressure of 13.94 N mThe functionalizationof GO with POSS was assessed by X-ray photoelectron spectroscopy (XPS)using an Axis Ultra spectrometer  with a monochromatic Al Kα source (1486.7 eV). The spectrawere analyzed using CasaXPS software.–1, and the step sizewas 0.5 cm–1.In addition, attenuatedtotal reflectance Fourier transform infraredspectroscopy (ATR-FTIR) was used to probe the GO functionalization.This was carried out using an iDS Nicolet iS5 spectrometer , with a Ge crystal as a background. The wavenumberrange was 650–4000 cmThe membranes were imagedusing scanning electron microscopy (SEM)  witha 15 kV acceleration voltage and a 2.5 mm spot size. To prepare thesamples, small pieces of each membrane were stuck onto SEM holdersusing carbon tape and were sputtered with gold (or platinum for thefouled membranes) with a layer thickness of 5–6 nm to renderthe samples electrically conductive.In conjunctionwith SEM imaging, elemental analysis of the membranes was performedwith EDX spectroscopy. This was used to analyze the components fromthe inorganic fouling experiments, and spectra were collected usingan Oxford Instruments X-Max detector and plotted using AZtec 3.3 SP1software.–1, and ultimate tensile strength and Young’s modulus valueswere calculated.The mechanical properties of the membraneswere investigated by tensile testing. Measurements were carried outusing an Instron 5542 tensiometer  with a 100 N loadcell under ambient conditions. Samples were prepared by cutting rectangularstrips of membranes (7 mm × 60 mm) and sandwiching each end betweentwo 10 mm squares of thin cardboard using double-sided sticky tape.The effective length of each sample was 40 mm, giving a length/widthratio of 5.71:1. Three identical samples were prepared for each membrane.The thickness of the membranes was measured with a digital micrometerscrew gauge in proximity to where the tensile strips were cut. Thetensile strips themselves were not measured as the compression fromthe micrometer may have affected the mechanical properties or induceda defect. Ten thickness measurements were taken for each membraneand averaged. The elongation rate was set up to 10 mm min2 permeability of the electrospun membranes were measuredby capillary flow porometry . Thisemployed the gas–liquid displacement method using perfluoropolyether as the wetting liquid as detailed in our previous work.62 The slope of the dry curve was used to calculatethe nitrogen permeability by dividing by the membrane thickness. Thistechnique was also used to measure the liquid entry pressure (LEP)of the membranes. Using a nonstandard method, 13 mm disks were cutfrom each membrane and inserted dry into the Porolux device. Then,0.3 mL of DI water was dropped onto the surface of the membrane, andthe compartment was closed by connecting the gas. The Porolux wasset to provide a maximum pressure of 1 bar over 50 steps, and the “fullporometry” program was executed. This gradually increased thepressure on the water sat atop the membrane. As the pressure continuedto increase, a sudden increase in gas flow was measured by the device,indicating that the water was forced through the membrane. The pressureat which this occurred was reported as the LEP. For all measurements,the reported values are averages of three samples taken from differentareas on the membrane.The poresize distributionsand N62 Briefly, 10 mm squares were cut out of the membranes (3 for eachmembrane) and weighed. Then these squares were immersed in the sameliquid used for porometry (Porefil 125) for 30 s to become fully wetted.One by one, the squares were removed from the wetting liquid and placedon tissue paper and were gently daubed, removing any residue fromthe surfaces. The samples were then weighed again in order to determinethe mass of wetting liquid, which was adsorbed by the pores. The membraneporosity was then calculated usingWw is the wet membrane weight and Wd is the dry membrane weight. The densities of Porefil125 (ρw) and the PVDF polymer (ρp) are 1.9 and 1.78g cm–3, respectively. The values reported were theaverages of three measurements.Membrane porosity, ε,was evaluatedusing the gravimetric method, as reported previously.63 Membrane strips were fixedto glass slides, which were then placed on the stage of an AttensionTheta optical tensiometer, and five drops were measured for each membraneand averaged.The wetting properties of the membraneswere evaluated using water contact angle (CA) measurements as describedpreviously.3+ and As5+ were quantifiedusing inductively coupled plasma mass spectroscopy  and Masshunter Version 5 software.The qualityof the permeateproduced from membrane distillation experiments was assessed in termsof conductivity using a Fisher Scientific Accumet XL200 conductivitymeter. In addition, AsFigure S5. The system is composed of two isolatedwater loops – one containing tap water used for cooling thecondenser plate inside the membrane module and one containing theheated feed water. The prepared synthetic solutions had concentrationsof inorganic arsenic and sodium chloride similar to the concentrationsof arsenic and conductivity recorded in water sources intended forhuman consumption in the rural area of the city of Tacna, Peru (LocumbaRiver and Sama River). The feed water contained 600 ppb sodium arsenatedibasic heptahydrate and sufficient NaCl to bring the feed conductivityup to 2500 μS cm–1 – similar to thatof the Locumba river. To prepare the feed solutions, a concentrated(5 mg/L) stock solution was prepared by dissolving 10 mg of sodiumarsenate dibasic heptahydrate in 500 mL of DI water. Then, 60 mL ofthis stock solution was added to 1940 mL of DI water to produce 2L of feed solution. Tests were carried out using a feed water volumeof 1 L. A test was also conducted on the commercial PTFE membraneto see if As3+ could be removed by AGMD. For this, a 300ppb solution of sodium meta-arsenite was prepared instead of sodiumarsenate dibasic heptahydrate by diluting a 1000 mg L–1 stock solution.Arsenic removal experimentswere performed using air gap membrane distillation, as depicted in Table S3 in the Supporting Information. The permeate samples were collectedin a measuring cylinder after 1 h of conditioning for each membrane.The flux was calculated by extrapolating the volume of permeate collectedover 30 min, given a membrane area of 27.33 cm2, and thesalt rejection was calculated from permeate conductivity values, asdescribed previously.56 For the inorganicfouling experiments, the normalized flux was calculated usingAfter prior optimization, the process conditionswere selected, as summarized in –1 calcium carbonate and 2 g L–1 iron(III) sulfateheptahydrate were added to the same arsenic and sodium chloride feedsolution used for prior experiments. This turned the water a terracottacolor as shown in 65 In these experiments, the massof the permeate was measured using a weighing scale (Adam HighlandHCB 3001) connected to a data logger collecting one data point every2 min in order to track any sudden changes in flux. The permeate wascollected for 3 h at the beginning and then was recirculated overnightand collected again for 22, 23, and 24 h of the 24 h experiment, duringwhich time the loss of the permeate resulted in the increased concentrationof the feed – the aim being to reach saturation conditions.At each hour of permeate collection, a sample was taken for conductivitymeasurements and then returned to the collection vessel. All otherprocess conditions were kept the same.Inorder to test themembranes’ propensity for inorganic fouling, 10 mg L