Poecilia sphenops), using immunohistochemistry and transmission electron microscopy (TEM). The hepatocytes’ sinusoidal structures of molly fish livers had taken two different forms; cord-like and tubular, while the biliary tract system showed two different types: isolated and biliary venous tract. The TEM showed that the hepatocytes possessed well-developed cytoplasmic organelles and numerous glycogen and lipid droplets of different sizes. Kupffer cells, Ito cells, aggregation of intrahepatic macrophages and melanomacrophages were also recognized. Melanomacrophages contained numerous phagosomes, many lysosomes, cytoplasmic vacuoles, and melanin pigments. Hepatocytes and Kupffer cells expressed immunoreactivity to APG5, indicating that these cells were involved in the process of autophagy. Telocytes (TCs) were also recognized in the liver of molly fish, and they shared the same morphological characteristics as those in mammals. However, TCs expressed strong immunoreactivity to APG5, TGF-β, and Nrf2, suggesting their possible role in cellular differentiation and regeneration, in addition to phagocytosis and autophagy. Both IL-1β and NF-KB showed immunoreactivity in the hepatocytes and in inflammatory cells (including intrahepatic macrophages and melanomacrophage center). Nrf2 and SOX9 showed immunoreactivity in hepatocytes, stem cells, and macrophages. The present study showed the spatial distribution of hepatic vascular-biliary tracts in molly fish. The liver of molly fish has unique functions in phagocytosis, autophagy, and cell regeneration. The expression of APG5 in hepatocytes, Kupffer cells, melanomacrophages, and telocytes supports the role of the liver in lymphocyte development and proliferation. The expression of TGF-β and NF-κB in hepatocytes, Kupffer cells, telocytes, and macrophages suggests the role of the liver in regulation of cell proliferation and immune response suppression. The expression of IL-1β and Sox9 in macrophages and melanomacrophages suggests the role of the liver in regulation of both innate and adaptive immunity, cell proliferation and apoptosis, in addition to stem cell maintenance.The liver of fish is considered an ideal model for studying the collaboration between environmental agents and the health state of the fish, where it gives good indications about aquatic ecosystem status. Therefore, this study presented immune roles for the liver in molly fish ( Poecilia sphenops  is a freshwater species of fish that is commonly named molly fish. They are natural inhabitants in the freshwater streams and the coastal brackish water of Mexico and Colombia. They mostly occur in swarms below the fluctuating vegetation as they feed principally on algae and other herbal resources. The mollies rank as one of the most popular feeder fish due to their high growth rate, birth size, reproduction, and brood number [d number .The liver plays a crucial role in several vital processes in the living body, mainly those related to its metabolism  that assume a dominant role in maintaining life, in addition to controlling some definite digestive processes , 4. MoreThe liver is known as an immunologically complex organ that contains huge, various populations of immune cells. These cells are responsible for the production of acute phase proteins, cytokines, and chemokines , 7. MoreIn general, fish species present two liver forms: either associated with pancreatic tissue (hepatopancreas) or not , 10. TheMany studies have been carried out on the fish liver as it is considered an ideal model for studying the collaboration between environmental agents and the health state of the fish, where it gives good indications about aquatic ecosystem status , 12. AltThe current work was performed in accordance with the Egyptian laws and University guidelines for animal care. The National Ethical Committee of the Faculty of Veterinary Medicine, Assiut University, Egypt, has approved all the procedures in the present study. The Ethical No is aun/vet/4/0015.Poecilia sphenops) (10 specimens for immunohistochemistry and eight specimens for electron microscopy examination). The fish were purchased from an ornamental fish shop. The specimens were 4.20 ± 4.0 cm in standard length and 10.60 ± 1.70 gm in body weight. Healthy fish were acclimated in the laboratory for 2 weeks in aerated water tanks with a natural light/dark cycle. The swimming and feeding behavior for all studied fish were observed. Fish were randomly collected from the tanks and euthanized with an overdose of MS-222 before tissue sampling.The materials employed in this study consisted of 18 randomly obtained adult male specimens of molly fish  for 24 h. The fixed materials were dehydrated in an ascending series of ethanol, cleared in methyl benzoate, and then embedded in paraffin wax. Tissue specimens were transverse sectioned at 3–5 μm thickness. These sections were prepared for immunohistochemical analysis using a Pierce Peroxidase Detection Kit . The sections were deparaffinized with xylene, rehydrated in graded ethanol, and washed with distilled water. The sections were heated for 15 min in sodium citrate buffer  in a microwave to increase epitope exposure. The sections were cooled at room temperature for 30 min, washed with wash buffer , and then were incubated for 30 min in peroxidase suppressor to quench endogenous peroxidase activity. The tissues were washed two times for 3 min with wash buffer and were blocked with universal blocker™ blocking buffer in TBS for 30 min at room temperature. The sections were incubated overnight at 4 °C with the following diluted (1:100) primary antibodies that showed reactivity in fish species: a rabbit polyclonal anti-autophagy protein 5 (APG5 or ATG5) , monoclonal anti-mouse TGF-β , a rabbit polyclonal anti-nuclear factor kappa B (NF-κB) , a rabbit polyclonal anti-nuclear factor erythroid 2-related factor 2 (Nrf2) , anti-Interleukin 1 beta (IL-1β) , and a goat polyclonal anti-SRY-Box transcription factor 9 (Sox9) . The slides were washed two times for 3 min with wash buffer and were incubated with diluted (1:1000) goat anti-rabbit IgG  or diluted (1:100) goat anti-mouse IgG  secondary antibodies for 30 min at room temperature. Following that, the slides were washed three times for 3 min each with wash buffer, and the tissues were incubated with the diluted (1:500) Avidin-HRP  in universal blocker blocking buffer for 30 min. The slides were then washed three times for 3 min each with wash buffer. The tissues were incubated with 1X metal enhanced DAB substrate working solution  for 5–15 min until the desired staining was achieved. Finally, the sections were washed two times for 3 min each with wash buffer, counterstained with Harris modified Hematoxylin, and mounted with mounting media.Small specimens of the molly fish liver were fixed in a solution of 2.5% paraformaldehyde- glutaraldehyde and left overnight for fixation . Then thTo increase the visual contrast between several structures in the same electron micrograph, we digitally colored specific elements as hepatocytes, Ito cells, macrophages, etc., to make them more visible for the readers. All the elements were carefully hand colored using Adobe Photoshop software version 6.The liver of molly fish appeared as a compound organ in the form of hepatopancreas. The hepatic parenchyma was not organized into lobules and was composed of hepatocytes interrupted with connective tissue that enclosed the hepatic vascular and biliary components Fig. A. The paThe hepatocytes sinusoidal structures of molly fish livers showed two different forms: cord-like and tubular. In the cord-like form, many hepatocytes were arranged in simple layers and the hepatic sinusoids were relatively straight and enlarged Fig. C, D. In According to the spatial distribution of vascular and biliary components, the biliary tract system in molly fish showed two different types: isolated and biliary venous tract (BVT). In the isolated type, the bile ductules were found independently within the hepatic parenchyma and were enclosed by connective tissue sheath Fig. A, B. In The intrahepatic bile ductules were lined by a single layer of high cuboidal or columnar cells that was enclosed by a thin layer of connective tissue followed by a thin layer of circularly arranged smooth muscle fibers. The duct cells showed a clearly distinct brush border Fig. B, D, F.Hepatocytes appeared as large polygonal cells with a distinct cell membrane. They contained large spherical euchromatic nuclei with distinct nucleoli. The cytoplasm of hepatocytes showed a wide variation depending on the functional activity. Some hepatocytes showed vacuolated cytoplasm containing abundant glycogen and lipid droplets Fig. A; meanwhThe hepatocytes showed a moderate to strong immunoreactivity to APG5 in the liver of molly fish Fig. A–C, whilBoth IL-1 β and NF-κB showed immunoreactivity in the hepatocytes and in inflammatory cells (including intrahepatic macrophages and melanomacrophage center) Fig. A–D. FurtHepatocytes appeared as large polygonal cells with a single large spherical euchromatic nuclei. Their cytoplasm contained abundant filamentous mitochondria, well-developed smooth endoplasmic reticulum and rough endoplasmic reticulum, many lysosomes, well-prominent Golgi complex, and numerous glycogen and lipid droplets of different sizes Fig. A, B. TypThese cells appeared as huge cells that were characterized by a large eccentric nucleus and numerous cytoplasmic processes. Their cytoplasm exhibited numerous phagocytic vacuoles, melanin-like granules, and large dense bodies containing heterogeneous material that represented phagosomes Fig. A.These cells were recognized between hepatocytes and presented numerous cytoplasmic processes. They contained large euchromatic nuclei showing clumps of heterochromatin. Their cytoplasm possessed numerous lipid droplets Figs.  A, 7B.They appeared as large stellate cells. They showed large heterochromatic nuclei and many cytoplasmic processes that projected into the lumen of hepatic sinusoids Figs. D and 7C.Bile canaliculi appeared as minute channels that were formed by the membranes of adjacent hepatocytes. Numerous microvilli were projected into the lumen of bile canaliculi Fig. C. The waThe current study revealed that the liver of molly fish appeared as a compound organ in the form of hepatopancreas. The hepatic parenchyma was not organized into lobules and was composed of continuous fields of hepatocytes interrupted by islands of connective tissue that enclosed the hepatic vascular and biliary components. These results agreed with those of Rocha et al. , 10 who The present study showed that the hepatocyte-sinusoidal structures of molly fish livers had taken two different forms: cord-like form and tubular form. Akiyoshi and Inoue  reportedBile is synthesized by hepatocytes and streams through the intrahepatic bile canaliculi, bile ductules, and bile ducts . The bilCtenopharyngodon idella and Atlantic salmon [Hepatocytes are the chief parenchymal cells in the liver, and they perform very essential roles in metabolism, detoxification, and protein synthesis , 17. Hepc salmon , 9, 11.An interesting finding in this study is the expression of APG5 in hepatocytes, Kupffer cells, melanomacrophages, and telocytes, indicating the involvement of these cells in the process of autophagy in the liver of molly fish. Autophagy is a highly synchronized biological process that is associated with stress adaptation induced by nutritional or trophic deficiencies to preserve cellular hemostasis by reprocessing the damaged organelles. APG5 is one of the essential players of the autophagy process , and it Another interesting finding in this study is the expression of TGF-β in hepatocytes, telocytes, and macrophages. Transforming growth factor-β (TGF-β) is a well-recognized, multifunctional cytokine transforming growth factor. TGF-β mediates hepatic stellate cell and fibroblast activation resulting in generation of myofibroblasts and deposition of extracellular matrix . It is aKareius bicoloratus [Salmo trutta fario [One of the most striking findings in this study is the recognition of Kupffer cells, which were typical to those of mammalian liver in their morphological features, Kupffer cells have not been recognized in many teleosts. These polymorphous cells are rare and hardly recognized in fish liver. When present, they show a strong phagocytic activity . Kupfferoloratus  or Salmota fario .Intrahepatic macrophages, Kupffer cells, and melanomacrophage centers are all present in the liver of molly fish. They can be distinguished by their topographical location and their morphology. The intrahepatic macrophages were located between the hepatocytes and characterized by many cytoplasmic processes. Their cytoplasm characteristically displayed many small dense vesicles (lysosomes), phagocytic vacuoles, and huge dense bodies with a heterogeneous content that represented phagososomes. Kupffer cells were pleomorphic cells, situated in the hepatic sinusoids. They projected slightly to the sinusoidal lumen and established close contact with endothelial cells. They possessed irregular cell surfaces and contained lysosomes, phagosomes in the form of vacuoles, as well as a few fat droplets. The nuclei of the Kupffer cells were indented and frequently eccentrically located. Clumps of heterochromatin were distributed throughout the nuclei and formed a distinct rim along the nuclear envelope. Melanomacrophage centers (MCs) were heterogeneous in composition, and widely distributed in the liver of molly fish: between hepatocytes, around bile ducts, in the adventitia of blood vessels, and associated with pancreatic–venous complexes, like those found in other fish species –34. MoreBy TEM, Kupffer cells showed large vesicular nuclei and many cytoplasmic processes that projected into the lumen of hepatic sinusoids in the liver of molly fish. In addition, they expressed strong immunoreactivity to APG5, TGF-β, and NF-kB. NF-kB responds to inflammatory and immune stimuli and regulates cell proliferation, adhesion, invasion, apoptosis, and angiogenesis in multiple cell types . MoreoveAggregations of intrahepatic macrophages and melanomacrophages were extensively distributed in the liver of molly fish. TEM revealed that the cytoplasm of these cells exhibited numerous phagocytic vacuoles, melanin-like granules and large, dense bodies containing heterogeneous materials that represented phagosomes. In addition, they expressed APG5, TGF-β, IL-1β, NF-κB, and SOX9. IL-1β is a critical early response proinflammatory cytokine that mediates immune regulation in both innate and adaptive immunity. It could be secreted by activated endothelial cells, tissue macrophages, blood monocytes, activated T lymphocytes, granulocytes, and other cell types . It affeAghaallaei et al. and Agius and Roberts , 43 addeTelocytes (TCs) are identified as a peculiar cell type of interstitial cells that is characterized by extremely long and thin cellular processes that are called telopodes (Tp) . These cThe present study showed the spatial distribution of hepatic vascular-biliary tracts in molly fish. The liver of molly fish is well organized with many types of active cellular elements. It has unique functions in phagocytosis, autophagy, and cell regeneration. The expression of APG5 in hepatocytes, Kupffer cells, melanomacrophages, and telocytes indicates the involvement of these cells in the process of autophagy and supports the possibility of a role of the liver in lymphocyte development and proliferation. The expression of TGF-β and NF-κB in hepatocytes, Kupffer cells, telocytes, and macrophages suggests the role of liver in regulation of cell proliferation, and immune response suppression. The expression of IL-1β and Sox9 in macrophages and melanomacrophages suggests a role of the liver in regulation of both innate and adaptive immunity, and in cell proliferation and apoptosis, in addition to stem cell maintenance. The expression of Nrf2 in the telocytes suggests a role of the liver in immunopotentiation and oxidative stress.


Acanthostyles buniifolius (Chirca) is one of the most important weeds in natural pasture areas widely distributed in southern South America and can adversely affect livestock production. The current study was designed to identify possible responses of Chirca to CO2 concentration ([CO2]) and drought that would indicate higher adaptation and potential proliferation within the Pampa Biome. Chirca plants were cultivated at two CO2 concentrations (400 (a[CO2]) and 700 (e[CO2]) µmol mol−1) and two water conditions . Besides growth parameters, we also determined water potential (ѱw), relative water contents (RWC), proline, glycine betaine, total soluble sugars, hydrogen peroxide, lipid peroxidation, superoxide dismutase (SOD), ascorbate peroxidase (APX) activity, chlorophyll A and B, carotenoids and root dry mass (RDM). Plants exposed to e[CO2] are more efficient in water use and have a greater increase in root dry mass, enabling greater adaptation to climate-induced droughts. Among the biochemical changes observed in the plants under drought stress, the accumulation of proline, glycine betaine, and total soluble sugars were the most evident mechanisms allowing plants to tolerate drought stress by osmotic adjustment.The differential growth and yield response of plant species to rising carbon dioxide concentrations and climatic change may alter species diversity within biomes. The Pampa Biome in South America is an important grassland biome of agronomic and environmental importance.  Such examinations are fundamental in determining the adaptive capacity of this species and its potential expansion within the Pampa. Therefore, the objective of this study was to assess the role of projected CO2 increases on the vegetative and reproductive response of Chirca to drought and identify the possible mechanisms that lead Chirca to adapt to an increase in CO2 and drought stress.Given the troublesome nature of Chirca in the Pampa Biome, the effect of rising ) at 400 ± 50 μmol mol−1 and elevated (e[CO2]) at 700 ± 50 μmol mol−1. Factor B represented water restriction conditions and the control (without drought). The water deficit consisted of irrigation suppression until the plants reached 15% of their pot capacity (Cw). Cw was determined according to Dos Santos et al. (2015), with some methodological adaptations. The Cw was calculated using the fresh mass of the soil after water saturation (Cfm) and the dry mass (Cdm) after drying the soil until it reached the constant weight and applied in the equation Cw = (Cfm − Cdm)/Cfm × 100.Seeds of Chirca were collected in Capão do Leão—RS, in a pasture area. Chirca seeds were sown in plastic boxes and plant growth chambers in a controlled environment at 28/25 °C (day/night) and 12 h photoperiod (day/night). Plants emerged seven days after sowing and were transplanted two weeks later from plots. The experimental units consisted of one plant and 8 L pots, with a volume of 0.042 me[CO2] and a[CO2], respectively. After the first stress, the plants underwent a recovery period until 246 days after emergence. For the second water stress, the plants were kept under water deficit until again reached 15% of pot capacity, a condition that lasted 7 and 6 days for e[CO2] and a[CO2], respectively.The plants were kept at pot capacity (full irrigation) until the beginning of the water deficit treatment imposed at 120 days after emergence and a second deficit imposed at 246 days after emergence, comprising the vegetative and reproductive stages, respectively . The begDuring the period of water deficit, the pots were monitored by daily weighing, at consistent daily times, and an additional reading was performed with the soil moisture sensor (TDR—Time Domain Reflectometer–Trace System). For the control treatment, water was replaced, assuming the maintenance of pot capacity at 15% and 100%, considering 1 mL = 1 g. After reaching 15% Cw, the plants were collected, and for the treatment of water stress, the total suspension of water was carried out. Two sampling times were carried out, on the day of water deficit (15% Cw) and rehydration  and compared to the fully irrigated control. After collection, the samples were stored in an ultra-freezer at −80 °C for analysis.The following parameters were measured following water stress and rehydration: water potential (ѱw), relative water contents (RWC), proline, glycine betaine, and total soluble sugars, hydrogen peroxide, lipid peroxidation, superoxide dismutase (SOD), ascorbate peroxidase (APX) activity, chlorophyll A and B, and carotenoids, such as root dry mass. A summary of the results of the variables analyzed at both stages of development are provided in the w:v). The homogeneous mixture was centrifuged , and the supernatant was used to determine the H2O2 content according to ref.  and at midday under drought stress. However, only in the predawn  was sufficient to reduce the relative water content of Chirca plants in activity . The rede[CO2]  the overproduction of ROS and lipid peroxidation of membranes . It was 2O2 generated by SOD in different cell organelles  E. We hyps of ROS . The accs of ROS . Carotent energy .e[CO2], and also after rehydration in the stage of the beginning of the reproductive cycle (Although plants showed significant responses of the enzymatic and non-enzymatic defense system, an increase in lipid peroxidation was observed in plants subjected to drought stress, even after rehydration, especially in the vegetative stage under ve cycle G,H. Howeve cycle C,E. Chlove cycle C,E.e[CO2] decreased the root growth rate by 0.035 g/day−1, while plants under water deficit increased by 0.053 g/day−1 from the vegetative period to the beginning of the reproductive period (a[CO2], plants showed an increase of 0.017 g/day−1, while plants exposed to water deficit decreased root dry mass accumulation by 0.020 g/day−1 from the vegetative period to the beginning of the reproductive period.Another important response mechanism when plants are exposed to water deficit is allocating resources for root growth, allowing the expansion of the water uptake area. Control plants of Chirca exposed to e period A,B. When2, especially in plants with C3 metabolism [e[CO2] may have an efficient regulation of the stomatal opening and, therefore, better efficiency in the assimilation of CO2. The increase in this efficiency under water deficit can support the higher allocation of photoassimilates to the root system  to e[CO2] stimulated growth and root dry massAmong the biochemical changes observed in the plants under drought stress, the accumulation of proline, glycine betaine, and total soluble sugars were the most evident mechanisms allowing plants to tolerate drought stress by osmotic adjustment. The increase in the atmospheric concentration of This result shows that climate change may favor this weed and may increase its presence and damage in the grasslands. Future research must evaluate the effect of climate change on this plant at the community level.


Obstructive sleep apnea (OSA) is a prevalent disease that is associated with an increased incidence of type II diabetes mellitus (DM) if left untreated. We aimed to determine the association between glycosylated hemoglobin (HbA1c) levels and both nocturnal hypoxemia and apnea-hypopnea index (AHI) among a Saudi patients with OSA.A cross-sectional study that enrolled 103 adult patients diagnosed with DM and confirmed to have OSA by full night attended polysomnography between 2018 and 2021. Those who presented with acute illness, chronic obstructive pulmonary disease (COPD)/restrictive lung diseases causing sleep-related hypoxemia, or no available HbA1c level within 6 months before polysomnography were excluded from the study. Univariate and multivariate linear regression analyses between HbA1c levels and parameters of interest were tested.Sixty-seven (65%) of the studied population had uncontrolled DM (HbA1c ≥7%). In univariate regression analysis, there was a significant positive association between HbA1c, and sleep time spent with an oxygen saturation below 90% (T90), female gender, and body mass index (BMI) (p<0.05) but not AHI, or associated comorbidities (p>0.05). In the multivariate analysis, HbA1c was positively associated with increasing T90 (p<0.05), and ODI (p<0.05), but not with AHI (p>0.05).Nocturnal hypoxemia could be an important factor affecting glycemic control in patients with OSA suffering from DM irrespective of the severity of both diseases. Therefore, we aimed to determine if there was an association between glycosylated hemoglobin (HbA1c) levels and nocturnal hypoxemia among Saudi diabetic patients presenting with OSA.22.1A cross-sectional, single-center study was conducted between November 2018 and December 2021 at the Sleep Laboratory of the Internal Medicine Department, King Fahd Hospital of the University, Imam Abdulrahman Bin Faisal University (IAU), Al-Khobar, Eastern Province, KSA, a tertiary referral hospital. To estimate the sample size needed we used Green’s rule of thumb  with the2.2The study included adult patients referred to the sleep clinic with symptoms suggestive of OSA. All patients who were adults (aged ≥ 18 years), had DM, and proved to have newly diagnosed OSA by polysomnography (PSG) were included in the current study. DM was defined as having a fasting plasma glucose of 126 mg/dl or more, an HbA1c of ≥6.5%, or a self-reported diagnosis of DM . Patient2.32), HbA1c levels, polysomnographic data, and information on comorbidities including DM, HTN, hypothyroidism, HF with a reduced ejection fraction, CAD, AF, and chronic kidney disease (CKD).Patients’ electronic records were reviewed after receiving the IRB approval. For each patient we collected the following data: age, gender, body mass index (BMI) calculated as weight in kilograms (kg) divided by the height in meters squared  using the American Academy of Sleep Medicine (AASM) scoring manual version 2.5  (type I sleep study)   regression to control for several confounders in three models. In model 1 we controlled for age and gender. In model 2 we added BMI, and in model 3 (full model) we added history of HTN, CAD, CKD, stroke, HF, and AF. We present the summary finding of the three models in the main paper and a detailed version in the 33.1A total of 293 patients with proven OSA were screened for eligibility criteria. One hundred and three patients (35%) were finally included in the study and further classified as controlled and uncontrolled DM Figure 13.2Univariate linear regression analysis with HbA1c level as the dependent variable showed a significant positive association with female gender , BMI , and LnT90  Table 2.3.3The results for the multivariate analysis after adjusting for known confounders are shown in 4In the current study, we found that nocturnal hypoxemia as detected by T90 as a marker of OSA severity  was assoAlthough several studies investigated the association between OSA and DM prevalence, few studies examined the effect of severity of OSA on glycemic control among individuals with DM . ThanaviNocturnal hypoxemia may play an important role in developing DM. Muraki et al.  found inStudies investigating the association between the prevalence of OSA among patients with DM indicate that the prevalence of OSA may be as high as 23% and that the prevalence of any form of SDB may be as high as 58% . Azman eNocturnal hypoxemia as assessed by T90 could be linked to the development of various complications of DM and early treatment could guard against organ damage. Xue et al.  found thWe found that females had increasing levels of HbA1c compared to males with higher levels of OSA when measured using ODI as an indicator even after controlling for age and BMI levels. Compared to other studies, Priou et al.  found thThe current study has some limitations. Firstly, our data were collected from patients following up at a single center sleep clinic as opposed to a community setting thus subjecting our findings to selection bias in which individuals with OSA and worse glycemic control might be more likely to be present in our study compared to individuals with OSA in a community setting. Secondly, we considered only one reading of HbA1c in our analysis rather than multiple readings at the time of OSA investigation. However, HbA1c is considered a marker of the glycemic state for an individual over several months and is a good predictor of diabetic complications . Thirdly5DM is commonly associated with OSA. Intermittent hypoxemia associated with obstructive events could be an important factor affecting glycemic control among patients with OSA suffering from DM irrespective of the severity of both diseases. However, this association could also be attributed to reverse causation in which glycemic control contributes to the severity of OSA.The original contributions presented in the study are included in the article/The study protocol was approved by the institutional review board (IRB 2020-01-136) of IAU according to the Helsinki regulations. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Elephas maximus borneensis). In this region, elephants range within forests, oil palm and community areas. The degree to which elephants are using these areas remains unclear. We used GPS telemetry data from 2010 to 2020 for 14 collared elephants to map their entire known ranges and highly used areas (hot spots) across four land use categories and estimate time spent within these. The use of land use types across elephants varied significantly. Typically, females had strong fidelity to forests, yet many of these forests are threatened with conversion. For the three males, and several females, they heavily used oil palm estates, and this may be due to decreased landscape permeability or foraging opportunities. At the pooled level, the entire range and hot spot extents, constituted 37% and 34% for protected areas, respectively, 8% and 11% for unprotected forests, 53% and 51% for oil palm estates, and 2% for community areas. Protecting all forested habitats and effectively managing areas outside of protected areas is necessary for the long-term survival of this population.In the Kinabatangan floodplain, Sabah, Malaysian Borneo, oil palm and settlements have reduced and fragmented lowland tropical forests, home to around 200 endangered Bornean elephants ( Such habitat loss is having significant negative impacts on biodiversity globally, with rates predicted to increase in tropical biomes and biological hot spots4. Deforestation and habitat conversion are major threats to large ranging species like elephants as they are increasingly left within landscapes with little and fragmented remaining natural habitat, often not large enough to sustain viable breeding populations5. Moreover, poorly planned land use changes and aggressive profit driven management of landscapes often result in a lack of habitat connectivity for elephants6. Elephants are increasingly roaming outside of the network of protected areas, and there is an urgent need to understand how they use these multiple-use landscapes to improve conservation and effective wildlife management7.Increasing human populations and greater demands for certain commodities have resulted in large-scale conversion of natural ecosystems to agriculture and other human land usesElephas maximus borneensis) is a prime example of a species that is threatened by habitat conversion of natural forest to agriculture and other types of man-made landscapes9. Bornean elephants are an endemic sub-species whose range is limited to approximately 5% of the island of Borneo, occurring largely in the central and south-eastern region of the Malaysian State of Sabah13. In Sabah, elephants are found within three major elephant managed ranges, the Lower Kinabatangan, Central Sabah and Tabin14. These ranges are isolated from each other due to large tracts of agricultural landscapes, linear infrastructures (roads) and human settlements15. In fact, Sabah has lost over 40% of its forest over the last 30 years, most of it being replaced with oil palm monoculture in the east and south-eastern lowlands16. Over the past 15 years or so, elephants have been increasingly observed within human landscapes, including oil palm estates and smallholdings, human settlements, and along linear infrastructures (roads), heightening human–elephant encounters and conflicts15. However, the frequency and extent to which elephants enter human-dominated landscapes have not been quantified yet. Around 200–250 elephants are currently found in the Lower Kinabatangan14. This population has the smallest range in Sabah, confined between the mangrove forests to the east and the Batu Putih Road Bridge to the west, which is located half-way up the Kinabatangan River and has proven to be an impassable barrier for elephant movements since the early 2000’s14.The Bornean elephant 31, are all factors that affect home ranges and seasonal migratory patterns of elephants. In fact, elephants are highly adaptable and can modify their behaviour, to some degree, to cope and adapt to habitat changes. For example, males, especially those that have formed long-term, stable all male groups, sometimes use potentially “risky” areas such as agricultural landscapes and settlements34. Females, on the other hand, are typically less adverse to risky areas that they tend to avoid; they have also been found to reduce vocalizations and stay in tighter groups whilst outside of forested areas to minimise detection35.Elephant spatial distributions are a reflection of ranging behaviour37. However, where a species utilizes landscapes dominated by monoculture crops with minimal heterogeneity, such as oil palm, model outputs may not produce meaningful results. Another approach is to apply hot spot analysis within a species geographic range  to map the frequency or weight of where a species is occurring across a landscape41. A hot spot is defined here as an area with statistically significant spatial clusters of either high or low values42. Hot spot analyses are useful in identifying important ecological biogeography patterns and habitat features, as well as anthropogenic characteristics associated with species distributions41.Habitat suitability models are commonly applied to determine the location of wildlife corridors or linkages in human-dominated landscapes44.In this study, we identify the distribution (entire known range) and hot spots of 14 GPS collared Bornean elephants  living in the Lower Kinabatangan, from 2010 to 2020, and consider these at the pooled, clustered, and individual levels. We develop spatial land use and land cover data for 2010 and 2015 to understand the extent and proportion of time spent within differing land use categories within the elephant’s hot spots and compare this with their known ranges. We also look at hot spots on, or near to, a planned highway alignment to understand the potential impact of this infrastructure development project. Lastly, we look at time spent by elephants in different oil palm estates to identify where better management strategies are needed to improve habitat permeability and reduce human–elephant conflicts45. Large-scale timber extraction, in this region, started in the late 1950s, followed by intense conversion of logged forests to large-scale oil palm estates from the 1980s onwards46. Today, the landscape is dominated with oil palm that is perforated with a number of small, fragmented protected areas (Wildlife Sanctuary and Virgin Forest Reserves), as well as unprotected forest fragments, roads, and human settlements47. The villages within this region, are largely dependent on income from their oil palm smallholdings as well as from wildlife-watching tourism. The region experiences mean monthly temperatures of 21–34 °C and average annual rainfall of 3000 mm48. Due to the high annual rainfall, the low lying floodplain experiences significant seasonal flooding events, which has been documented to effect elephant range areas9.We restricted the study area to the general geographical extent of the Kinabatangan elephant population in eastern Sabah, a Malaysian state located in north Borneo Fig. . The KinElephas maximus), listed in Schedule 1  of the Sabah Wildlife Conservation Enactment 1997, with a total population size estimated to be below 2000 individuals14. As a result of restricted distribution ranges10, habitat fragmentation9, and a lack of linkages within the landscape49, current Bornean elephant populations are subjected to high levels of inbreeding7. In the Lower Kinabatangan floodplain, the elephant population size is estimated between 200 and 250 individuals, with population density estimates of 2.15 elephants/km250.The Bornean elephant is an endangered sub-species of the Asian elephant  and carried out in accordance with the current laws of Malaysia and Sabah Wildlife Department’s Standard Operation Procedures on Animal Capture, Anaesthesia and Welfare. Animals were anaesthetised by a sedative agent (Xylazil-100) administered by a wildlife veterinarian. Once sedated, the elephant was kept in a standing position by using small poles pointed at the back of its ears and the collar with the tracking device was attached around its neck. To minimize unnecessary time sedated, we used a reversal administered intra-veinously (Yohimbine) as soon as the collaring was completed.10. For each elephant social unit, we collared only one individual. Males were solitary bulls except for one (Gading) who was often found traveling with various social units. All GPS units were set to record the location of the elephants at two-hour intervals, totalling 12 equally spaced GPS locations every day.Satellite Global Positioning Systems collars (IR SAT (GPS/UHF), iridium, 12 D cells) from Africa Wildlife Tracking™  were fitted to 14 adult individuals  for differing time periods between 2010 and 2020  that linked the most exterior points of the dataset54. Then, we undertook a hot spot analyses (Getis-Ord Gi*) to identify where spatial clustering occurred within the datasets41. Hot spot analyses calculates a z-score based on the Getis-Ord Gi* statistic that tells where features (in this case location points) have significantly high or low values that cluster spatially55. A larger z-score indicates a tendency towards more intense clustering of high values (hot spots) while a negative z-score indicates a tendency towards more intense clustering of low values (cold spots) (see Table To better understand the areas most frequented by elephants (hot spots), we used a Moran’s I test to identify if, and to what degree, each collared dataset was clustered 10. The resulting output feature map displayed ‘hot spots’ and ‘cold spots’ along with respective confidence levels (Gi_Bin) and z-scores. We extracted the ‘hot spot’ points that had a 95% Confidence Interval or above , we performed Moran’s I  tool for spatial autocorrelation using the layers from the Collect Events tool. Next, we used the Hot Spot Analysis (Getis-Ord Gi*) tool, using the Collect Events output layers, and selected a fixed-distance band value of 2 km, which represented the average daily distance travelled by elephants in the Lower Kinabatangan floodplain47. This 2010 data included detailed forest and oil palm spatial layers that defined forest types and age/productivity of oil palm areas47. For this analyses, however, we pooled all of the forest types into one generic forest area, and for the oil palm, we reclassified the extent into two classes, one consisting of oil palm within large industrial estates, and the other, oil palm in smallholdings. We demarcated these two classes based on the use of official land title information and distinctive visual cues  seen in high resolution satellite imagery (SPOT 5 2.5 m) and Google Earth. For the oil palm smallholdings class, we expanded this layer by including village areas and dwellings as these land uses were often aggregated together. For the 2015 land use/land cover dataset, we used the 2010 layers and modified areas that had changed between the periods of 2010 to 2015, and used high resolution SPOT 5 1.5 m imagery to detect any changes in the landscape.To understand land use and land cover within elephant ranges, we overlaid elephant ranges with land use/land cover data for 2010 or 2015, depending on when each elephant was collared  within ArcGIS 10.8.1 and imported these into Google Earth. Each point was assessed and assigned a class  for those points that covered high resolution tiles for their appropriate years (2010 and 2015). The assigned classes were then compared to the land use/land cover layers developed and an error confusion matrix table was used to assess data accuracy, and kappa statistics were calculated57.Classification accuracy was determined through the use of an error confusion matrix method and kappa statisticsWe used government-provided spatial data to identify the protected areas within the region. These data included a number of protected areas and forest reserves under different designations including Wildlife Sanctuary (Sabah Wildlife Department), Class I Protection Forest Reserve, Class VI Virgin Jungle Reserve and Class VII Wildlife Reserve (Sabah Forestry Department), all named herein as ‘protected areas’ Fig. . We used58.We also digitised the main roads in the landscape, using the SPOT 5 images to understand where these linear features are. To understand potential threats from other planned new roads/highways, we georeferenced and digitised relevant maps from the Sabah Structure Plan 2033, which is an overarching policy/planning document for the stateWe investigated the percentage of time that each collared elephant spent in their hot spots versus their entire range by calculating the proportion of GPS points  within their hot spots. Further, we assessed the proportion of time spent for each collared elephant within our four land use/land cover classes, for their entire extent and for their hot spots. Additionally, for those elephants who had a hot spot that intersected with the existing or planned new roads/highway, we calculated time spent within those hot spots.We undertook four sets of statistical assessments of these data described above. First, we calculated Pearson correlation coefficients and conducted associated t-tests for independence to assess the pairwise (linear) relationships between the extent of an elephant’s entire range, the extent of the hot spot, the extent of the hot spot as a percentage of the entire extent, and the proportion of time spent in the hot spot.Second, we undertook similar tests to evaluate the impact of the existing road and the planned road/highway. Specifically, we assessed correlations between an elephant’s: (a) entire extent and the proportion of time spent in hot spots that intersect the existing road and planned new road/highway; and, (b) proportion of time spent in hot spots and the proportion of time in hot spots that interested the existing road and planned new road/highway.Third, to understand the variation between land use/land cover types within collared elephants entire ranges and hot spots, we conducted an analysis of variance (ANOVA) to assess whether the average proportion of extents for protected forest, unprotected forest, and oil palm estates were the same at the entire range, and hot spot levels. Only three land use types were used, as the use of oil palm smallholdings/villages were comparatively very small, and therefore somewhat obvious in regards to their significance. Further, if the four groups were included in these analyses then the proportions would add up to 1, which distorts the assumption of independent groups.Fourth, we conducted a k-means cluster analysis to assess similarities and differences between elephants with respect to the proportion of time spent in the four types of land use/land cover across the entire range time, and in hot spots. Only values of k = 2 and 3 were considered, based on the sample size of 14 elephants. The choice of k was determined by comparing the between SS/total SS measure.http://www.jtuwma.net/) in January 2017. Through online searches for company information and reports, and by using the GeoRSPO online platform (https://rspo.org/members/georspo) we collated company level data on land title ownership, to the best of our ability, and incorporated this information into the cadastral (land title) layer. Additionally, we used the full extent of the cadastral layer, along with the extent of the protected areas data, to identify areas used by elephants that may be on state lands, meaning lands that have not yet been allocated  under title, and therefore may be available for the government to easily include in their protected area network.To enable us to understand the corporate stakeholders within elephant entire ranges and hot spots, we used available cadastral data that was ascertained from the Land and Survey Department’s online platform , with nine out of 14 elephants having over 2 years of data, and four individuals having less than one year  and varied from 7.07 to 72.53 km2 in size (averaging 33.44 km2) , indicating that elephants with smaller entire range extents also have smaller sized hot spots.Individually, entire known range extents averaged 263.37 km2) Table  and SI 2p = 0.99) nor the absolute size of the hot spots . Instead, it was significantly positively associated with the relative size of hot spots . Scatter plots indicated no alternative nonlinear or nonstandard relationships between the pairs of variables.Hot spots made up 4 to 20% (averaging 12%) of an individual’s entire range, however, time spent within these was high, ranging from 10 to 60%  of the pooled entire known range extent  (Table p = 0.20). However, there was a highly significant correlation between the proportion of time spent by an elephant in the hot spots and the proportion of time spent in hot spots intersecting roads.Nine, out of the 14, elephants had hot spots that intersected or boarded the existing main road and/or planned new road/highway. Hot spot extents ranged from 3.64 to 43.5 km59.Land use and land cover classification accuracy was 97.3%, with a Kappa statistic of 0.96 using an error matrix method and 226 points for 2010, and for 2015 the classification accuracy was 97.7%, with a Kappa statistic of 0.96 with 221 points Tables  and 3. BAt the individual level: forest habitats (protected and unprotected) were important and constituted 45 to 89% of elephants entire known range (averaging 66%); and from 0 to 91% (averaging 53%) within their hot spot extents Fig. . Oil paldf = 2, 39; p = 7.6E−12). We also found an almost perfect negative linear association between the proportion of protected area extent versus oil palm estate extents across the entire range . For hot spots, we found a substantive but not significant difference between the average proportion of extents of protected areas, unprotected forest, and oil palm estates . The correlation between the proportion of protected area extent versus oil palm extent within the hot spots was also significantly negative .There was a highly significant difference between the average proportion of extents of protected areas, unprotected forest, and oil palm estates within elephants entire range , only 37% (231.35 km2) was in protected areas, 8% (49.09 km2) was in unprotected forests, 53% (331.08 km2) was in oil palm estates, and 2% (9.64 km2) was in oil palm smallholdings/villages  was protected, 11% (29.12 km2) was in unprotected forests, 51% (136.88 km2) was in oil palm estates, and 2% (5.24 km2) was in oil palm smallholdings/village areas , 24.01 km2 was identified as potentially being on state land, with the remaining being on various land titles. For the unprotected forests identified in hot spots (29.12 km2), 15.83 km2 was identified as potentially being on state land.For the pooled entire known range  ranged from 34 to 95% (averaging 65%) for their entire range; and from 0 to 98% (averaging 54%) within their hot spots  and unprotected forest (26%), and very little time in oil palm estates (7%); three elephants were grouped into cluster 2 also spending a lot of time in protected areas 57%) and unprotected forest (16%) but also in oil palm estates (26%); and, six elephants were in cluster 3, spending over half their time (average 57%) in oil palm estates and much less time in protected areas (29%) and unprotected forest (12%)  in oil palm estates; whereas elephants in the other two clusters spent around 10% of their time in this land use/land cover type. Five animals spent over half their time on average in protected areas (59%) and only a third of their time (31%) in unprotected forest, whereas the other cluster of two animals spent less time in protected areas (13%) and much more time in unprotected forest (52%) and oil palm smallholdings/villages (26%).2 (or 41%) of the 627.95 km2 pooled entire range, and nine corporate land owners for 115.84 km2 (or 43%) of the 266.89 km2 pooled hot spot extents (Table We identified eleven corporate land owners for 259.82 km2 or 1% of the2 (or 16%) of the pooled entire range, and 38.56 km2 (or 14%) of the pooled hot spot extents that had alienated titles (native titles and country lease titles) but ownership could not be identified. Further to this, 33.95 km2 (or 5%) of the pooled entire known range extent, and 21.32 km2 (8%) of the pooled hot spot extents could possibly be on state lands.There were 102.83 km2 . Our results showed strong fidelity of certain elephants to these forested habitats. Our k-means cluster analysis found that within elephant entire ranges and hot spots, two out of the three cluster groups had high or very high usage of forests. Both cluster 1, for the entire range, and cluster 1 for hot spots extents, had five females that on average used forest environments 90% of their time, with protected areas being used 64% and 59%, and unprotected forested being used on average 26% and 31%, respectively  of forest identified within the pooled entire known range were not protected, with half potentially being on state land, and the remaining half on land titles of various types (Table 2) of the total extent, with 54% being potentially on State land and 46% on land titles , before going through oil palm estates then through another protected area to the south and through the Tabin elephant population range. For the Kinabatangan, creating a public highway will cut the elephant population range into two parts  and/or the planned road/highway alignment  and their respective social units, also seemed to have high levels of oil palm use, while other individuals had zero or very little use of oil palm , IOI Corporation , Genting Plantations , Sime Darby Plantation , and Karangan Agriculture  Table ; Fig. 4.9. A previous study identified 20 bottlenecks in the Lower Kinabatangan with the two main ones (of 9 km and 6.5 km in length) found around the village of Sukau9. In addition, the unplanned and chaotic erection of electric fences by large estates and smallholdings has disrupted significantly elephant movement patterns and resulted in artificial hot spots for certain individuals 72. Electric fences have widely been used to mitigate human–elephant conflicts. The establishment of fences rarely consider the traditional elephant routes nor the location of existing fences in neighbouring estates. If elephants manage to enter such areas, they often become trapped and experience difficulties in returning to nearby forests, exacerbating conflicts with people35.Linear features like major highways, electric fences and drainage ditches hamper elephant movements within the Lower KinabatanganCertain estates such as Melangking Oil Palm Plantation have allowed elephants to roam freely in their estate . Since 2017, this plantation has shown a drastic reduction in damages to their oil palms following the removal of their permanent electric fences surrounding their entire estate. Instead, this plantation is using a temporary electric fencing regime around newly planted palm areas. Concurrently, they now do not push elephants out of their estate, which can explain why Melangking Oil Palm Plantation is a significant hotspot in the region.66, the Bornean elephant has been observed doing the opposite, which may explain some of the hot spots within oil palm estates. This movement strategy may allow for better vigilance as seen on a few occasions when elephants spent 2–5 days in the Bukit Melapi-Yu Kwang Corridor, near the village of Sukau, before leaving the area .Another reason why elephant ranges incorporate oil palm estates is to move between forest patches that are becoming completely isolated following forest conversion, as is the case close to Sukau Fig. ; Fig. 1.73. Elephants are known to eat the shoots of newly planted oil palms, often killing the palms and causing significant economic damages35. Since 2010, many estates located in the Lower Kinabatangan have started a new palm rotation. Palms are replanted every 25 years. A new rotation includes land clearing, bole and root mass removal, and the shredding or chipping of felled palms. Elephants are attracted to the shredded palm hearts since it gives them easy access to one of their favourite food72. This particular behaviour does not cause economic damage, and some estate managers allow the elephants to stay and forage in the chipping areas. This was documented for several collared elephants, whose hot spots and time spent were particularly high within oil palm . Once the shredded palms have dried, however, elephants will leave these areas and move elsewhere. Within oil palm estates, some elephants have been found to travel more directly and rapidly suggesting ‘exploratory’ behaviour, which could be associated with searching for young palms or areas of palm felling and chipping of palm hearts15.Hot spots in the oil palm landscape can also be explained by feeding opportunities, since elephants feed on palm shoots, leaves and hearts63.Lastly, elephants may still be using their historical range that used to be covered with forest before conversion to oil palm. Other factors potentially explaining the relatively high use of oil palm estates include seasonal variations of ranging patterns. Indeed events of drought or floods limit the access to various parts of the floodplain and will tend to confine the animals in some areas76. Deaths from non-natural causes are largely due to poisoning , gunshot wounds, poaching for tusks and other body parts, and snares35. Stopping killing and enabling a safe coexistence between people and elephants within multiple-use landscapes that are dominated by oil palm is one of the key strategies developed in the Bornean Elephant Action Plan for Sabah (2020–2029), which was endorsed by the State14. Based on our results in Lower Kinabatangan, a series of recommendations are proposed.In Sabah the state authorities have recorded at least 200 elephant deaths from the year 2010 to 2021 and most of these have occurred on, or near, oil palm estates46.This study underscores the importance of remaining forested areas for the Lower Kinabatangan elephant population. Full protection of all forest fragments left in the Lower Kinabatangan is urgently needed. Several official mechanisms are available to fulfil this request that has been proposed for the past 20 years by various organizations23.The current network of forests available in the Lower Kinabatangan is too small and fragmented to sustain a viable elephant population. Forest corridors must be created across the landscape through reforestation exercises, whilst concurrently undertaking enrichment planting of native understory forage within forested areas as this may minimize the need for elephants to search for easily accessible food in high-risk oil palm landscapes67.Current governmental plans to build a road bridge and public road/highway linking the southern bank of the Kinabatangan River to Tabin Wildlife Reserve to the south will irreversibly impact the Lower Kinabatangan elephant population by cutting the current range into two isolated parts. This will impact the elephants ranging patterns, potentially even fragmenting the already small population into two groups, and potentially leading to elephant deaths by vehicle collisions , and increase the risk of poaching activities, all resulting in a decrease in the genetic diversity of the, already small and isolated, population14.Eventually, the future of the Kinabatangan elephant population resides in improving land use and management practices within oil palm estates currently used by elephants. We recommend that priority should be given at improving elephant movements in oil palm estates by removing unnecessary man-made barriers and only cautiously installing temporary electric fences to protect sensitive areas. For example, the use of electric fences around mature oil palm and areas whereby palms are being removed and chipped could be prohibited, and electric fences permitted solely for protecting oil palm nurseries, new plantings and young oil palms (e.g. up to 7–8 years old), and staff and office quarters. This would greatly allow for landscape permeability for elephants, and other species that need to cross the landscape for their ecological and biological needs77. However, there is a need for a more comprehensive set of guidelines, which delineate better practices with the aim to increase the protection of people and elephants outside protected areas. Guidelines should specify “do’s” and “don’ts” (based on best available data and knowledge) of actions needed before, during and after elephants visit oil palm estates and smallholdings.A handful of guidelines exist to assist oil palm managers and staff in managing elephant populations in their respective estates80. Sabah government can use this platform to build an integrated landscape level approach to better manage landscapes within known elephant ranges  to allow for a safe and permeable movement through the landscape.Sabah now is in an interesting transition within their palm oil sector. On the 21st October 2015, the Sabah State Cabinet committed to produce 100% certified sustainable palm oil, by 2025, under the Roundtable for Sustainable palm Oil (RSPO) Jurisdictional Certification approach. Under this approach, areas of High Conservation Value and areas identified within the High Carbon Stock Approach need specific management and monitoring, in order to comply with RSPO principles and criteriaEventually, long-term survival of the Bornean elephant will mainly depend on how people and elephants can co-exist. It is our hope that this study illustrates the importance of protecting all forested habitat and effectively managing areas outside of protected areas to allow for long-term elephant coexistence with humans in this landscape.Supplementary Information.


Thyroid-associated ophthalmopathy (TAO) is a chronic autoimmune disease. In this study, high-throughput sequencing was used to investigate the diversity and composition of the ocular microbiota in patients with TAO.Patients with TAO did not receive treatment for the disease and did not have exposed keratitis. Patients with TAO (TAO group) and healthy individuals (control group) were compared. All samples were swabbed at the conjunctival vault of the lower eyelid. The V3 to V4 region of the 16S rDNA was amplified using polymerase chain reaction and sequenced on the Illumina HiSeq 2500 Sequencing Platform. Statistical analysis was performed to analyze the differences between the groups and the correlation between ocular surface microbiota and the disease. The ocular surface microbiota of patients and healthy individuals were cultured.Bacillus and Brevundimonas increased significantly in the TAO group. Corynebacterium had a significantly decreased relative abundance (P<0.05). Paracoccus, Haemophilus, Lactobacillus, and Bifidobacterium were positively correlated with the severity of clinical manifestations or disease activity (P<0.05). Bacillus cereus and other opportunistic pathogens were obtained by culture from TAO patients.The ocular surface microbiota structure of TAO patients changed significantly. The average relative abundance of Bacillus, Brevundimonas, Paracoccus, and Haemophilus in TAO patients, increase the potential risk of ocular surface infection. The findings of this study provide a new avenue of research into the mechanism of ocular surface in TAO patients.This study found that the composition of ocular microbiota in patients with TAO was significantly different from that in healthy individuals. The ocular surface opportunistic pathogens, such as  Corynebacterium mastitidis can induce interleukin-17 production and inhibit the growth of Candida albicans or Pseudomonas aeruginosa by stimulating T cells in the mucosa of healthy human eyes  is one of the most common orbital disorders in adults. It is generally accepted that TAO is a chronic autoimmune disease. The thyrotropin receptor expressed by orbital fibroblasts and fat cells are the target of intraorbital autoimmunity, and the anti–thyrotropin-receptor antibodies are generated during the cellular immunity process . These aTherefore, in this study, high-throughput sequencing technology was used to explore the diversity and composition of ocular surface flora in TAO patients without exposed keratitis.A total of 70 TAO patients without exposure keratitis and 77 healthy volunteers were recruited from 2019 to 2022 during their first visit to Shanxi Eye Hospital. At the same time, 13 unsampled swabs were retained during the sampling process as blank controls for the environment.99m-DTPA scintigraphy for TAO diagnosis .Samples were collected by qualified ophthalmologists in a clean disposal room following the established procedures in batches. Before sampling, hands were disinfected, and disposable gloves were worn. Subsequently, the patients were subjected to local eye anesthesia with proparacaine hydrochloride eye drops . After 1–3 min, the patients were instructed to look upward, and a sterile swab  was wiped from the inner canthus of the patient’s subconjunctival fornix to the outer canthus twice. At the end of each sampling batch, sterile swabs from the same production batch without nucleic acid sampling were used as blank control samples.® Kit .The sample swab was sealed in a sterile centrifuge tube. Swabs from all subjects were placed in ice boxes and sent to the laboratory for storage at -80°C for centralized nucleic acid extraction. Genomic DNA was extracted from all samples using the MO-Bio PowerSoilhttps://www.ncbi.nlm.nih.gov).At the same time, swabs from the other eye of 43 TAO patients and healthy individuals were placed in a bacteria-preserving solution for conventional culture. After enrichment, those samples were inoculated in Brain-Heart Infusion medium  supplemented with sheep blood and cultured in oxygen (30°C), 5% carbon dioxide (35°C), and anaerobic (35°C) environment. The approximate full-length 16S rRNA gene sequence was returned by sequencing  and was compared online with the GeneBank database  was used to generate the sequencing library and add indexes. Library quality assessment was performed using the Qubit@2.0 Fluorometer  and Agilent Bioanalyzer 2100 . Library sequencing was performed on the NovaSeq platform (Illumina).Specific primers (341F-806R) were used to amplify 16S rRNA in the corresponding regions (16S V3-V4). Polymerase chain reaction (PCR) was performed using approximately 10 ng of template DNA. The PCR process included 15 μL of Phusion High-Fidelity PCR Master Mix  and 0.2 μM of forward and reverse primers. Thermal cycling consisted of initial denaturation at 98°C for 1 min; 30 cycles of denaturation at 98°C for 10 s, annealing at 50°C for 30 s, and elongation at 72°C for 30 s; and final extension at 72°C for 5 min. The PCR products were mixed in equimolar ratios. Purified PCR products were extracted using the Qiagen Gel Extraction Kit (Qiagen). The TruSeqhttp://ccb.jhu.edu/software/FLASH/) was used to read and merge paired-end reads. QIIME  was used for quality filtering of raw tags. The UCHIME algorithm  was used to detect and remove chimeric sequences after comparing tags with the reference databases  sequence. Then, the tags were compared with the OTU sequence to obtain the OTU abundance distribution table for each sample. Mothur software was used for species annotation analysis using the SILVA database, and the confidence threshold was set at 0.8. Due to the low biomass of eye surface samples, environmental contamination can be easily introduced in sample collection, DNA extraction, library construction, and sequencing. Blank control samples were used as the standard to monitor the contamination of nucleic acid extraction samples in the same batch. By treating specific microorganisms in blank control samples as spike-ins, we estimated sample contamination and excluded three samples from the TAO group and 13 samples from the control group from subsequent analysis. The accession code of the microbiome raw sequence data in the NCBI Sequence Read Archive is PRJNA807746 (SRR18065410-SRR18065514).Paired-end reads were assigned to samples based on their unique barcode. Unique barcode and primer sequences were truncated. FLASH . Intra-group differences were analyzed using t-test dichotomous traits and t-test regression coefficients. Spearman’s correlation coefficients were used to assess the correlation between microbiota and disease. A total of 89 ocular surface microbe samples were included , yielding 9,059,610 sequences Table 1.Corynebacterium, Cutibacterium, Staphylococcus, Pseudomonas, Escherichia-Shigella, and Streptococcus. Among them, Corynebacterium, Cutibacterium, and Staphylococcus were present in all samples of both groups   ((P<0.05) Table 2.Ligilactobacillus, Lactobacillus, Limosilactobacillus, and Bifidobacterium were positively correlated with CAS     with dry eye, and healthy individuals (control) without dry eye (P<0.05)  was significantly higher than that in the control group (5.9%). TAO group belonging to Corynebacterium are Corynebacterium accolens, Corynebacterium macginleyi, Corynebacterium suicordis, and Corynebacterium tuberculostearicum; belonging to Staphylococcus are Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus aureus. In addition, Bacillus cereus, Enterococcus faecalis, and Stenotrophomonas maltophilia were also detected.Among the 43 patients with TAO, 29 samples were successfully cultured with one or more bacteria, and 11 different species of bacteria were detected. Bacteria were cultured in 17 of 43 healthy subjects under the same culture conditions, and a total of six bacteria were detected , which also occurred in traumatic corneal ulcer . Among them, Bacillus is one of the main causes of traumatic endophthalmitis  and Staphylococcus , in all ocular surface samples of TAO, are easier to obtain pure strains under suitable conditions, and the detection rates of Corynebacterium and Staphylococcus were 36.4% and 27.3%, respectively. These two species have also been detected in previous studies on TAO-exposed keratitis, with the detection rates of Corynebacterium and Staphylococcus being 25% and 17.7%, respectively . Previous studies have suggested that some Lactobacillus and Bifidobacterium species have significant structural homology with thyroid peroxidase and anti-thyroglobulin and can competitively bind thyroid peroxidase antibodies and anti-thyroglobulin antibodies to induce autoimmune thyroid disease through molecular mimicry  can be found in the article/Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.BS and JX supervised this study. XJ, KD and ZZ conceived and designed the experiments. XJ, XN, QM and ZK collected the samples. JP, JY and XJ analyzed the data. XJ, KD and BS wrote the manuscript. The final manuscript was read and approved by all authors.This work was supported by grants from the Research Units of Discovery of Unknown Bacteria and Function (2018RU010); the State Key Laboratory of Infectious Disease Prevention and Control (2019SKLID313) and (2019SKLID312); the Scientific Research Project of Shanxi Provincial Health Commission (2019076), (2020XM07), (2020SYS12) and (2021XM46); the Natural Science Research Project of Shanxi Provincial Department of Science and Technology (201903D311009), (20210302123343) and (202104010910013); the Shanxi Provincial Eye Hospital Innovation Fund Project (C201901).We express our sincere thanks to Shanxi Eye Hospital, the State Key Laboratory of Infectious Disease Prevention and Control and all of the participants who recruited patients in this study.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Gallionella and Mariprofundus and by sulfur oxidation from Sulfuricurvum and Thiobacillus with an additional contribution of nitrite oxidation from Nitrospira. Metagenomic analysis showed that cbbM genes were identified as Gallionella and that aclB genes were identified as Nitrospira, further supporting these taxa as autotrophic drivers of the community. The detection of several taxa containing arsC and nirK genes suggests that arsenic detoxification and denitrification processes are likely co-occurring in addition to at least two modes of carbon fixation. These data link the importance of the detected autotrophic metabolisms driven by fluids derived from benthic hydrothermal springs to Crater Lake’s entire lentic ecosystem.Crater Lake, Oregon is an oligotrophic freshwater caldera lake fed by thermally and chemically enriched hydrothermal springs. These vents distinguish Crater Lake from other freshwater systems and provide a unique ecosystem for study. This study examines the community structure of benthic microbial mats occurring with Crater Lake hydrothermal springs. Small subunit rRNA gene amplicon sequencing from eight bacterial mats was used to assess community structure. These revealed a relatively homogeneous, yet diverse bacterial community. High alpha diversity and low beta diversity indicate that these communities are likely fueled by homogeneous hydrothermal fluids. An examination of autotrophic taxa abundance indicates the potential importance of iron and sulfur inputs to the primary productivity of these mats. Chemoautotrophic potential within the mats was dominated by iron oxidation from  Crater Lake is an ultra-oligotrophic freshwater caldera lake in south-central Oregon at the crest of the Cascade Mountain range. It formed in the crater of Mount Mazama left behind after a volcanic eruption approximately 7,000 years ago, yet volcanic activity has taken place as recently as 4,000 years ago . Crater 2 compared to bulk lake water  rRNA gene has been shown to be an efficacious tool to accurately identify community structure and diversity . SSU rRNbacteria . From thDeep Rover’s mechanical arm that was inserted into bacterial mat features. Water temperatures were collected using conductivity, temperature, and depth or “CTD” instrument carried on the submersible. Bacterial mat samples were collected using either push cores or Go-Flo bottles to collect mats and fluids with a minimum of lake water admixture (Deep Rover Dive number (216 to 230) followed by sample number .Microbial mat and adjacent mat fluid collection occurred in the south-central and north-east regions of Crater Lake during 16 HOV Deep Rover dives in August 1989. Samples were collected from two primary areas: seven from Llao’s Bath/Brain Mat complex in the south-central and one from Palisades Point Pools in the north-east regions of Crater Lake. Sample names, sites, and map locations along with geochemical profiles of respective mat and pool fluids are described in dmixture . ProcessDNA extractions were performed on mat and pool fluid samples using the FastDNA Spin Kit for Soil  protocol, which was followed according to the manufacturer’s instructions. Lysis was performed with two rounds of bead beating for 45 s at a setting of 5.5 using the FastPrep instrument with samples being placed on ice between runs. DNA was eluted in 100 μl of 1.0 mm Tris pH 8.0. Total DNA was quantified with a Qubit 2.0 fluorometer using the dsDNA HS kit .2, 200 μm each dNTPs, 1 μm (each) forward and reverse primers, and molecular-grade water to a total volume of 50 μl. The following conditions were used for the amplification process: an initial 8-min hot start at 95°C, followed by 25 to 30 cycles of denaturation (94°C for 1 min), annealing (58°C for 90 s), and elongation (72°C for 3 min). This was followed by a final elongation step at 72°C for 7 min. Amplicons were sized by 1% agarose gel electrophoresis against a 1-kb ladder. Negative controls were maintained throughout. The five replicate PCR mixtures were pooled, concentrated, and desalted. The desalted PCR amplicons were then cloned with a Topo-TA cloning kit following the manufacturer’s instructions (Thermo Fisher Scientific). All putative clones were streaked for isolation, and the inserts were assayed for correct size using PCR with M13F and M13R primers prior to sequencing  and the 1492R reverse primer (5’ dKGdP TAC CTT GTT ACG ACT T 3′), where dK is a purine analog, dP is a pyrimidine analog, and dN is an equal mixture of dK and dP . Five replicate PCRs were performed using ~50 ng of gDNA template, 5 U of Taq polymerase, 2.5 mm MgClquencing .via polymerase chain reaction (PCR) from all samples using bacterial primers 340F-CCTACGGGNGGCWGCAG and 784R-GGACTACHVGGGTATCTAATCC  for calculation of Good’s coverage, Abundance-based Coverage Estimator (ACE) richness , Chao-1 Mariprofundus were further assessed using the program ZetaHunter  in the SILVA v123 SSU reference database. Zetaproteobacteria reads across all eight mat communities with an abundance >10 reads were isolated, identified, and assessed using ZetaHunter to further classify these Zeta OTUs  prior to metagenomic sequencing. Libraries were prepared with the Nextera DNA library kit  for Illumina sequencing with 2 × 300 bp paired-end reads. Sequenced reads were quality checked using FastQC  and qualThe quality filtered metagenomic reads were assembled into contigs using the program MegaHIT . Genes wThe results of the pipeline were imported into R, version 3.6.1 using the SQMtools R package, version 0.7.0 . This paLlao’s Bath and Brain Mat Complex lies in the south-central area of the lake . It is i2, SO4, Fe, Mn, and NO3 compared to bulk lake water, which are consistent with fluids of hydrothermal origin , respectively.An initial clone library of nearly full-length SSU genes was constructed from sample 226S3  that yielded a total of 76 clones, of these 45 clones were detected at least twice  and comprised 16 operational taxonomic units (OTUs), which were then chosen to be fully sequenced. Of these OTUs, 40% were identified as putative chemoautotrophs, with most of these identified as iron-oxidizing bacteria and were represented by three OTUs belonging to the genus Amplicon sequencing was done from seven Llao’s Bath and Brain Mat Complex and one Palisades Point Pool bacterial mat samples. In total 6,646,366 raw, paired-end sequences covering the V3-V4 regions of the SSU rRNA gene were generated. Quality filtering in mothur , resulteThe highest number of observed OTUs, as determined by 97% sequence similarity, occurred in community 226S2 from the Llao’s Bath/Brain Mat Complex . The 226Acidobacteria, Actinobacteria, Bacteroidetes, Candidatus Saccharibacteria, Chloroflexi, Gemmatimonadetes, Ignavibacteriae, Latescibacteria, Parcubacteria, Planctomycetes, and Verrucomicrobia.Although many samples had elevated Fe and Mn concentrations, the bacterial community structure was revealed to be similar across all eight samples that were examined. Non-metric multidimensional scaling in three dimensions revealed even distributions among all communities with no obvious clustering, indicating a low differential in beta diversity (data not shown). The majority of reads from all communities were from the Proteobacteria  were unable to be classified representing novel Zetaproteobacteria phylotypes , nirK (nitrite reductase), and nifH (nitrogenase) had variable abundances. Ammonia monooxygenase was identified in the bacterial phylum Nitrospirota and in the class Nitrospira. However, amoA was also detected in the archaeal unclassified Thaumarchaeota at relatively high abundances. Nitrite reductase genes were identified across most of the major classes of autotrophic organisms and in the archaeal class Nitrososphaeria, whereas nitrogenase was only identified in three taxa at minimal levels. Genes involved in the sulfur cycle were more taxonomically restricted in comparison to nitrite reductase. The Betaproteobacteria contained genes encoding dsrAB and soxAB, suggesting both dissimilatory sulfate reduction and sulfur oxidation were important in respiratory pathways. Additionally, both the oxidative and reductive forms of dsrAB were found to be present. Betaproteobacteria, a taxon found to be most prevalent with SSU rRNA analysis, had nearly all the investigated functional genes except for aclB and nifH. Arsenic detoxification gene arsC (arsenate reductase) was identified in only a few taxa and was most abundant within the Alphaproteobacteria  dominated by the genus Sulfuricurvum was also observed across all eight bacterial mats and was also most highly enriched at Palisades Point Pool comprising ~1.4% of the community. Sulfuricurvum spp. are chemolithoautotrophs which oxidize sulfur and fix carbon via the rTCA cycle, which allows them to be early colonizers of hydrothermal systems  and sulfur oxidase (soxAB). The most abundant gene involved in sulfur metabolism is the dissimilatory sulfur reductase gene dsrAB, followed closely by the sulfur oxidation gene soxAB, both identified in the highest abundances as Betaproteobacteria. The co-occurrence of dsrAB and soxAB genes in Betaproteobacteria is likely due to these sulfur-oxidizing autotrophs utilizing both sets of genes in the oxidative direction; some sulfur-oxidizing bacteria have been found to express both dsrAB and soxAB genes , Alphaproteobacteria , and Nitrospirota (soxB). Detecting sulfur-oxidizing soxB genes in Nitrospirota may indicate that these organisms are oxidizing reduced sulfur compounds to fuel nitrite reduction. Previous studies have shown sulfate concentrations below 0.4 mm in freshwater oligotrophic lakes  can be found at: CM and HF conceived, designed, and supervised the analysis of the experiments. AS, LM, HF, and CM wrote the manuscript. AS conducted SSU amplicon sequencing analysis. LM conducted metagenomic sequence analysis. All authors contributed to the article and approved the submitted version.This work was funded in part by Western Washington University’s Office of Research and Sponsored Programs to CM, AS, and LM and by the Biology Alumni Student Research Fellowship to AS and LM, and by the Fraser Family Endowment summer research fund to AS. Funds were also provided by the Fouts Foundation for the Enhancement of Student Research Experiences to CM and the College of Charleston Department of Biology Research and Development fund to HF.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


CATN through the Menshutkin reactionand evaluate its potential as an impedance sensor for Escherichia coli cells. On account of its positivesurface charge  and pyridinium moieties, CATN is expected to interact favorably with bacteria thatpossess a negatively charged cell surface through electrostatic interactions.The interdigitated electrode arrays were coated with CATN using a simple yet non-traditional method of electrophoresis andthen used in two-electrode electrochemical impedance spectroscopy(EIS) measurements. The impedance response showed a linear relationshipwith the increasing concentration of E. coli. The system was sensitive to bacterial concentrations as low as∼30 CFU mL–1, which is far below the concentrationconsidered to cause illnesses. The calculated limit of detection wasas low as 2 CFU mL–1. This work is a rare exampleof a COP used in this type of bacteria sensing and is anticipatedto stimulate further interest in the synthesis of organic polymersfor EIS-based sensors.Numerous species of bacteria pose a serious threat tohuman healthand cause several million deaths annually. It is therefore essentialto have quick, efficient, and easily operable methods of bacterialcell detection. Herein, we synthesize a novel cationic covalent organicpolymer (COP) named  They are laborious, time-consuming,and expensive, require trained operators, and fail to detect microorganismsin real time or outside the laboratory.5 Thus, there is a pressing need for inexpensive, easily operable,rapid, label-free, and portable detection methods that give a quantitativereadout.The primary infections of drug-resistantbacteria cause approximately five million deaths annually, makingsuch infections the third leading cause of mortality globally.6 EIS measurements are facile to perform, do not require labeling,exhibit compatibility with complex samples, and offer high reproducibility.7 For details on the working principle of EIS,the reader is directed to the literature.8 EIS sensors register changes in the electrical properties at theirsurface (capacitance or resistance) that are caused by the interactionbetween the analyte  and the recognition elementon the surface of the electrode.9 A crucialpoint in using EIS for bacterial detection is therefore the preparationof the sensor electrodes.Electrochemical impedance spectroscopy (EIS) is a powerfultechniquethat meets the abovementioned requirements.10 Materials containing recognitionelements of interest for the EIS sensor applications are commonlydeposited onto the electrodes by drop casting,11 spin coating,12 vapor deposition,13 or cyclic voltammetry (CV).14 These methods face several challenges: some result in unevenlydeposited materials or demand high levels of sample stability, whileothers require the materials to be conductive. Herein, we presentan alternative electrophoresis method15 for IDEA preparation in which a fine suspension of particles issubjected to an external electric field. The potential applied acrossthe electrodes causes the particles from the suspension to depositonto one of the electrodes, depending on their surface charge. Thisapproach allows simple deposition on geometrically defined IDEAs withoutusing a mask. Unlike conventional methods, it is suitable for non-conductiveand insoluble materials. Given these strengths of electrophoresis,we used it to deposit a novel porphyrin-based cationic covalent organicpolymer (COP) onto a commercially available IDEA.Recently, interdigitated electrodearrays (IDEAs) have gained intereston account of their enhanced sensitivity and miniaturization of sensorplatforms., including inorganic nanoparticles,16 metal–organic frameworks (MOFs),17 molecularly imprinted polymers (MIPs),18 and block copolymers.11 These materials have achieved fairly low limits of detection (LODs),but each class faces its own challenges. MOFs often suffer from poorstability in aqueous media.19 The reliabilityof MIPs has been questioned due to nonspecific interactions with thenonimprinted surfaces.20 Antibodies usedas components of some sensors are laborious to produce and purify,and they have limited stability.21 In contrast,insoluble COPs and covalent organic frameworks (COFs) have so farnot been explored as EIS sensors for bacteria, yet they can overcomemany of the challenges other classes of materials are facing.24 They are highly thermally and chemically stable, possess tunablestructures, and have low toxicity due to purely organic structures.Thus, we herein synthesize and characterize a novel cationic COP basedon porphyrin for bacterial cell sensing. We utilize the Menshutkinreaction between the pyridyl derivative of porphyrin and 1,3,5-tris(bromomethyl)benzeneto gain a positively charged material that is anticipated to interactwith the negatively charged bacterial surface. By means of electrophoresis,we deposit the material onto a simple commerciallyavailable IDEAs with spacing between the sensing and reference electrodein the range of 100 μm such that we obtain a coated sensingelectrode and an uncoated reference electrode. Finally, this systemis used as an EIS sensor for a model bacterium, Escherichiacoli DH5α. An excellent level of sensitivityis observed with LOD calculated at 2 CFU mL–1. Incomparison with other reported systems for EIS detection of E. coli, our material exhibits one of the lowestLODs, a property that speaks to its potential.Various materialshave been tested as EIS sensors for bacterialcells25 In the current work, weutilized the pyridyl derivative of porphyrin as a core and 1,3,5-tris(bromomethyl)benzene as a linker to generatea cationic network  for 96 h. The solids formed during the reaction were then purifiedby Soxhlet extraction using first DMF and then chloroform as extractionsolvents. For achieving the best purity of CATN, it wassoaked in CHCl3 overnight. After drying at 45 °C overnight,the CATN material was fully characterized.The Menshutkin reaction is acommon reaction in organic chemistrythat uses a tertiary amine and an alkyl halide to generate a quaternaryammonium salt.rk CATN, 1a. The r13C NMR spectroscopy. TheFT-IR spectra were recorded for both monomers and CATN (S1 and S2). We note that the C–Br stretchingand the C–H wagging from the CH2Br group are presentin the linker at 578 and 1208 cm–1, respectively,but they do not appear in CATN, suggesting that the functionalgroup has reacted. The C=N stretching appears both in the porphyrinderivative and in CATN at 1592 cm–1, as expected, due to the preservation of the porphyrin core structure.However, CATN exhibits an additional bond vibration at1634 cm–1 that was ascribed to the C–N bondswithin the quaternized pyridyl ring of the porphyrin. To further confirmthe COP formation, we resorted to CP/MAS 13C NMR spectroscopy.26 Two ofthe signals correspond to the linker moiety, namely, a peak at 128ppm assigned to the benzene ring and a peak at 63 ppm that correspondsto the aliphatic C atoms. Combined, the techniques of FT-IR and NMRspectroscopy confirmed the formation of CATN with a structureshown in Molecular-level characterization was performedwith Fourier-transforminfrared (FT-IR) spectroscopy and a cross-polarization magic-anglespinning (CP/MAS) solid state troscopy1c. The rCATN . The measurements revealed that the polymerized network is thermallystable up to ∼300 °C, which is comparable to other covalentnetwork structures.27 Powder X-ray diffraction(PXRD) measurements showed that the material is amorphous in nature. The amorphous nature of thematerial was further confirmed by a selected area electron diffractionmeasurement in a transmission electron microscope . Scanning electron microscopy (SEM)revealed a spherical shape of CATN particles (S6). The particles had a diameter of ∼300nm and formed larger clusters with a size of several microns. TEMimaging was performed to investigate the nature of these spheres . Images taken at different magnificationsconfirmed the spherical morphology and revealed that the CATN particles were solid spheres.Thermogravimetric analysis (TGA) was also performed with the buildingblocks and articles 1b and S6CATN would exhibit apositive surface charge. To confirm this,we performed ζ-potential measurements in water  followedby two rounds of centrifugation at 5000 rpm for 1 min. The thus-preparedsuspension was stable and had a narrow particle size distributionin DLS measurements with a half-width of 20.1 nm . Based on 10 consecutive measurements, an averageparticle size was ∼300 nm ± 20 nm. Photographs of thesuspensions demonstrate that the stability was enhanced with the centrifugationstep, as the finest particles remained in the supernatant . The suspension was used in a custom-madeTeflon electrophoretic cell with a volume of 9.5 mL . We note that all the major peakscorresponding to the network also appear in the coated IDEAs.The coated-to-be IDEA served as one electrode,and a strip of Cufoil served as the opposite electrode. A potential difference of 3.0kV was applied, and the electric field formed in the cell caused CATN-coatedIDEA in the detection of bacterial cells, E. coli (E. coli) strain DH5α was selectedas a model organism. Since the negative surface charge is a generalfeature of any bacterial cell, we selected E. coli as a model on account of its wide availability.29 The bacterial colonies were cultured overnight until theiroptical density (O.D.) reached 1.0. The pellet was then collectedand washed with phosphate buffered saline (PBS), and dilutions inthe range of 10–9 to 10–1 wereprepared (details in the Supporting Information). E. coli detection. Thefaradaic approach using Fe2+/3+ as a redox probe was selectedover the non-faradaic one, as it is generally considered more sensitive.30 A two-electrode system was used in the potentiostaticEIS mode, and a potential of 150 mV was applied between the S andRE electrodes, corresponding to the Fe2+ oxidation potential. Although the applied voltagecan damage the bacterial cell membranes, this only happens at muchhigher voltages than those used in the current experiments.31 The EIS data for IDEA dipped in PBS are presentedwith the Nyquist and the Bode plots in Table S1 along with their relative errors;the residual plot of relative and imaginary part of the impedanceis given in Figure S11b. The PBS solutionwas modeled as a resistor , and its resistivity wasfound to be 44 Ω. The formation of the double layer on CATN is presented with a resistor and constant phase elementin parallel . The uncoated part of the electrode,which was directly accessible to electrolyte ions, was modeled asa resistor and constant phase element in parallel . The resistivity of the CATN layer is almost 100-foldhigher than that of the uncoated part at 232 kΩ versus 3 kΩ,respectively. The processes arising from the Fe2+/3+ redox probe at the electrode interface are presentedwith the Randles equivalent circuit as a capacitor in parallel witha series of a resistor for a charge transfer resistivity and a Warburgimpedance element (C|(Rct-W); labeled IV). The charge transfer resistancefor oxidation of Fe2+ to Fe3+ in the absenceof bacteria was found to be 650 Ω, which agreeswith previously reported values.33To evaluate the performance of the prepared otentialS11a. AltotentialS11a. AltE. coli. are presented in the Bodeplot in 2+/3+ redox probe at the electrode/electrolyteinterface and indicates that bacteria’s absorption to the electrodesurface, as expected, suppresses the charge transfer. The change inthe absolute impedance at 10 Hz shows a linear trend with respectto the logarithm of E. coli concentrationexpressed in CFU mL–1 (S12). Ascan be noted in the graph, our system responded to concentrationsof bacteria down to ∼30 CFU mL–1 and reachedLOD of just 2 CFU mL–1. This value is well belowwhat is considered an illness-causing concentration for various speciesof bacteria.34 It should be noted thatonly 5 min was required for each reading to stabilize which demonstratesthe quick response time of our system.EIS measurements in the presence of different concentrationof E. coli cells (Table S2). The majority of reported systems utilize bacterium-specificantibodies and are thus suitable for the sensing of a particular speciesof bacterium only. In contrast, CATN shows the abilityto detect various bacterial species regardless of their Gram-positiveor Gram-negative nature . Most of the reported systems have higher limits of detection than CATN. Furthermore, antibodies are laborious to produce andpurify, and they may have limited stability.36 Other systems with aptamers, carbohydrates, and lectins have alsobeen reported with similarly higher limits of detection, but thereis a severe lack of polymer systems explored for this type of application.Several examples of materialshave been reported for EIS sensingof 1 coated onto an IDEA in a similar fashion as CATN (detailsin the Supporting Information, E. coli cells in PBS was carried out. In contrast to CATN,the porphyrin monomer 1 performed poorly (2+ ions ofthe redox mediator in the solution.37 Oncethe pyridyl sites are saturated, the response is no longer changingwith an increasing E. coli concentration.An experiment was also performed with the monomer d poorly 4b, as itCATN was synthesized and fully characterizedwith molecular-level and macroscopic techniques. Second, electrophoresisas an alternative to the traditional methods of electrode preparationis demonstrated for an IDEA. It is found to be an efficient methodthat produces evenly coated surfaces even in miniaturized setups.Finally, EIS measurements with varying concentrations of E. coli bacterial cells showed a consistent responsewith a short stabilization time of 5 min and sensitivity for bacteriain very dilute solutions down to 30 CFU mL–1. Itis anticipated that this study will stimulate further interest inthe development of organic polymer-based electrochemical biosensorsand in further exploring alternative methods of electrode preparation.In summary, this work introduced severalnovel concepts to thefield of electrochemical sensing fabrication of bacteria. First, anew COP