Ks) is one of the most important soil properties that determines water flow behavior in terrestrial ecosystems. However, the Ks of forest soils is difficult to predict due to multiple interactions, such as anthropological and geomorphic processes. In this study, we examined the impacts of vegetation type on Ks and associated mechanisms. We found that Ks differed with vegetation type and soil depth, and the impact of vegetation type on Ks was dependent on soil depth. Ks did not differ among vegetation types at soil depths of 0–10 and 20–30 cm, but was significantly lower in managed forest types  than native evergreen broadleaf forests at a depth of 10–20 cm. Boosted regression tree analysis indicated that total porosity, non-capillary porosity, and macro water-stable aggregates were the primary factors that influenced Ks. Our results suggested that vegetation type was a key factor that influences hydraulic properties in subtropical forest soils through the alteration of soil properties, such as porosity and macro water-stable aggregates.Saturated hydraulic conductivity ( Ks) is one of the most important soil properties that determines the behavior of water flow systems1. A detailed understanding of Ks is critical in the assessment of irrigation practices, infiltration rates, runoff, groundwater recharge rates, and drainage processes, which makes it of particular concern in forest management2.Saturated hydraulic conductivity  models are typically applied to the prediction of Ks19. Among a number of physical parameters, soil porosity, texture, and bulk density are determinants for Ks in the PTF23. The chemical characteristics of soils such as soil organic carbon (SOC) or soil organic matter (SOM) are also important predictors for Ks in the PTF26. The effects of soil aggregate dimensions on Ks were investigated and it was found that higher Ks associated with higher SOM was positively associated with soil aggregate size27. Ks has also been found to be affected by pore dimensions and distribution28. At the field scale, however, the physical and chemical parameters of soils are not always significantly correlated with Ks29.Vegetation is expected to be an important factor that influences the hydraulic properties of soil by affecting its physical and chemical characteristicsKs remains poorly understood. Moreover, the contribution and importance of specific soil parameters on the resulting Ks is not always certain. Here, our objectives are to test: (1) whether differences in forest vegetation, resulting from changes in management objectives, affect soil Ks across multiple soil depths and; (2) how changes in soil Ks might be associated with the physicochemical attributes of soil. To address our first objective, we used analysis of variance to test the effect of forest vegetation types on soil Ks. For the second objective, we used boosted regression tree (BRT) analysis, which resembles an additive regression model and can achieve higher accuracy and less bias in predictions than traditional multiple regression models30. In particular, BRT analysis is good for handling multi-collinearity concerns and violations of parametric assumptions32.Despite the drastic effects of forest vegetation type shifts that occur frequently at a global scale, how changes in forest vegetation types affect Ks and other characteristics (Table Ks was significantly dependent on soil depth as indicated by the significant interaction effect between vegetation type and soil depth (P = 0.03). In the top soil layer (0–10 cm), soil Ks was higher in bamboo forests and tea gardens than in native and mixed forests; in the deep soil layers (10–20 cm and 20–30 cm), native forests had significantly higher soil Ks than other vegetation types, with the lowest values in tea gardens . A similar trend was seen for total porosity and capillary porosity, which were impacted by vegetation type and soil depth, but not by their interaction. Total soil nitrogen was impacted considerably by changes in vegetation type with P = 0.01, while the impacts of soil depth and the interaction between vegetation type and soil depth were not significant. The non-capillary porosity of soil was impacted by the interaction between vegetation type and soil depth with P = 0.09, but not by these individual factors . Meso and micro water-stable aggregates were impacted only by soil depth with P = 0.04 and P = 0.02, respectively . Root surface area density was impacted by changes in vegetation type and soil depth , but not by their interaction (P = 0.59). Root length density of bamboo forests was higher than other vegetation types in all soil depths, while root surface area density was lower in 0–10 cm soil depth. The root length density of bamboo forests in 0–10 cm and 10–30 cm was lower  and other soil physicochemical characteristics4. This stimulates belowground microbial biomass and rhizospheres43, and the effects of the physicochemical attributes of soil on Ks via microbial community activities46. So to alter the physiochemical characteristics of soil, by means of producing solid, gas, and gel phases in order to adjust the fraction of the total spatial volume that is available for water flow, and hence the Ks.The roots distribution characteristics also affect the soil texture by adjusting litter input from the soil or its surface over timeKs differed with vegetation types though Ks in native forests did not vary with soil depth. This is in alignment with prior researches15, which may have been the result of distinct vertical distributions of the physicochemical characteristics of soil and root distribution14. This might partly explain the increasing trend in soil depth from 10–20 cm to 20–30 cm. The decreasing root length and surface area densities were weakened by the effects of roots via mechanical forces or litter input characteristics. The higher Ks in 0–10 cm might be attributed to the great probability in contacting a fresh litter of leaves or branches.The effects of soil depth on soil Ks value was higher in the tea garden. This may be attributed to the higher distribution density of the tea stems and the complexity of the root distribution underground, which could affect the Ks value37. We also observed that the Ks value at the 10–20 cm depth for the vegetation types other than native forests was lower than at the other soil depths. We attributed this to soil disturbances during vegetation conversion at that time, which might have had the effect of compacting the 0–20 cm depth layer. However, since the relation between Ks and root distribution was not clarified in this study, we propose to explore this area further in future research.In this study, the variation of the Ks. A key parameter in this study was bulk density, from which the calculations on total soil porosity were derived. This was similar to a study in which differences in Ks between samples were found to be correlated with bulk density and macro porosity47. The characteristics of pores in soils, such as their dimensions, distribution, and interconnections have been known to impact Ks. It was found in many studies that lower bulk density was aligned with higher Ks, and vice versa49, while water stable macro aggregates were positively correlated with Ks50. It was found that the Ks values were reduced in soils with smaller aggregates in contrast to those with large aggregates12. This was likely attributed to the impacts of different fresh organic matter, which were produced by different vegetation types51. Vegetation generated litter may simulate soil aggregation52, which subsequently influences bulk density and porosity54, while bulk density and porosity are closely correlated to adjustments in Ks56. It remains a scientific challenge to describe in detail the complex continuous soil space28. However, we may conclude that soil pore characteristics are important factors.In this study, total soil porosity, non-capillary porosity, and macro water-stable aggregates were the principal factors that influenced Ks significantly, and the effects of soil depth on Ks varied for different types of vegetation. The Ks of native forests did not significantly differ at soil depths from 0–30 cm. For the other vegetation types, the Ks at the 10–20 cm depth was significantly lower than that at 0–10 cm and 20–30 cm depths. There are multiple factors that impact Ks; however, total soil porosity, non-capillary porosity, and macro water-stable aggregates comprised the primary factors in this study. Soil Ks is strongly influenced by changes in vegetation type, indicating that shifts in aboveground vegetation may strongly impact the water dynamics of soil. Based on our data, we suggest that the restoration of the native evergreen broad-leafed forests will assist in the retention and maintenance of soil hydrologic properties. Additional research will be required to confirm other factors and mechanisms that influence Ks, such as the role of root systems and microbial communities in the processes that follow changes in vegetation species.In conclusion, our results show that change in vegetation type is a driving factor that strongly influences the hydraulic properties of soils in subtropical forests. Vegetation type, soil depth, and their interaction were observed to influence Camellia japonica Linn., Cyclobalanopsis glauca (Thunberg) Oersted, Eurya japonica Thunb., and Rhododendron simsii Planch.). Intensive selective cutting and reforestation was conducted during 1971–1973, and portions of the forests were converted to mixed evergreen broad-leaved and coniferous forests (henceforth referred to as mixed forests), primarily consisting of Schima superba Gardn. et Champ., Rhododendron simsii, and Pinus taiwanensis Hayata). Subsequent to clear-cutting, pure plantations with Cunninghamia lanceolata (Lamb.) Hook., Cryptomeria fortune Hooibrenk ex Otto et Dietr., bamboo forests (Phyllostachys heterocycla (Carr.) Mitford cv. Pubescens Mazel ex H.de leh.), or tea gardens were established. Following the establishment of the nature reserve in 1975, the entire study area, including the tea gardens, was protected from anthropogenic disturbances. The roots characteristics of different vegetation types were shown in Supplementary Figure.Major forest conversion is occurring globally. In China, vegetation change from native evergreen broadleaf forests to mixed evergreen broadleaf and coniferous forests or other vegetation types is common. This study was conducted at the Fengyang Mountain Nature Reserve, Zhejiang Province, China , which comprised a land area of 15,171 ha. This nature reserve is characterized as having a subtropical climate, with an annual average temperature of 12.3 °C, and annual rainfall of 2,400 mm. Prior to 1970, this area was dominated by native evergreen broad-leaved forests  at each sampling point61, which resulted in a total of 171 samples .In June 2013, we randomly sampled five native evergreen broad-leaved forest stands, five mixed forest stands, five bamboo forest stands, and four tea garden stands at elevations ranging from 1250 to 1450 m, which resulted in a total of 19 sampled stands. All of the sample stands resided on well-drained mesic sites with slopes inclines of less than 5% to minimize the effects of inherent site conditions on soil characteristicsKs, we did supplementary sampling of soil with a metal corer (5.5 cm in diameter × 5 cm in height) in December 2018. Similar to the early sampling, we randomly sampled five native evergreen broad-leaved forest stands, four mixed forest stands, three bamboo forest stands, and three tea garden stands at elevations ranging from 1250 to 1450 m, which resulted in a total of 15 supplementary sampled stands.To further understand how roots affect the impact of vegetation type on Ks was determined based on the constant hydraulic head method by imposing a stable hydraulic head to the top of the cores that were sampled at each of the sampling points, which were saturated with water prior to experiments in the laboratory1.58. Soil samples for other physicochemical analyses were air-dried, sieved (2 mm mesh) in the laboratory, and then stored in air-tight plastic bags. Total organic carbon (TOC) content was measured using the sulfuric acid-potassium external heating method62. Total nitrogen and total phosphorus were simultaneously determined using a Bran + Luebbe Autoanalyser 3 Continuous Flow Analyzer . Root length density and root surface area density were analyzed with the Win RHIZO root system . Before the analysis, all roots were washed out from the metal corer and then scanned with EPSON LA .Soil bulk density was determined by drying the samples in an oven at 105 °C until a constant weight was attained, and then adjusting for root and stone volume63:Pt is the total soil porosity (%); 100 is the unit conversion factor; Db is the soil bulk density (g cm−1); and Dp is the soil particle density (g cm−1), which was assumed to be 2.65 g cm−1 according to China’s standard64. The soil capillary porosity was determined based on the water suction method, with the surface of the water located just below the tops of the soil cores63. Each soil core was initially weighed and placed onto a salver via filter paper until it attained a constant weight. Following weighing, the soil samples were allowed to drain completely under gravity. The soil samples were subsequently weighed again; their capillary water contents were determined by the differences in weight between the saturated and drained states.Pc is the capillary porosity (%); Pn is the non-capillary porosity (%); 100 is the unit conversion factor; Wc is the soil capillary water content (%); V is the volume of the soil core (cm3).The total porosity was calculated using the following equation65. Briefly, for each soil sample, 200 g of air-dried soil was placed on a series of sieves to determine the dry aggregate size distribution  prior to wet-sieving. Subsequently, 50 g samples were prepared according to their dry-sieving percentages by the weight of aggregates at each size distribution for wet-sieving. The samples were immersed in water for 10 minutes and then placed under oscillation at 30 rpm for 30 min. The aggregate fractions that remained on each sieve were removed with aqua distillate into aluminum bins, to be oven-dried at 105 °C for 24 h. The aggregate fractions were then weighed to calculate the aggregate weights from each size class66.Water stable aggregate was measured using a routine wet-sieve method via a mechanical sieving procedureKs and other soil characteristics, an analysis of variance (ANOVA) was performed following a split plot design, with soil layers nested within the sample plot. We modelled the fixed effects of vegetation type, soil layer, and their interaction on Ks with plot as the random factor using maximum likelihood with the lme4 package67. ANOVA assumption tests were done with the lmerTest package68. Shapiro –Wilk’s test was conducted. In this study, the Shapiro –Wilk’s test involving capillary porosity and non-capillary porosity failed, so a Box-Cox transformation was performed by the following equation69:Vtrans is the transformed value of capillary porosity or non-capillary porosity; Vorigin is original value of capillary porosity or non-capillary porosity; and λ is the parameter of box-cox.To examine the impact of land use type and soil depth on the Ks was potentially affected by soil physicochemical characteristics. Furthermore, we examined Pearson’s correlation between potential factors and Ks to reduce the fitting predictors. Then we fitted all BRT models using the adjusted settings for ecological modeling: tree complexity = 5, learning rate = 0.0001, bag fraction = 0.7. All analyses were performed using BRT with the R package gbm70.We used boosted regression tree analysis (BRT) to elucidate how Supplementary information


In recent decades, there has been an increase in the research for the development and improvement of dye sensitized solar cells (DSSCs), owing to their singular advantages such as greater efficiency in energy conversion and overall performance in adverse environmental conditions. Therefore, work is carried out to enhance the energy efficiency of the components of the DSSCs: photoanode, counter-electrode, electrolyte, and dye sensitizer layer. Electrospun nanofibers in particular, have showed to be a novel alternative as components in DSSCs, mainly for energy conversion and as collector materials due in part to their tridimensional structure, high contact surface area and conductivity. Moreover, the incorporation of metallic compounds into nanofibers is advantageously employed in the electrospinning technique, owing to their conductivity and optical properties. Therefore, the present work consists of a detailed recompilation of the use of electrospun nanofibers loaded with metallic compounds and their application in DSSCs. The functionality of the components of DSSCs, parameters and experimental conditions of electrospinning, such as the intrinsic aspects in the polymer solution, are discussed and applied to the photoanode, counter-electrode and electrolyte of the DSSC. Lastly, the use of the electrospinning technique in combination with the use of metallic compounds could provide a great approach for the developing of DSSCs, with superior efficiency, high stability and durability. For the last 20 years, advancements in the generation of energy through solar cells has been remarkable, not only due to increased energy efficiency, but also because of improvements which can be seen in its design and manufacturing . CurrentAn advantage in the design of DSSCs is the use of flexible and lighter materials, which consequently increases the number of applications in which the DSSCs can be used. Furthermore, DSSCs bear certain advantages compared to the first and second generation solar cells: a lower manufacturing cost in conjunction with a market low cost of the components, makes the cost of a DSSC about 10–20% of the manufacturing cost of silicon cells . The inc2) layer with a Ruthenium (II) complex as a photoanode, and with this change in the manufacturing process an increase to 7% in the efficiency of the DSSC was achieved [The first DSSC solar cells date back to 1972, when cells were prepared using chlorophyll as a sensitizing dye and zinc oxide as a photoanode, however, efficiency was very low (2.4%) due to its low surface area . In 1991achieved .The structure of the DSSC is illustrated in −2 S·cm−1), optical transparency and 75% of transmittance approximately [2) is the most common semiconductor used in depositions in the TCO of photoanodes for DSSC [The photoanode consists of a metal oxide semiconductor deposited on the surface of a transparent conductive oxide substrate (TCO). The TCO allows the collection and transfer of electrons to an external circuit. Usually, a fluoride doped tin oxide (FTO) is used as the TCO; this is due to its excellent electrical conductivity 5.8 × 10−2 S·cm−1ximately ,14. Titafor DSSC ,10,11.The dye sensitizer in the DSSC is responsible for absorbing the light generated from the electron current through photo excitation and is thus affixed to the photoanode .The redox electrolyte in the DSSC works as a transport path to transfer electrons from the counter-electrode to the oxidized dye, thus compensating for the loss of electrons in the dye. The electrolyte in the DSSC can be classified as a: liquid, quasi-solid and solid. The most common electrolyte used in DSSC are in the liquid state; usually a iodide/triiodide based compounds ,16,17.The counter electrode is responsible for taking the electrons from an external circuit and dispersing them in the electrolyte . ConsequThe operating principle of a DSSC can be understood by the following ,21,22,23DSSCs have made their way into the world of solar cells, due to their advantages over first- and second-generation solar cells; not only for their low cost, but for operating in non-standardized conditions, making them ideal for places where the environmental conditions vary greatly. DSSC for example, are efficient in low light environments, e.g., at dusk, on cloudy days or even indoors. Therefore, DSSC can be applied to many applications in areas such as panels, tiles, facades, solar roofs and smart sensors in automotive and nautical industries, among many others .Electrospinning is a technique used for the formation of fibers and manufacture of fibrous scaffolds; its applications have increased substantially in recent years; particularly in the fields of medicine and engineering (R&D). The electrospinning technology is based on the injection of a conductive polymer solution, electrically charged and directed towards a collector, against an electrode that is grounded closing the circuit. The solution then passes through a field of electrostatic forces ranging from 20 to 50 kV, which produces a stretching effect that forms fibers from one nanometer to one micrometer . “Upon eThe parameters to mainly consider are: applied voltage, flow velocity, type of collector, and distance between the collector and the tip of the dispenser of the polymeric solution. The experimental conditions are ideally carried out in a controlled atmosphere  in the electrospinning chamber, as well as, “air velocity inside the chamber” . An impoIn principle, the selection of the polymer should be made according to the characteristics that match according to the application. In the case of the photovoltaic industry, important parameters such as the free flow of electrons, characteristic of the conductivity of the material and mobility between empty spaces, are some aspects that should be considered in the synthesis of solar cells. Generally, the conductivity mechanism in conducting polymers is based on the motion of charged defects within the conjugated framework. These defects can be positive (p-type) or negative (n-type) and result from the oxidation or reduction of the polymer, respectively .2, CaCO3, fumed silica, carbon black, iron and nickel, with an average particle diameters of less than 100 nm and fiber size on the order of 200–2000 nm [Particle or suspension electrospinning refers to dispersing particles in the polymer solution and electrospinning the mixture. Various nanoparticles have been electrospun at low loadings including magnetite, TiO–2000 nm .Despite the multiple advantages presented by the DSSC, there is a problem with their low efficiency; consequently, in recent years there have been many studies where strategies are proposed to increase their efficiency. One way to achieve this goal is to develop electrospun components for the DSSC, such as photoanodes, counter-electrodes and electrolytes .In the photoanode of a DSSC, morphology and structure play a very important role in the performance of electron transport . Photoan2 is the most widely used semiconductor, however, compounds such as Sn (IV), Zn (II), Ni (II), have also been studied for the manufacture of electrospun nanofibers used as photoanode [TiOotoanode ,38,39,402 nanofibers are not the only scaffolds used as photoanode. ZnO/poly (vinyl acetate), composite nanofiber mats  illumination [TiOber mats , entry 1mination .2 from electrospun nanofibers  reported for the first time the fabrication of vertical nanowires of TiOnofibers , entry 2ic forms , entry 3thesized .2 nanofibers . A higher amount of graphene oxide, results in a decrease in the power conversion efficiency [Graphene oxide (GO)-incorporated into TiOnofibers , entry 4ficiency .2 nanofibers to enhance the conversion efficiency of ZnO-based DSSC. According to the authors, the system TiO2/ZnO .Li et al. (2014) , also usTiO2/ZnO , entry 52 nanofibers-TiO2 (SnO2 NF-TiO2) was ~4.61% (2 nanoparticles films in (P25 film) DSSCs.Gao et al. (2012) , reportes ~4.61% , entry 62/Nb2O5 core–sheath nanofibers (NFs)  , entry 7 ca. 30% .2 nanofibers that have been doped with silver  into TiO2 films. The electron lifetime of DSSCs with ATN increased from 0.29 to 0.34 s and the electron recombination was reduced [TiOh silver , entry 8 reduced .2 electrospun fibers  with a reported efficiency of 6.86% [Finally, the most efficient nanofibrous system is fabricated with TiO2-Graphene nanofibers , as well as the low onset potential and high current density of the composite electrode. Moreover, these fibers were effective, providing stable electro catalytic activity (ECA) and conductivity, indicating that the composite can improve catalytic activity in triiodide reduction [Cobalt-titanium carbide nanoparticles (Co-TiC NPs) embedded on electrospun carbon nanofibers were studied , entry 1eduction .2ZnSnS4 (CZTS) nanofibers were fabricated by electrospinning process  , reporteIn 2015, Saranya et al. , studied−2), higher short-circuit current density (14.77 mA·cm−2), and higher PCE (6.23%) which is comparable to a commercial Pt counter electrode. The improved photovoltaic performance of the counter electrode in the DSSC was attributed to the combined effects of small grain size, which resulted in a high number of electrochemical sites, a high electrical conductivity that lead to improved electro catalytic activity, and a unique network structure that allowed for rapid electron transfer and rapid diffusion of the electrolyte [Rare earth elements have also been used for the fabrication of electrospun nanofibers used as CE, Ru(III) nanofibers consisting of nano-sized grains are used instead of the typical Pt counter electrode for DSSCs , entry 6ctrolyte . The effctrolyte .2S4, NCS) particles were prepared through sulfurization of NiCo2O4 (NCO) electrospun nanofibers  composite nanofibers (2/C nanofibers as the counter electrode (CE) delivered a photoelectric conversion efficiency of 7.23%, which was higher than the efficiencies of DSSCs assembled, using the samples without graphene (6.48%) and with the CuInS2/C nanofibers (5.45%). It was also much higher than that of the DSSC with a Pt CE (6.34%). The excellent photoelectric performance of the p-GN@CuInS2/C CE was attributed to its special hierarchical porous structure, which facilitated permeation of the liquid electrolytes and provided additional active catalytic sites for the oxidation reaction of the electrolytic (I−/I3−). The doping of reduced graphene oxide (RGO) resulted in the well-dispersed growth of CuInS2 nanocrystals in the carbon nanofibers, which further increased the number of active catalytic sites and promoted electron and ion transfer [Another example of the use of rare earth elements in DSSC manufacturing is the porous graphene-doped copper indium disulfide/carbon  nanofibers  , Table , entry 9nofibers , entry 9ct) of the Ni-CNT-CNF composite electrode was 0.71 Ω·cm2, much lower than that of the Pt electrode (1.81 Ω·cm2). Such a low value of Rct indicated that the Ni-CNT-CNF composite carried a higher catalytic activity than the traditional Pt CE. By mixing with CNTs and Ni nanoparticles, the series resistance (Rs) of the Ni-CNT-CNF electrode was measured at 5.96 Ω·cm2, which was close to the Rs of 5.77 Ω·cm2 of the Pt electrode. The DSSCs based on the Ni-CNT-CNF composite CEs yielded an efficiency of 7.96% with a short circuit current density (Jsc) of 15.83 mA·cm−2, open circuit voltage (Voc) of 0.80 V, and a fill factor (FF) of 0.63, which was comparable to the device based on Pt, that exhibited an efficiency of 8.32% with Jsc of 15.01 mA·cm−2, Voc of 0.83, and FF of 0.67 [Finally, a nickel incorporated carbon nanotube/nanofiber composite (Ni-CNT-CNF) was used as a low-cost alternative to Pt as counter electrode (CE) for dye-sensitized solar cells (DSSCs)  , elabora2   SiO2 , entry 3ectrodes . As mentodes [2) , entry 4In an effort to improve the performance of DSSCs, electrospun compounds of polyvinylidene fluoride (PVDF) have been used as a quasi-solid electrolyte , entry 7The efficiency obtained in PVDF-HFP nanofibers , entry 7In another study conducted by S.J. Seo et al. (2011) , an elecV. Murugadoss et al. (2018) , improve−3 S·cm−1 was obtained. Another factor that was significantly modified with the presence of PEG in the PMMA-PVDF solution was the average fiber diameter; which decreased from 500 nm to 223 nm [M. Fathy et al. (2016) , createdo 223 nm .−2 S·cm−1 whereas PAN nanofibers was only 3.2 × 10−3 S·cm−1. In comparison, the reported conductivity for a conventional polymeric electrolyte, is 7.37 × 10−3 S·cm−1 [Continuing with electrospun compounds based on PVDF, I.A. Sahito et al. (2017) , added a3 S·cm−1 . This da2 nanofibers are the most commonly used, due to their high efficiency, fast electron transport, slow interfacial electron recombination and large specific surface area. Other systems with ZnO have been proposed, but most of the strategies maintain a TiO2 base; nonetheless adding graphene to TiO2 nanofibers considerably improved the electron transport efficiency, achieving the highest efficiency of all of the fibrous scaffolds discussed. In the case of the counter electrode, the main idea behind the use of nanofibers is the reduction of platinum (Pt) due to its high cost. Researchers are using rare earth elements and graphene oxides as replacements and are obtaining promising results, and Ni-Carbon fibers exhibited the best results. Finally, in the electrolyte component, the idea is to minimize the use of liquid solutions because of its disadvantages such as spills and delicate handling, PVDF-LiCl nanofibers presented the best results for its use in this DSSC component. Furthermore, electrospinning is a versatile, reproducible and low-cost technique which is commonly used in various applications. Electrospun nanofibers offers several advantages for the fabrication of DSSC, such as high surface area, a tridimensional structure, stability, and adequate mechanical properties. Hence, these fibers are particularly convenient in the manufacture of flexible DSSC, moreover, fibers occupy a smaller space in the components, so final DSSC can be fabricated with a tiny thickness. Still many challenges need to be overcome to completely replace the standards components in DSSC with electrospun nanofibers, such as the reproducible concentration of metals over the polymeric nanofibers, equal or higher efficiencies compared to the conventional DSSC, as well as the high cost of some polymers and solvents used in the electrospinning technique.In this review, the use of electrospun composite nanofibers as components for dye-sensitized solar cell (DSSC) was discussed. Nanofibers can be applied in three different components in the structure of DSSC: photoanode, counter electrode and electrolyte. In the case of the photoanode component, TiO


The glycogen storage diseases (GSDs) are a group of inherited metabolic disorders that result from a defect in any one of several enzymes required for either glycogen synthesis or glycogen degradation. The traditional diagnostic approach is based on the invasive hepatic or muscle biopsies, which are neither cost effective nor convenient. Molecular (gene testing) has emerged over the course of past few years as a robust alternative diagnostic tool, which not only confirms the diagnosis of GSDs but also clearly differentiates the types of GSDs allowing the initiation of the type-specific appropriate treatment for the particular type of GSDs. The aim of this update is to highlight the limitations of undertaking a liver biopsy for the diagnosis of GSDs; and to further describe the pros of the molecular testing for better patient centered care. A distinct group of inherited metabolic disorders caused by a defect of the enzymes involved in glycogen synthesis, degradation, or regulation are classified under the umbrella term of glycogen storage diseases (GSDs). Several studies have reported the overall GSD incidence to be approximately 1 case per 20000-43000 live births.1Glycogen is the storage form of glucose and the chief source of non-oxidative glucose disposal. It is most copiously present in the liver and muscle cells, in addition smaller amounts are present in brain, heart, kidney, adipose tissue and erythrocytes. It serves to maintain glucose homeostasis in the liver whereas provides substrates for the adenosine triphosphate (ATP) in the muscles.2The GSDs can be classified on the basis of organ affected and the enzyme deficiency involved. On the basis of target organ involved GSDs are comprised of three broad groups including; hepatic type of GSDs, muscles type of GSDs and the group affecting both liver and muscles. Each group has several different types; caused by defect of a different enzyme or a transporter, which is encoded by a different gene .From a clinician’s perspective the hepatic group of GSDs often has an easily recognizable clinical phenotype usually apparent as doll-like facies, failure to thrive/short stature, hepatomegaly and the biochemical features of fasting ketotic hypoglycemia, lactic academia, raised alanine transaminase with or without hypertriglyceridemia and hyper-uricemia.In Pakistan the conventional diagnostic approach towards hepatic types of GSDs includes recognition of clinical symptoms followed by biochemical work up and ultimately the more invasive liver biopsy to reveal features consistent with fatty change, nuclear hyper-glycogenation and fibrosis. Additional electron microscopy and enzyme studies on the liver tissue are required to specify the type of GSDs. Neither the electron microscopy nor the enzyme assays for GSDs are locally available, which compromises the diagnostic yield of liver biopsy for diagnosing GSD in local setting. The liver biopsy has the following limitations, some of which are specific to Pakistan:• Controversial diagnostic accuracy for GSDs with lack of specificity based on light microscopy only.• The procedure is invasive with serious complications including pain, hemorrhage, bile peritonitis, penetration of abdominal viscera, pneumothorax, and even death.4• The high cost including multiple clinic visits, anesthesia reviews and procedure cost.• Lack of centers with trained staff with sufficient experience for performing liver biopsy, further adds to the constraints especially for patients in smaller cities with limited technical expertise available.• Usually 30–40 mg of tissue or four cores of hepatic tissue including about 15 mg of snap-frozen hepatic tissue in liquid nitrogen is required for all the studies necessary including light microscopy, electron microscopy and enzymes analysis to make a definitive diagnosis.5• Specialized laboratories offering enzyme studies for hepatic GSDs are not available in Pakistan thus liver tissues are needed to be out-sourced to overseas laboratories in completely frozen condition with very vigilant temperature control, which is extremely challenging and chances of losing the precious hepatic tissue collected after an invasive procedure are very high.• Electron microscopy on the hepatic tissue, which is required for the diagnosis of GSDs is not readily available at most diagnostic centers in the country owing to its high cost.• The treatment is aimed at specific type of GSDs, which is not possible based on only histo-pathological findings of hepatic tissue on light microscopy without the enzyme testing, which is not available in Pakistan.On the other molecular testing, which was previously only performed to supplement enzyme activity analysis and confirm equivocal results has now become the principal diagnostic tool for GSDs.• Non-invasive compared to the liver biopsy.• High diagnostic accuracy as molecular methods minimize false positive test results by targeting the specific gene of interest.• Molecular testing clearly differentiates between different types of GSDs allowing physicians to initiate specific type-based treatment of GSDs and organizing the specific type-based surveillance plan, which varies significantly for various GSDs.• Ease of sample transportation to the laboratory without vigilant temperature control.• Automated analyzers are available and a single trained pathologist can report numerous samples.• Short turnaround times, which is 48 hours.• Cost-effective, as the cost of next-generation sequencing (NGS) allowing analysis of multiple genes in a single DNA sample is around $278.• Provision of reliable prenatal diagnosis and carrier testing by offering targeted familial variant testing to at-risk couples and carrier testing for family members.Early diagnosis of GSDs is imperative for initiation of appropriate treatment and achieving better prognosis. Owing to the nonspecific clinical presentation of GSDs and the lack of specific biomarkers to differentiate various types of GSD, NGS has become the first line diagnostic tool for the evaluation of GSD. NGS depends on massive molecular parallelization and allows analyzing multiple genes at the same time in a single DNA sample, it is a rapid and a much cost-effective way of not only diagnosing GSD but also differentiate different types of GSDs in absence of the invasive procedure of liver biopsy.In context of NGS instrumentation availability, a wide variety exists e.g. MiniSeq and Miseq from Illumina, Ion Torrent from Life Technologies Thermo Fischer Scientific and MinION from Oxford Nanopore etc. These differ primarily in terms of cost, capacity, principle chemistry and read length, DNA library preparation and run time. On the basis of feasibility, the Illumina MiSeq is the most popular sequencer due to its relatively low cost, significantly low error rate and capacity to deliver the moderate throughput required by most centers.7From a local perspective, measurement of enzyme activity suffers from logistic issues and is technically more challenging alongside a dearth of laboratory expertise. Furthermore, enzyme analysis is usually not reliable in detecting heterozygous carriers of a disease. These testing modalities are often very laborious, time consuming and require a pre-selection by clinical phenotype for targeting the specific enzyme.In line with the best practices, the undertaking of molecular testing for suspected cases with GSDs, by outsourcing the samples abroad to accredited laboratories has been a standard practice at our institute. For this purpose, the extracted DNA samples from peripheral (whole) blood specimen is outsourced for NGS based GSDs panels, with ease of transportation at room temperature.The capacity and capability to perform molecular testing using NGS is available in Pakistan. NGS equipment and proficient molecular pathologists and molecular geneticists are present at few centers locally, but molecular testing for GSDs is currently not offered in the country. In most clinical settings, the pediatricians and pediatric metabolic geneticists outsource NGS molecular testing to various accredited centers abroad, offering GSDs panels. However, the expertise of local molecular pathologists and molecular geneticists can be easily nurtured in this respect; and the widespread use of molecular testing of GSD can be achieved with its added distinct advantages.BA conceived, designed the idea and did final review of the manuscript, is responsible for integrity of research.SA did literature review and manuscript writing.


Antimicrobial resistance (AMR) is a global concern, pertaining not only to human health but also to the health of industry and the environment. AMR research has traditionally focused on genetic exchange mechanisms and abiotic environmental constraints, leaving important aspects of microbial ecology unresolved. The genetic and ecological aspects of AMR, however, not only contribute separately to the problem but also are interrelated. For example, mutualistic associations among microbes such as biofilms can both serve as a barrier to antibiotic penetration and a breeding ground for horizontal exchange of antimicrobial resistance genes (ARGs). In this review, we elucidate how species interactions promote and impede the establishment, maintenance, and spread of ARGs and indicate how management initiatives might benefit from leveraging the principles and tools of community ecology to better understand and manipulate the processes underlying AMR. As pathogens and other microbes become increasingly and more frequently resistant to antibiotics, concern is growing world-wide that the use of antibiotics for treating and preventing diseases in humans, animals, and plants is rapidly becoming less effective and unsustainable . Antimicvia horizontal gene transfer  purpose of antimicrobial resistance genes (ARGs). ARGs occur naturally in the environment, where they have been shown or posited to confer protection against toxins such as heavy metals and host-produced biocides and to perform important roles in cellular processes such as quorum sensing  and biosynthesis  that deter predators such as the amphipod Gondogeneia antarctica, as well as protozoan parasites of the genus Leishmania  and has been shown to be effective under certain circumstances , includihia coli , as doesishmania . AntibioLeptospermum scoparium relies on endophytic bacteria to produce antibiotics such as phenazine and 2,4-diacetylphloroglucinol, which inhibit infection of the plant by pathogens such as Pseudomonas syringae pv. actinidiae , these pairwise relationships do not exist in a vacuum. In the context of real-world ecological communities, mutualisms can enable predators and parasites to subdue their prey or hosts , enable tinidiae . Wicaksobacteria , bacteribacteria . Fungus-cuticles . There icuticles  and thatcuticles . In a stXenopsylla cheopis may have been the cause of AMR in Yersinia pestis strains isolated from bubonic plague patients in Madagascar.Antibiotics and ARGs associated with symbionts of macroscopic hosts seem, at first glance, unlikely to be an important source of antibiotics and ARGs in clinical, industrial, or water treatment settings. If they have shared a long co-evolutionary history with their macroscopic hosts, the symbionts will likely have lost certain functional traits that are important to the fitness of their free-living counterparts  and haveWhile using methods such as -omics to comprehensively profile microbial communities both in and out of clinical settings is still a major priority, the biological data produced through these methods must ultimately be interpreted and synthesized for there to be progress in addressing the problem. The frameworks for this synthesis currently exist within community ecology. Two that are well-established and appropriate for the task are “integrated pest management” and “ecological succession.”Integrated pest management (IPM) is a strategy designed to safeguard human health and the environment while avoiding, attenuating, or delaying pest outbreaks or associated damages . HistoriBiological control is the use of living organisms to control pests. As previously stated, microbes, including those possessing ARGs, have natural enemies such as predators and parasites. A few studies highlight the potential of using these natural enemies to control pathogens responsible for post-harvest diseases of fruits and vegetables , tree anUnfortunately, evidence is conflicting as to whether there are tradeoffs between AMR and defense against natural enemies. For example, whereas via sampling and manipulative experiments and be used to establish the host/prey specificity of putative biological control agents or select the most appropriate stage of infection or population growth at which to introduce biological control agents.Due to these concerns, biological control of pathogens, especially within humans and domesticated animals, requires forethought and the means to ensure that “ecological release” of the biological control agents and other potential adverse outcomes do not occur within or on the patients . Barrattvia apparent competition , invasion of the ecological community by exotic species, and changes in abiotic conditions due to seasonality or pollution. They can also often be autochthonous, as in the case of pioneer lichen creating soil from bare rock . Patternategists . Howeverategists  and of mategists . Throughategists .via mixed diets), and host immune responses, natural enemies, and competitors of various kinds may also be present. Furthermore, as stated previously, microbes can obtain exogenous or emergent AMR via exploitation of ARG-possessing microbes, potentially promoting Black Queen coevolutionary dynamics (see Competition subsection of Species Interactions Involving AMR). Depending on the level of impact it has on the ARG-possessing microbes, this could allow for coexistence or be analogous to successional replacement of “pioneer” and “nurse” plants (However, real-world microbial succession is typically more complicated . Within ” plants . Concept” plants  and that” plants . The lat” plants , posits ” plants  could bevia mechanistic models and experimental tests of ecological theory.AMR is a global human health concern, exacerbated by human population growth and socioeconomic inequity , climateAmong the many questions still needing to be addressed regarding AMR ecology is what impact ARGs have on the longevity and infectivity of pathogens in the environment, outside of their hosts. Although many studies have traced ARGs introduced through human activities such as the generation of wastewaters, few have examined how the microbes associated with these ARGs persist and interact with other species in their new surroundings . KnowingAB provided the premise and framework of the manuscript and contributed the bulk of the writing and literature review pertaining to ecological theory and applications. MJ, MH, NB, and SK each contributed to the background regarding molecular/genetic aspects of AMR and AMR-related exposure risks, assisted in proof-reading, and helped to finalize the overall scope of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A mixed composition of these organic and inorganic materials was electro-spun to produce composite nanofibers. Morphological investigation indicated that smooth and uniform nanofibers could be produced via this technique. The average of the nanofiber diameters was slightly increased from 190 to 265 nm with the β-TCP content but some agglomeration of particles began to impede in the fiber at a higher content of β-TCP. It was observed that the fibers were damaged at a higher content of β-TCP nanoparticles. With the presence of higher β-TCP, the wettability of the PLLA was also improved, as indicated by the water contact angle measurement from 127.3° to 118°. The crystallization in the composite decreased, as shown in the changes in glass transition (Tg) and melting temperature (Tm) by differential scanning calorimeter (DSC) and X-ray diffraction analysis. Increases in β-TCP contributed to weaker mechanical strength, from 8.5 to 5.7 MPa, due to imperfect fiber structure.A natural polymer of carboxymethyl starch (CMS) was used in combination with the inorganic mineral of β-Tricalcium Phosphate (β-TCP) and Poly  There are numerous techniques to fabricate nanofibers, including drawing, phase separations, template synthesis, self-assembly, and electrospinning ,2,3,4. I22+ ions from water [Presently, numerous types of composite nanofibers have been developed, such as synthetic–synthetic polymers ,23, natuom water . Sharma om water  derived In improving the properties, the nanofibers are not relying on a single-phase polymer but can be mixed with other polymers or composed with inorganic mineral. In the case of tissue engineering applications, cell interaction has become a major factor to scaffolds beside the structural integrity. Besides the blend between synthetic to synthetic polymers, natural polymers are starting to get greater attention as some synthetic polymers are hydrophobic. Natural polymers are becoming an alternative to improve the hydrophilicity of the scaffold. However, natural polymers have no structural integrity in their nature and are not spinnable to the nanofiber form. Ming He  has prodIn GBR applications, a number of natural polymers have being used with inorganic materials such as collagen and hydroxyapatite (HA). The combination of polymer with HA in nanofiber fabrication has shown good bioresorbability and osteoconductivity but low toughness and plasticity . ComposiIn this work, we used the natural polymer of carboxymethyl starch (CMS) derived from local sago starch with PLLA and the inorganic mineral of β-TCP. The effect of CMS on the PLLA was reported in our previous work . CMS wasl-lactide acid) with the inherent viscosity of 2.32 dL/g was obtained from BioInvigor . β-TCP nanopowder was purchased from Berkeley Advanced Biomaterials Inc. , with an average particle size of approximately 250 nm. Carboxymethyl starch was prepared from local sago starch. Ten grams of sago starch was stirred in 300 mL isopropanol  with an addition of 30 wt % NaOH in a reactor flask equipped with a reflux condenser and burette. In this work, the carboxymethylation process of sago starch was prepared as described by Yaacob et al. [l-lactide acid) solutions with 7 wt % concentration were prepared by dissolving the granule PLLA in dichloromethane by stirring the mixture for 24 h using the magnetic stirrer. CMS solution was prepared at 10 wt % concentration and then mixed with PLLA in 5% v/v of CMS content in PLLA of a total of 5 mL PLLA/CMS solution. A different ratio of β-TCP of 0.25. 0.5, 0.75, and 1 wt % were mixed to PLLA/CMS solutions. Sodium dodecyl sulfate (0.2 wt %) was added to prepare PLLA/CMS solutions to improve the homogeneity of the mixtures. The samples were ultrasonically dispersed, stirred for 72 h, and rotate in circular motion to homogenize the mixture.Poly. The fabrication routes from the sago to the composite nanofibers are summarized in The composite PLLA/CMS/β-TCP polymer solution was placed into a 1 mL syringe with a 0.6 mm diameter of a blunt needle tip. Distance between the needle and collector was set to 12 cm and connected to a high voltage source . Voltage was set to 10 kV, and the syringe pump  was placed vertically. The flow rate of the polymer jet was set to 0.006 mL/min, and the nanofiber mat was collected on an aluminum foil. The surface of fibers was characterized by using a scanning electron microscope . The images were collected at ×2000 and ×10,000 magnification at 10 kV. The fiber diameter and surface texture were analyzed and calculated by using an image J and StatGraphic Plus software  spectroscopy . The FTIR spectrum was measured in the spectral range of 400 to 4000 cm−1 of nitrogen gas was blown into the sample to avoid the oxidation during heating.Changes in thermal properties were investigated by using the differential scanning calorimeter . About 2.35 mg samples were heated from 20 to 250 °C at a heating rate of 10 °C/min. Approximately 35 mL minX-ray source was from the CuKα with the wavelength of 1.5140. The Xpert High Score Plus software was used to analyze the XRD. Fitting of the XRD curves was carried out using the Gaussian equation by Gnuplot software to determine the full wave half maximum (FWHM). The Debye–Scherrer equations were applied to determine the changes in crystallite size in samples.The crystallinity and amorphous regions were investigated using an XRD instrument . Samples were scan from 5° to 60° of 2θ angle with a step size of 0.02°/sec. The Wettability of the nanofiber surfaces was determined by water contact angle . 2 μL of distilled water was dropped on the surface of the nanofibers. 130 of WCA data points were collected in 12 s.−1 of the crosshead velocity. The tensile strength was taken as the maximum stress of the stress–strain curve.The composite PLLA/CMS/β-TCP nanofibers were formed into a rectangular shape (5 × 15 mm) with 10 mm of the gauge length for a tensile test. Samples were removed from the aluminum foil using a paper frame with double-sided tape attached to it. The frame provided additional support to the sample for handling during the testing process . TensileThe distribution of nanofiber diameter generally changed in the range of 20 to 400 nm for PLLA/CMS with the content of 0.25 to 0.75 wt % of β-TCP. The diameter distribution was increased to approximately 500 nm when the concentration was raised to 1 wt %. The distribution was shifted to the right, as shown, indicating that the diameter of nanofibers was increased with the increase in the composition of β-TCP particles. The increased formation of the cluster due to agglomeration contributes to this phenomenon. −1 that rises from the stretching of symmetry and anti-symmetry of P–O bonding, respectively. The O–P–O bond was indicated at 601 and 547 cm−1 of the absorption band. The low intensity peak of 1085 cm−1 was rising from the portion of crystal structure in the amorphous region [−1, and was attributed to the COO−Na+; whereas the absorption peaks of 2995 and to 2945 cm−1 corresponded to C–H in PLLA. Peaks at 1767, 1453, and 1383 attributed to C=O presence in PLLA. The peak of 1023 cm−1 was observed to budge at 1036 cm−1 after the addition of β-TCP into PLLA/CMS  and cold crystallization (Tcc) temperatures for composite nanofibers for two different compositions of β-TCP.Changes in composite nanofibers properties with regards to temperature were analyzed using DSC room temperature up to 250 °C. The sample without β-TCP, low and high composition of β-TCP particles were analyzed to investigate the influence of β-TCP on the composite nanofibers. Tg increased from 53 to 56 °C after 0.25 wt % of β-TCP was added to PLLA/CMS nanofibers. A slight increase in Tg indicated the movement of macromolecules of the polymer chain which was obstructed by the particles during heating. There was no significant change in Tg after more of β-TCP was added to the mixture to up to 1%, suggesting that the damage in the polymer structure due the particles increases and eases the movement of polymer chains. Gay et al. [Tg was reported to increase after HA particles were added to PLLA nanofibers. However, there were no significant changes in Tg observed by Siquiera et al. [Tg values when β-TCP particles were added in PLLA nanofibers that related to semi hydrolysis of polymer chains with the present of β-TCP. y et al.  observeda et al.  with thea et al.  reportedTm) decreased from 170 to 166 °C at the concentration of 1% of β-TCP, while there were no significant changes at lower concentrations (0.25 wt %)  b. Changetion, Xc , was fouu et al. , summariu et al.  during tX-ray diffractions spectrum (XRD) of PLLA/CMS/β-TCP composite nanofibers are depicted in The intensity of the XRD peak of PLLA/HA nanofibers also exhibited a similar pattern after the addition of the HA particle via electrospinning . Kim et 2/PMMA composite film. The hydrophilic and hydrophobic properties of the composite nanofibers were analyzed by the water contact angle (WCA). Several studies have indicated that a hydrophilic surface encourages cell adhesion compared to a hydrophobic surface . There wA small change in WCA of PLLA/CMS/β-TCP was related to the existence of a high porosity that caused macroscopic roughness at the surface. Changes in surface macroscopic level at different scales impede the improvement to a hydrophilic surface. The β-TCP particles in nanoscale also contribute to the increase in the surface roughness of the composite nanofibers, thus reducing the hydrophilic properties.Mechanical strength of the composite nanofibers was measured by the tensile strength test. Generally, the mechanical strength of composite nanofibers decrease with the increase in the β-TCP concentration. There was a slight increase in tensile strength at 0.25% of β-TCP concentration and a decrease from 8.5 down to 5.7 MPa after increasing the content to up to 1%. At lower content, the β-TCP acts as a barrier in molecular movement during the tensile test which results in slight increase a. The weSeveral observations have reported the relationship of a decrease in tensile strength in nanofibers to the presence of particles in the polymeric structure of either HA or TCP particles ,61. HoweIn this study, we prepared and optimized a composite of synthetic–natural–inorganic mineral nanofibers with the potential to be applied as biomedical membranes, such as guided bone regeneration (GBR) membranes. The composite nanofiber mat was successfully electrospun to uniform non-woven nanofibers. The combination of natural polymers and β-TCP mineral improved the hydrophilic behavior of the PLLA but a higher concentration of the β-TCP led to decreases in mechanical strength and an increment in fiber size. Agglomeration may restrain the performance of the uniform and smooth nanofibers. In comparison to the properties of other research works in the area of nanofibers that have undergone cell studies, overall, the engineered electrospun nanofibers prepared in this works are promising candidates for bone tissue engineering applications such as the GBR membrane. Based on the presented work, this research topic has a great potential to be further investigated for degradation behavior, cell and materials interactions, and in vivo studies towards the bone tissue engineering applications.


Insufficient purification and incomplete characterization pose a serious problem for attributing photoluminescence properties to carbogenic nanodots, especially those synthesized by bottom-up approaches. Here, we provide a roadmap for the successful future of these nanodots. Recent research suggests that the fluorescence associated with CNDs may originate significantly from molecular fluorophores and/or their aggregates, quasi CNDs (molecular fluorophores attached to the core of CNDs) or polymer dots. These are produced as by-products , because of their superior optical properties, high photostability, quantum yield, aqueous solubility and low toxicity, have tremendous applications in optoelectronics, light harvesting, photovoltaics, drug delivery, bioimaging, catalysis and sensors. CNDs are very small in size  and show excitation dependent photoluminescence (i.e. the emission maximum peak shifts with the excitation wavelength). While the real chemical structure and the corresponding photoluminescence mechanism of these CNDs are still elusive, recent reports on bottom-up-synthesized CNDs pose another critical issue2 and showed that an organic molecular fluorophore  was actually responsible for the origin of fluorescence in the as-synthesized CNDs solution4. The above conclusion was drawn when they were able to synthesize the same fluorophore separately by reacting the same precursor molecules but in slightly milder experimental conditions. The fluorophore was purified and its chemical structure was confirmed by high resolution mass spectrometry (HRMS), nuclear magnetic resonance (NMR) and single crystal X-ray diffraction (XRD). Interestingly, all of the optical properties of this fluorophore, such as UV-VIS absorption, photoluminescence, quantum yield (QY) and fluorescence lifetime, were found to be similar to those of the as-synthesized CNDs solution. In addition, the excitation dependent photoluminescence typical of CNDs was neither present in the fluorophore nor in the CNDs solution. Finally, the authors confirmed the presence of large quantities of the fluorophore in the CNDs solution by dialyzing the solution using a 1000 Dalton molecular weight cut off filter and analyzing the optical properties of the dialysate and retentate. Interestingly, the dialysate, which contained the fluorophore, showed the same optical behavior as that of the as-synthesized CNDs solution, including UV-VIS absorption spectrum, high fluorescence QY, fluorescence excitation and emission spectra as well as excitation independent fluorescence. Meanwhile, the retentate, which contained the actual CNDs, displayed characteristic excitation dependent photoluminescence but with a very low QY (0.038%). The dominant peak in the HRMS spectrum was also attributed to the fluorophore. The authors concluded that the amount of real CNDs (with excitation dependent photoluminescence) produced in the synthesized solution was very small.For example, Shi et al. synthesized CNDs using citric acid and L-cysteine as the precursor molecules by a typical hydrothermal method5 synthesized by the hydrothermal method using citric acid and ethylenediamine as the precursor molecules. They proposed the formation of the fluorophore (with high QY and excitation independent photoluminescence) at a lower temperature, followed by the formation of polymer dots at a moderate temperature, and finally the formation of carbon cores (with very low QY and excitation dependent photoluminescence) at a higher temperature. They performed the dialysis and liquid chromatography-mass spectrometric (LC-MS) purification of the synthesized sample to confirm the presence of the small molecular fluorophore. The partial conversion from low molecular weight fluorophore to the π-conjugated carbon core with increasing temperature suggested that the unconverted fluorophore may bind to the larger π-conjugated domains to give rise to so-called quasi CNDs (CNDs coated with molecular fluorophores). This observation was supported by a few other recently studied CNDs systems8.Song et al. identified a different molecular fluorophore in a CNDs solution9. The aggregated structure had the same topology and size as that of real reported CNDs. Reckmeier et al. showed that supposed CNDs synthesized by the hydrothermal treatment of ammonia and citric acid were actually the amorphous aggregates of the derivatives of citrazinic acid (a molecular fluorophore)10. A concentration-dependent aggregation of citrazinic acid was observed by ageing the solution at ambient conditions. With an increase in the concentration of citrazinic acid, the photoluminescence of the solution changed from excitation independent to excitation dependent. Interestingly the measured particle size (4.5 nm) of the aggregated structure using transmission electron microscopy (TEM) was found to be similar to that of real CNDs. On the other hand, Khan et al. reported the molecular fluorophore as a sole product during CNDs synthesis using only citric acid as precursor molecule in a hydrothermal synthesis11. The fluorophore underwent drying mediated hydrogen-bonded crystalline aggregation to show CND-like properties. They identified the fluorophore by solving the single crystal XRD and by measuring the diffusion coefficient using fluorescence correlation spectroscopy (FCS).On a different note, several evidences suggest that presumed CNDs are actually aggregates of molecular fluorophores, either in ordered crystalline or amorphous form. For example, by performing HRMS analysis of the gel permeation chromatography-purified fluorophore that was obtained as a byproduct in CNDs synthesis in a hydrothermal method using citric acid and ethlylenetriamine, Zhang et al. concluded that the supramolecular aggregation of the fluorophores via hydrogen bonding showed excitation independent fluorescence with high QY5. Nevertheless, dialysis alone may not be a satisfactory method of purification, as it separates only a certain fraction of molecular weights that may contain different species within that cutoff range. Liquid chromatography purification, especially high performance liquid chromatography (HPLC) and gel permeation chromatography (GPC), which separate each component in a sample mixture based on polarity or size/molecular weight difference, will be extremely beneficial. As a result, the molecular fluorophore and its aggregated structure, polymer dots, and quasi CNDs can easily be separated from actual CNDs as ultrapure components.It is clear from the above reports that molecular fluorophores are often synthesized either as a sole product or in parallel to CNDs in the bottom-up hydrothermal synthesis. The synthesized molecular fluorophore, as an impurity, can contribute extensively to the fluorescence properties of the synthesized CNDs solution. As a result, it is extremely important to conduct a thorough purification and characterization to avoid any erroneous conclusions in CNDs research. The CNDs or quasi CNDs, considering their large mass difference from the small organic fluorophore molecule , can be separated easily using 1–3.5 kDa dialysis membrane. The similarity in the optical properties and the weight percentage of the dialysate (containing molecular fluorophore) with the retentate (containing CNDs) will provide insight into the contribution of the fluorophore to the optical and chemical properties of the as-synthesized CNDs14. Table 13. Raman spectroscopy, a non-destructive versatile technique, has become the benchmark to identify the nature of graphene based materials15. Different forms of graphene have been identified by the well-known D, G and 2D bands. The D band (~1360 cm−1) characterizes the defect or the amorphous nature, the G band (~1580 cm−1) characterizes the in-plane vibration of the sp2 graphitic carbon, and the 2D band (~2680 cm−1) corresponds to the double resonance band. For a crystalline multilayered graphitic carbon core, in general one should not observe a D band; rather, an intense G band and a broad envelop of 2D band will be observed with the intensity ratio of the G/2D as 1:416. Considering the edge defects created by the attachment of the functional groups, it is not surprising to observe a lesser intense D band (compared to G band). On the other hand, neither the interlayer spacing nor lattice fringes will be observed for an amorphous carbon core. In addition, a broad peak at Bragg’s angle (2θ) in XRD and a broad defective band in Raman spectroscopy will be observed for the amorphous CNDs core structure.After complete separation of the individual species, the characterization of the proposed CNDs can be done by performing high resolution transmission electron microscopy (HR-TEM) and analysing its selected area electron diffraction (SAED) pattern, XRD, Raman spectroscopy and atomic force microscopy (AFM). The CNDs’ core, by its definition, can be both single crystalline multilayered graphitic carbon (with an interlayer spacing of ~0.34 nm for the 002-plane and 0.21 nm for the 001-plane) or it may consist of amorphous carbon17. Considering the fact that the organic fluorophore will be volatile and have a much lower melting temperature than the graphitic core, the fluorophore will be destroyed (without disturbing the core) at its melting temperature and the total mass loss of the sample divided by the mass of the fluorophore will provide the number of fluorophores attached to the core in quasi CNDs. On the other hand, although there might be slight differences in either TEM or Raman spectroscopy data for aggregated molecular fluorophores, it will be difficult to distinguish them from CNDs. TGA again can be useful to differentiate between aggregated fluorophores and CNDs. It is obvious that the aggregated structure will have less thermal stability than the graphitic core in real CNDs and hence will show the mass loss at a lower temperature than CNDs core. X-ray photoelectron spectroscopy (XPS) can provide information about the surface functional groups and hence can distinguish the surface composition of CNDs, quasi CNDs and aggregated molecular fluorophores18. Finally, quantitative information about the contribution of the fluorophore in the optical properties in quasi CNDs can be obtained by in situ temperature dependent  photoluminescence measurements using confocal Raman spectroscopy19. The destruction of the fluorophore of the quasi CNDs will decrease the photoluminescence substantially. At a later stage, when all the fluorophore moieties are destroyed, the remaining photoluminescence should originate only from the core structure of the CNDs. On the other hand, complete quenching of fluorescence should be observed for the aggregated molecular fluorophore.It will be difficult to distinguish between CNDs and quasi CNDs by the above characterization as both of them contain similar carbon cores. However, the number of fluorophore units in quasi CNDs can be quantitatively obtained by thermogravimetric analysis (TGA), which works on the principle of mass loss with increasing temperatureWe hope that the above discussion will provide some guidance for the identification and characterization of CNDs and their associated photoluminescence properties in the bottom-up-synthesized complex products. It can be concluded that sufficient and adequate purification and characterization of the synthesized complex products are extremely crucial for the safe and successful future of the CNDs. While we discussed the key aspects of a few reported bottom-up hydrothermally synthesized CNDs, other hydrothermal or bottom-up syntheses using saccharides , food waste, biomass, or other reagents still must be verified.


The purpose of this case report is to describe a conjunctiva and eyelid Kaposi’s sarcoma (KS) as the initial manifestation of acquired immunodeficiency syndrome (AIDS), which led to the diagnosis of HIV infection. There are only 3 reported cases of ocular KS as an initial manifestation of HIV infection.3 and viral RNA load 427,000/ml. Further systemic evaluation showed a diffuse sarcoma.A 32-year old white man presented to our department with a 1 month history of eye redness. The patient had an enlarged violet-coloured mass on the right superior eyelid which had evolved over the course of 1 week. There was also a mobile bulbar conjunctival lesion with a bright red colour, approximately 5 mm × 5 mm, in the superior temporal quadrant of his left eye. The lesions looked like a chalazion and a subconjunctival haemorrhage, respectivly. Presumed KS diagnosis was confirmed with HIV-1 positive testing and histopathology from tissue biopsy. The patient’s CD4 count was 23/mmThis case report demonstrates the importance of recognizing the ocular manifestations of AIDS in establishing the correct diagnosis of KS and subsequently diagnosing occult HIV infection. Although ocular KS as the initial manifestation of HIV-AIDS is an extremely rare event, a proper diagnosis may contribute to prompt management with personal and social relevance. Kaposi’s sarcoma (KS) is the most common tumour in patients with the human immunodeficiency virus (HIV) infection and fully developed acquired immunodeficiency syndrome (AIDS). KS is a multifocal systemic disease related to the human herpes-virus 8 infection, found in patients with a low CD4 cell count: less than 500cell/μl, typically below 200. However, ocular involvement of this endothelial malignant tumour reported as AIDS-defining illness, is an exceptionally rare event , 2.In the literature, there is no evidence-based algorithm for the treatment of ocular KS . Neverth3 and the viral RNA load of 427,000/ml. Pathologic examination confirmed KS diagnosis of both lesions. Postoperative evaluations were uneventful and no signs of recurrence were noticed during the 6-month follow-up.A 32-year old white man presented to our department with a 1 month history of eye redness (left eye). He had already been observed by a general practitioner who advised the patient to seek ophthalmological advice if the lesion would not resolve within 3 weeks. In the first ophthalmic evaluation, the patient presented with 2 ocular lesions. These were best observed in biomicroscopy. There was an enlarged violet-coloured mass on the right superior eyelid which had evolved over the course of 1 week (Fig. Further multiple disciplinary assessments showed a diffuse KS with skin and supraglottis involvement. The patient was offered treatment for both AIDS and diffused KS. In order to achieve disease control, patient initiated systemic antiretroviral therapy – HAART - and systemic chemotherapy under medical supervision with regression of the tumour.Kaposi’s sarcoma is the most common neoplasm in AIDS patients , 2. HoweFortunately, after HAART introduction in 1997, HIV-AIDS patients with KS are not seen as often in occidental societies . This unThis case report shows the importance of accurately identifying AIDS ocular involvement. The knowledge about ocular lesions in AIDS led to the correct diagnosis of KS and subsequently the identification of occult HIV infection. Although ocular KS as the first clinical sign of HIV-AIDS is an extremely rare event, a prompt diagnosis can lead to a vital intervention in the patient’s own health and utmost social relevance.


Levels of desmethylimipramine-insensitive mazindol-sensitive [3H]mazindol binding were significantly higher in BA 10 from patients with schizophrenia (p = 0.004) and significantly lower in the dorsal striatum  from those with the disorder. There were no differences in levels of desmethylimipramine-insensitive [3H]mazindol binding in BA 17 or nucleus accumbens. These data raise the possibility that high levels of DAT in BA 10 could be contributing to lower synaptic cortical dopamine, whereas lower levels of DAT could be contributing to a hyperdopaminergic state in the dorsal striatum.The dopamine hypothesis proposes that there is a hypodopaminergic state in the prefrontal cortex and a hyperdopaminergic state in the striatum of patients with schizophrenia. Evidence suggests the hyperdopaminergic state in the striatum is due to synaptic dopamine elevation, particularly in the dorsal striatum. However, the molecular mechanisms causing disrupted dopaminergic function in schizophrenia remains unclear. We postulated that the dopamine transporter (DAT), which regulates intra-synaptic dopamine concentrations by transporting dopamine from the synaptic cleft into the pre-synaptic neuron, could be involved in dopaminergic dysfunction in schizophrenia. Therefore, we measured levels of DAT in the cortex and striatum from patients with schizophrenia and controls using postmortem human brain tissue. Levels of desmethylimipramine-insensitive mazindol-sensitive [ Significantly, the molecular basis of schizophrenia has yet to be fully elucidated but one long-standing proposal is the dopamine hypothesis, which posits there is a hyperdopaminergic state in the mesolimbic system that is critical in generating the positive symptoms of schizophrenia.2 In addition, there is a hypodopaminergic state in the mesocortical pathway, which is thought to contribute to negative and cognitive symptoms.Schizophrenia has a wide range of symptoms that includes hallucinations, delusions, disorganized thinking, anhedonia, and apathy.123I](S)-(-)-3-iodo-2-hydroxy-6-methoxy-N-[(1-ethyl-2-pyrrolidinyl) methyl]benzamide ([123I]IBZM) binding to the dopamine D2 receptor as an indirect measure of dopamine release following the administration of amphetamine, reported increased levels of dopamine release in patients with schizophrenia.4 This hypothesis has been supported by a meta-analysis5 and the increase in dopamine release in schizophrenia is of a relatively high effect size.6 Another study used positron emission tomography (PET) and 3,4-dihydroxy-6-[(18)F]fluoro-l-phenylalanine ((18)F-DOPA) to measure dopamine synthesis capacity to show that there is an elevated dopamine synthesis capacity in the striatum, and in particular the associative but not the limbic striatum, in those at ultra-high risk for psychosis.7 These data suggested the associate striatum as a key region for dopaminergic dysregulation very early in the onset of the disorder. In a follow-up study it was shown that individuals that developed a psychotic disorder had the greatest dopamine synthesis capacity in the associative striatum and that there was a positive correlation between dopamine synthesis capacity and symptom severity in those that developed a psychotic disorder.8 These data supported the notion of a hyperdopaminergic state in the striatum as being important in the genesis of psychotic symptoms in schizophrenia. Another PET neuroimaging study used carbon 11-labeled FLB 457 binding to dopamine D2-like receptors9 after an amphetamine challenge to show a reduced release of dopamine in the dorsolateral prefrontal cortex in patients with schizophrenia.10 These data support the proposition of a hypodopaminergic state in the cortex in patients with the disorder.A role for a hyperdopaminergic state in the mesolimbic system is supported by data from neuroimaging studies. Thus, studies using single photon emission computerized tomography, which took the reduction mazindol binding and autoradiography, in the frontal pole (BA 10) and the striatum from patients with schizophrenia. We also measured levels of DAT in the visual cortex (BA 17) as a negative control as this is a cortical region involved in the pathophysiology of schizophrenia28 but there is no strong evidence for disrupted cortical neurotransmission in that cortical region.29 We chose to use [3H]mazindol, rather than another radioligand that would bind to DAT, so we could more readily relate our findings to our previous studies on DAT in schizophrenia in which we used that radioligand.18 The outcome of our cortical studies would be of added interest because our expression microarray experiments lacked the sensitivity to detected DAT mRNA.Given the important role of DAT in maintaining dopamine homeostasis, the anatomical complexity of the striatum and our recent data showing abundant changes in gene expression in the frontal pole from patients with schizophrenia,There were no statistically significant differences in age, gender frequency, PMI or CNS pH with diagnoses Table .Table 1A3H]mazindol binding was shown to have a normal distribution  and, compared to controls, the level of desmethylimipramine-insensitive mazindol-sensitive [3H]mazindol binding was higher in BA 10 , but not BA 17 , from the patients with schizophrenia . There were no correlations between the binding of desmethylimipramine-insensitive mazindol-sensitive [3H]mazindol in BA 10 with that in BA 17, dorsal caudate or nucleus accumbens but there was a weak correlation between desmethylimipramine-insensitive mazindol-sensitive in BA 10 and the dorsal putamen  except for the data from the dorsal putamen from patients with schizophrenia, which was not normally distributed . Given the overwhelming amount of our data was normally distributed we decided to analyze all data using parametric statistics. Thus, striatal desmethylimipramine-insensitive mazindol-sensitive [3H]mazindol binding was lower in the dorsal putamen , dorsal caudate,  but not the nucleus accumbens , from patients with schizophrenia  and that, whilst the levels of desmethylimipramine-insensitive mazindol-sensitive [3H]mazindol in nucleus accumbens between patients with schizophrenia and controls did differ (ANCOVA p < 0.048), this difference did not survive a Bonferroni correction. By contrast, whilst FRADD (ANCOVA p = 0.001) and LEAP (ANCOVA p < 0.001) were both significant covariates, the difference between desmethylimipramine-insensitive mazindol-sensitive [3H]mazindol in BA 10 from subjects with schizophrenia remained significantly different from that in controls (ANCOVA p < 0.0001 for both FRADD and LEAP), a level of significance that survived Bonferroni correction. Notably, these data add to the notion that, in small cohorts where relationship are measured using linear regression, relationships where r2 < 0.49 does not warrant a secondary analysis.Exploring data for potential confounds, it was notable that correlations between desmethylimipramine-insensitive mazindol-sensitive Hmazindolols Fig. , as wellADD Fig. , as wellADD Fig.  approach30The liquid intake by all groups of rats was consistent with their receiving the expected daily dose of antipsychotic drugs administered in drinking water.3H]mazindol binding to the frontal cortex of rats treated with varying doses of haloperidol for 3 months , as well as lifetime exposure to antipsychotic drugs (LEAP) and DAT in BA 10. Thus, it is possible that differences in levels of DAT between patients with schizophrenia and controls could be greater in BA 10 from medication naïve individuals, a notion supported by an increase in the significance in the difference between desmethylimipramine-insensitive mazindol-sensitive [3H]mazindol binding in the striatum of rats treated with 0.01 mg/kg/day, but not higher doses, of haloperidol. Our findings have some similarities with an earlier study that found no change in the levels of [3H]GBR 12,935 to the dopamine transporter in the striatum of rats treated with haloperidol at 1 mg/kg/day for 21 days,42 and a study that showed no changes in [125I]RTI-121 binding to the striatal dopamine transporter after treating rats with either 1 mg/kg haloperidol or 20 mg/kg clozapine per day.43 These data are consistent with the hypothesis that treating rats with “clinically equivalent” doses of haloperidol44 does not affect level of radioligand binding to the dopamine transporter in the striatum. Notably, in our study, the dose of haloperidol that lowered levels of DAT in rat striatum would be viewed as being equivalent to a sub-clinical dose in humans46 and therefore may not be relevant to doses received by the patients with schizophrenia in this study. This posit is supported by an imaging study which reported no change in DAT in patients with schizophrenia after 6 months of treatment with haloperidol,47 and another study that showed existing lower striatal DAT levels in patients with schizophrenia did not change after 4-week treatment with antipsychotic drugs.48 Thus, overall current data from rat studies suggests that antipsychotic drug treatment is not likely to be a major confound in our study. However, an in vivo electrophysiology study has reported that acute treatment with 10 µM haloperidol, but not clozapine, reduced striatal dopamine uptake in the striatum.49 In addition, studies using synaptosomes from rodent striatum have reported that metabolites of haloperidol, but not haloperidol itself, can inhibit dopamine uptake.51 Thus, whilst treatment with drugs such as haloperidol may not alter levels of the dopamine transporter, it could alter levels of dopamine uptake by the transporter.In this study, we also reported higher levels of desmethylimipramine-insensitive mazindol-sensitive mazindol binding in schizophrenia.18All human tissues were sourced through the Victorian Brain Bank Network, the Florey Institute for Neuroscience and Mental Health, Australia. Tissue, from the left hemisphere, was collected from BA 10 , BA 17  and striatum from 15 patients with schizophrenia and 15 individuals with no history of psychiatric or neurological illness (controls) (Table 30 groups of 6-week-old Sprague-Dawley rats (n = 5–7 per group) were treated with vehicle , haloperidol  or thioridazine  for 3 months in drinking water. From the recorded consumption of water per cage and the number of rats per cage, it was calculated that the dose of drug received by each rat would have been within 10% of the target daily intake. These two antipsychotics were used because they have different affinity to dopamine D2 receptor,63 and the majority of patients from whom tissue was collected for this study had been treated with first generation antipsychotic drugs or the second generation antipsychotic drug, risperidone, both of which have a limited selective pharmacology, which includes a high affinity for the dopamine D2-like receptors.64 Rats were euthanized by sodium pentobarbitone overdose (1.5 ml i.p. injection of 60 mg/ml solution) and brains were rapidly collected, frozen, and stored at −80 °C until required.Using a well-described protocol,65). All sections were thaw mounted on to gelatinized slides. Prior to being incubated with the radioligand, tissue sections were incubated in 50 mM Tris buffer containing 300 mM NaCl, 5 mM KCl for 60 min at 4 °C.66Using a cryostat, 20 µm frozen sections were cut from human BA 10, BA 17, and striatum. In addition, rodent CNS was sectioned to obtain frozen sections with frontal cortex and striatum  binding to DAT (TB: three sections) was taken as the binding of radioligand in the presence of 0.3 µM desmethylimipramine (DMI), a drug concentration which completely displaces [3H]mazindol binding from the noradrenaline31 and the serotonin32 transporter. The difference between the binding of [3H]mazindol in the presence of DMI and in the absence and presence of mazindol (10–6 M)  was taken as specific binding to DAT.33 After exposure to radioligand, all sections were washed twice in ice-cold assay buffer, dipped into ice-cold distilled water, and then dried thoroughly. The sections where then partially post-fixed overnight in paraformaldehyde fumes before being apposed to a BAS-TR2025 plate  with [3H]microscalesTM  for 7 days. All plates were scanned in a BAS5000 high-resolution phosphoimager . The resulting images were analyzed using AIS imaging software .Total [To measure the intensity of radioligand binding in the cortex, we took an integrated measure of the intensity of radioligand binding across Lamina I–VI, as binding was homogenous across those laminae. However, because the border between gray and white matter was not clearly visible due to the low intensity of DAT in prefrontal cortex Fig. , the cytFor Klüver–Barrera staining, sections were fixed in 10% formalin in phosphate-buffered saline for 1 h at room temperature. Sections were immersed in 95% ethanol to hydrate and subsequently in 0.1% Luxol Fast Blue overnight at 37 °C and then rinsed with 95% ethanol. After rinsing in distilled water, the slides were alternately immersed in 0.05% lithium carbonate for 5–20 s, and in 70% ethanol for 1–2 min to differentiate the stain until the borderline was clear and white matter was sharply defined.Sections were counterstained with 0.1% Cresyl Violet for 20 min at 37 °C and rinsed in 70% ethanol and 95% ethanol, then dehydrated through two changes of xylene each for 2 min. Sections were mounted in DPX and imaged using a light microscope with a digital camera attached.67 For normally distributed experimental data, outliers were identified using the Grubb’s test which allows the exclusion of a single outlier per data set.68First, all groups of data were interrogated with the d’Agostino and Pearson omnibus normality test as this is best at determining data distribution in small cohorts.t-tests were performed to identify variation in data between measures, with p < 0.05 being taken as indicating a significant variation. Gender frequency was compared using the Fisher’s exact test. Cohen’s d was calculate at https://www.socscistatistics.com/effectsize/default3.aspx.For normally distributed data, Student’s p < 0.05 was considered significant.The low number of animals in our rat study meant it was not possible to accurately assess the distribution of the data. Therefore, an ANOVA was used to identify significant variance and a post hoc Bonferroni comparing all drug doses within CNS region and correcting for multiple comparisons was used to identify the source of any variance; p < 0.05. Such an outcome was taken as showing some covariance between two factors, which could require a secondary analysis of variance designed to assess the impact of covariance. Next, the percentage of the variance described by the linear regression line, as shown by the Pearson’s product–moment, was assessed. Notably, there are now significant data showing that in studies such as this that have relatively small cohort size relationships between variables, where r2 that does not exceed 0.49 does not impact on the outcome of a primary analysis of variance.70 Therefore, in this study only relationships between variables were r2 > 0.49 that would be included in an ANCOVA to determine if relationships were impacting on the primary analyses of variance.Relationships between experimental data and between experimental data and demographic, pharmacological and CNS collection data were identified using linear regressions. A linear regression was accepted as deviating from the horizontal when When data were not normally distributed, equivalent non-parametric tests would be used with no attempt being made to identify outliers.All analyses were conducted using GraphPad Prism, except for ANCOVAs, which were conducted using Minitab 18.Further information on research design is available in the Reporting SummarySupplementary Infomation