The prefrontal dopamine D1 receptor (D1R) is involved in cognitive processes. Viral overexpression of this receptor in rats further increases the reward-related behaviors and even its termination induces anhedonia and helplessness. In this study, we investigated the risky decision-making during D1R overexpression and its termination. Rats conducted the rodent version of the Iowa gambling task daily. In addition, the methyl CpG–binding protein-2 (MeCP2), one regulator connecting the dopaminergic system, cognitive processes, and mood-related behavior, was investigated after completion of the behavioral tasks. D1R overexpressing subjects exhibited maladaptive risky decision-making and risky decisions returned to control levels following termination of D1R overexpression; however, after termination, animals earned less reward compared to control subjects. In this phase, MeCP2-positive cells were elevated in the right amygdala. Our results extend the previously reported behavioral changes in the D1R-manipulated animal model to increased risk-taking and revealed differential MeCP2 expression adding further evidence for a bipolar disorder-like phenotype of this model. Viral oOne aspect of cognition is decision-making, the process of making a specific choice between various alternatives. Hereby the alternatives can vary in risk and/or amount of a specific outcome. One possibility to access decision-making in humans is the Iowa gambling task (IGT) ,8. In shIn rodents, an adapted version of the IGT, the so called rat gambling task (RGT) has been used . The RGTThe goal of the present study was to investigate risky decision-making in the D1R-manipulated rat model and to identify if MeCP2 expression is altered. The RGT is based on operant conditioning and can be evaluated daily. Therefore, decision-making could be observed during D1R overexpression and after the termination within one animal.22.1ad libitum in constant temperature and humidity conditions (22 ± 2°C and 55 ± 25%) on an inverted 12 h light/dark cycle (light on at 11 p.m.). Behavioral measures began no earlier than 1 h after the beginning of the dark phase of the cycle. One week before behavioral procedures, the animals were allowed to acclimate to the animal facility. During behavioral procedures, rats were food restricted to 35–40 g/cage, available after each training and testing session. Animal weight was controlled daily to maintain their weight at constant level.A total of 13 adult, male Sprague Dawley rats were obtained from Charles River Laboratories . Rats were pair-housed in a standard cage with water available Ethical approval: The research related to animals’ use has been complied with all the relevant national regulations and institutional policies for the care and use of animals.2.22.2.1DRD1 gene) or for control condition for the red fluorescent protein dsRed. A calmodulin Kinase II subunit alpha (CamKIIα) promoter resulted in constitutive regulation of rtTA3 expression and dsRed or D1R expression was regulated by the tetracycline derivative doxycycline (DOX). Virus production was performed by the Viral Core Facility, Charité Universitätsmedizin Berlin based on the protocol of Stewart and colleagues [The used lentiviral construct was described previously . Brieflylleagues  with the2.2.27 transducing units per µL) per hemisphere was bilaterally injected into the mPFC at stereotaxic coordinates AP: + 2.7, ML: ±0.4, DV: −2.8 according to the rat brain atlas [20 min before surgery rats received metamizole (100 mg/kg), were anesthetized with isoflurane, and 1 µL of virus (2 × 10in atlas .2.2.3Virus expression was activated via 0.5 g/l DOX (Sigma-Aldrich) administration in the drinking water to produce an “ON” state. Animals received 7 continuous days of DOX (“ON” state) followed by 7 days of DOX withdrawal (“OFF” state).2.32.3.1Behavioral testing took place in an operant chamber (30 cm × 30 cm × 30 cm). Operant chamber was controlled by a Raspberry Pi 3B + connected to a touchscreen and feeder to deliver sugar pellets automatically. Custom RGT training and testing programs were written in MatLab  in combination with the Biopsychology-Toolbox .2.3.2Prior to surgery, rats learned to touch a wide white strip on the touchscreen to receive a reward. In the next training session, subjects were trained to associate three continuous hits on the wide strip to be rewarded with a sugar pellet. Rats had an answer time of 10 s to interact with the touchscreen, followed by an inter-trial interval (ITI) of 10 s. Each animal was trained for 60 min each day for at least 5 days in each training phase. The last training block consisted of the training of the actual RGT. RGT test procedures were adapted from previously established protocols . Two con2.3.3Surgery was performed after the subjects successfully learned the RGT task. One animal had to be excluded because it did not learn the RGT task. Therefore, the final sample size amounted to six animals for each group. After surgery, the animals were allowed a recovery phase of 3 days without RGT testing. Afterwards, the rats performed the RGT paradigm for 3 continuous days, referred to as baseline measurement, before DOX treatment started, and the assessment of risky decision-making behavior took place over a period of 14 days .The test consisted of a single 1 h session each day. Subjects had free access to the previously learned symbols , but each choice was associated with different consequences, which resulted in an overall advantageous or disadvantageous decision-making . Choices2.4Rats were deeply anesthetized with a ketamine/xylazine mixture (100/10 mg/kg) and intracardially perfused with 4% paraformaldehyde as previously described . Brains 2.52), 25 mM MgCl2, 10 mM dNTPs, Taq-Polymerase, and 70 ng DNA were filled up to a final volume of 52.8 µL with H2O. The PCR protocol consisted of an initiation denaturation at 95°C for 5 min, followed by 50 cycles at 95°C for 30 s, annealing for 30 s at 60°C, and primer extension for 1 min at 72°C, and a final extension at 72°C for 10 min followed by holding at 4°C in the cycler (Eppendorf). All amplified results were analyzed via agarose gel electrophoresis, staining with Midori Green (NIPPON Genetics), and visualization of DNA bands using an UV transilluminator (Vilber). The synthesized amplicon for the rtTA3 had a size of 264 bp and determined by DNA Ladder . Only subjects with a positive detection of the rtTA3 PCR product were used for data analysis.Virus placement was verified via PCR after behavioral testing. Genomic DNA was isolated from mPFC sections via QIAamp DNA FFPE Tissue Kit (Qiagen). The following primers were used to evaluate the virus placement against rtTA3 with the previously purified DNA: forward primer 5′GGAGGAACAGGAGCATCAAG3′ and reverse primer 5′GGCAGCATATCAAGGTCAAAG3′. 10× PCR-Buffer  were analyzed with repeated measures ANOVA with three factors: RGT testing day, virus group (D1R and dsRed), and virus state (“ON” and “OFF”). IHC of MeCP2-positive and DAPI-positive cells were analyzed for each region via ANOVA with virus group, region (mPFC and BLA), and hemisphere (left and right) as between factors. Correlation between MeCP2 immunoreactivity and behavior was assessed with Pearson correlation coefficients. In all cases, 33.1p = 0.27, testing day X virus group X virus state interaction, F = 1.6, p = 0.2).All animals were included for data analysis due to a positive verification of viral placement. Manipulation of the mPFC via overexpression of D1R or dsRed and its termination had no effect on the general ability to perform the task. The number of responses during the 14 days of RGT testing did not differ between the groups  = 1.4, p = 0.5). Therefore, baseline measurement could be used to account for interindividual differences between single subjects and calculate the number of disadvantageous decisions during testing condition in relation to baseline by subtracting the number of disadvantageous decisions for each test day from the mean number of disadvantageous decisions during baseline. However, raw data is illustrated in p = 0.018) was found. To determine at what virus state the risky decision-making was affected, both virus states (“ON” and “OFF”) were investigated separately (p = 0.038) was found. In the “OFF” state after termination of viral overexpression, risky decision-making did not significantly differ  = 1.5, p = 0.25). Statistical analysis testing day for each testing day revealed that D1R subjects choose more disadvantageous decisions compared to dsRed animals on day 3, day 8, and day 9 . Individual stimuli were analyzed to determine if D1R manipulation influenced a specific pattern of choice. No significant interaction of testing day X virus group X virus state was found for symbol A  = 0.6, p = 0.7), B  = 0.9, p = 0.5), C  = 1.7, p = 0.13), or D  = 0.6, p = 0.7). To further investigate the pattern of decision-making, the responses to a long delay were investigated. Manipulation had no significant effect on number of safe choices  = 0.6, p = 0.6) or risky choices  = 1.4, p = 0.27) following a long penalty.Baseline measurements of decision-making in the RGT took place after surgery and prior DOX treatment. Surgery had no significant effect on disadvantageous decisions during baseline measurements  = 0.6, parately . During p = 0.9) was present prior to DOX treatment. During testing, no significant interaction of testing day X virus group X virus state  = 1, p = 0.4) was found. However, DOX treatment and therefore virus state significantly affected the amount of earned pellets  = 7.8, p = 0.019). But this effect is only observable in dsRed subjects  = 6.3, p = 0.05) and not in D1R animals  = 1.5, p = 0.27), with dsRed animals earning more pellets during the OFF phase compared to the ON phase (t(12) = −3.6, p = 0.003).In addition, the amount of earned pellets was investigated throughout the experiment. Baseline measurements revealed that no significant interaction of testing day X virus group  = 0.1, 3.2p = 0.008). Therefore, the brain region and hemispheres were investigated separately in the following part. D1R manipulation did not affect the amount of MeCP2-positive cells in the whole mPFC  = 0.4, p = 0.7), neither in the left  = 0.1, p = 0.7) nor right hemispheres  = 0.06, p = 0.8) (p = 0.013). This effect is exclusively driven by changes in the amount of MeCP2-positive cells in the right  = 14.8, p = 0.003) and not the left hemisphere  = 0.09, p = 0.7). Post-hoc comparison revealed that the number of MeCP2-positive cells was significantly increased in the whole BLA, especially the right BLA in D1R subjects compared to dsRed animals .D1R manipulation had a significant virus group X brain region X hemisphere interaction  = 10.9, p = 0.8) . Howeverp = 0.28), nor left  = 0.2, p = 0.6) or right hemispheres  = 1.7, p = 0.2) of the BLA.To determine if changes in MeCP2-positive cells are dependent on total cell numbers, DAPI-positive cells were analyzed. D1R manipulation did not affect the amount of DAPI-positive cells within the whole BLA  = 1.5, 3.3r = −0.54, p = 0.07). Furthermore, the amount of earned pellets and MeCP2 expression were analyzed. Indeed, analysis revealed a strong trend for an association between the number of earned pellets and MeCP2 expression in the right BLA .To further analyze if MeCP2 and decision-making are connected, a correlation between disadvantageous decisions and MeCP2-positive cells was performed. Analysis revealed a trend for a connection between the elevated number of MeCP2-positive cells in the mPFC and less disadvantageous decisions  until 2 days after DOX removal. The observed risky decision-making in D1R subjects beyond day 7, the last day of DOX administration, might be due to properties of the inducible lentiviral construct. The Tet.On system needs rned off . It was Irregularities, namely similar decision-making of both groups on day 4 to day 7 might be due to inter-individual differences and intra-individual differences between days. Risky decision-making increased from day 3. This specific amount of time is necessary until viral constructs with a Tet.On system change the morphology of the magnitude of cells  or lead After termination of viral overexpression, no differences in risky decision-making could be observed between D1R transduced subjects and controls. However, while the number of earned pellets over time increased in control animals, this effect could not be observed in D1R manipulated animals. As in the DOX “OFF” phase, neither risky decisions nor number of completed trials differed between groups, it cannot be interpreted as a general increase in motivation or decrease in risky decisions in the control group, but a combination of both. D1R-manipulated animals in contrast did not increase the number of earned pellets. Previous studies reported depressive-like behavior of D1R “OFF” subjects indicated via increased anxiety, learned helplessness, reduced sucrose preference, and decreased locomotor activity ,4. The rThe role of prefrontal D1R in depression is in line with the fact that the antidepressant effect of levo-stepholidine depends on its function as an D1R agonist within the mPFC .One potential mechanism connecting the dopamine manipulation, risky decision-making, and BD is MeCP2 and its key role in affecting gene transcription. After 7 days of DOX administration followed by 7 days of DOX withdrawal, D1R-manipulated animals showed an elevated number of MeCP2-positive cells in the right BLA. No differences in the total cell numbers were found between groups indicating that altered number of MeCeP2-positive cells are not based on overall cell quantity differences. Interestingly, during this phase, no differences in risky decision-making were observed between groups. Deng et al. (2018) observed a decreased MeCP2 expression in the BLA in animals that had performed a decision-making task compared to animals that had not performed.Mecp2-null mice display reduced dopamine within the striatum [It has been previously reported that manipulation of MeCP2 influences neurotransmitter systems, such as GABA , dopaminstriatum . Here westriatum ,62 and astriatum . Even thstriatum . Interesstriatum . Amygdalstriatum . Hemisphstriatum . Severalstriatum ,69. Thisstriatum . Those fIn the present study, a potential new animal model for BD was further characterized and risky decision-making was investigated in a longitudinal manner. Increased risk-taking during the proposed mania-like phase could be reduced to baseline with the induced switch to the depressive-like phase. However, the standard deviation was high due to a low number of animals per group. Therefore, an increase and decrease in risky decisions over time were not clearly detectable. Using operant conditioning but with a less complex task might result in a more pronounced switch. An additional limiting factor of the study is the fact that MeCP2 was investigated when no difference in risky decision-making was observed between the groups. During the “ON” phase, additional differences in MeCP2 expression comparable to that reported by Deng et al. might have been observed. Another limitation is that only males were involved in this study. Differential D1R signaling between sexes may contribute to sex specific vulnerability to consequences of social withdrawal, anxiety-, and depressive-like behaviors ,72.5In conclusion, D1R overexpression within the mPFC-induced maladaptive risky decision-making is an indication for mania-like behavior. Termination of viral overexpression resulted in return to control the levels of risky decision-making, but at the same time reduced motivation to earn reward was also observed. The previously found bipolar-like behavior after D1R manipulation in the mPFC  was confBDbipolar disorderBLAbasolateral amygdalaCaMKIIcalmodulin kinase IIDAPI4′,6-diamidino-2-phenylindoleDOXdoxycyclineD1Rdopamine D1 receptorIGTiowa gambling taskIHCimmunohistochemistryANOVAanalysis of varianceMeCP2methyl CpG–binding protein-2mPFCmedial PFCRDTrisky decision-making taskRGTrat gambling taskTet.Ontetracycline-on inducible lentiviral vector system


In the early stages of diabetic retinopathy (DR), subtle biochemical and functional alterations occur in Müller cells, which are one of the components of the blood–retinal barrier (BRB). Müller cells are the principal glia of the retina and have shown a strong involvement in the maintenance of homeostasis and the development of retinal tissue. Their functional abnormalities and eventual loss have been correlated with a decrease in the tight junctions between endothelial cells and a consequent breakdown of the BRB, leading to the development of DR. We demonstrated that the endothelium reticulum (ER) triggers Müller cell death and that nuclear accumulation of glyceraldehyde 3-phosphate dehydrogenase is closely associated with ER-induced Müller cell death. In addition, induction of ER stress in Müller cells increased vascular endothelial growth factor expression but decreased pigment-epithelium-derived factor (PEDF) expression in Müller cells. We found that nobiletin, a polymethoxylated flavone from citrus explants, exerts protective action against ER-stress-induced Müller cell death. In addition, nobiletin was found to augment PEDF expression in Müller cells, which may lead to the protection of BRB integrity. These results suggest that nobiletin can be an attractive candidate for the protection of the BRB from breakdown in DR. Müller cells are one of glial cells in the retina and the signature cells to spread through the retina and are in contact with retinal vessels and neurons ,2. MülleWe have reported that MMP-9 activity and the secretion of tissue inhibitors of metalloproteinase, known as endogenic MMP inhibitors, are mediated by protein kinase C in a human Müller cell line (MIO-M1) . In addiEndothelium reticulum (ER) stress is a potential cause of retinal vascular and neuronal cell death in retinal diseases ,20,21,22Small interfering RNAs (siRNAs) were synthesized by Thermo Fisher Scientific, Inc. . We obtained antibodies against C/EBP homologous protein (CHOP) (L63F7), BiP, and caspase-3 from Cell Signaling Technology, Inc. . We also obtained antibodies against GAPDH and beta-actin (β-actin), tunicamycin (Tm), thapsigargin (Tg), and other chemicals from Sigma-Aldrich . Nobiletin  was obtained from FUJIFILM Wako Pure Chemical Co. . Following the previously reported protocol ,25,26, wv/v) fetal bovine serum , 100 U/mL of penicillin, and 100 µg/mL of streptomycin at 37 °C in a humidified 5% CO2 atmosphere until sub-confluence. After passage culture, the cells were treated for up to 72 h with ER stress inducer Tm (0.5 µg/mL) or Tg (1 µM) in the presence or absence of nobiletin (4–64 µM) or nobiletin congeners (64 µM).We received the human retinal Müller cell line (MIO-M1) from Dr. G. Astrid Limb  . MIO-M1 ® reagent  in accordance with the manufacturer’s instructions. Briefly, MIO-M1 cells were counted and seeded at 1 × 105 cells/well in a 96-well plate and cultured for 48 h with or without Tm (0.5 µg/mL) or Tg (1 µM). Following treatment, the cells were stained with alamarBlue® dye, the fluorescence intensity of the incorporated reagent was measured at 530 nm (excitation) and 590 nm (emission), and the values were calculated in relation to those of the controls considered as 100%.Cell viability was evaluated using alamarBlue5 cells/well in a 6-well plate and cultured for 48 h with or without the tested compounds. Following treatment, the cells were subjected to trypsin digestion followed by centrifugation at 400× g for 5 min to collect the cells. Collected cells were re-suspended with Ca2+- and Mg2+-free Dulbecco’s phosphate buffered saline (PBS) (−), and then, we mixed even amounts (100 µL) of cell-suspending and 0.4% trypan blue solutions . We analyzed blinded specimens using a hemocytometer and determined the quantity of cell death as the number of blue cells/total cells. The specimens were then unblinded, and the numerical values from four independent tests were averaged. We obtained digital images using an OLYMPUS CKX53 microscope  at a magnification of 40×.We conducted cell viability analysis using trypan blue. Briefly, MIO-M1 cells were counted and seeded at 1 × 10v/v) Triton® X-100, 10 mM ethylenediaminetetraacetic acid (EDTA), and 10 mM Tris-HCl (pH 7.4), followed by centrifugation at 20400× g for 7 min to extract total DNA. The supernatants including DNA fragments were incubated with 5 µg/mL of RNase, DNase-free  for 1 h at 37 °C and then treated with 4 µg/mL of proteinase K for 30 min at 50 °C to deactivate RNase activity. The purified DNA solution was incubated with 1M NaCl and 50% isopropanol overnight at −20 °C. After centrifugation at 20,400× g for 20 min, the isolated DNA fragments were re-dissolved with loading buffer containing 0.05% (w/v) bromophenol blue and 6.7% (w/v) sucrose and then subjected to electrophoresis with 2% (w/v) agarose. After electrophoresis, we visualized the DNA fragments by staining them with ethidium bromide.For DNA fragmentation analysis, we cultured MIO-M1 cells in a 100 mm dish until sub-confluence. Following treatment with Tm (0.5 µg/mL) or Tg (1 µM), adherent cells were harvested by scraping in lysis buffer containing 0.5% (5 cells/well in a 6-well plate. Following treatment with Tm (0.5 µg/mL) in the presence or absence of nobiletin (64 µM), cell fractions were collected using Blue Loading Buffer  containing 62.5 mM Tris-HCl (pH 6.8), 2% (w/v) sodium dodecyl sulfate (SDS), 10% glycerol, 0.01% (w/v) bromophenol blue, and 41.6 mM dithiothreitol. The isolated cellular proteins were subjected to Western blot analysis using specific rabbit antibodies against CHOP (L63F7) (1:2000 dilution), BiP (1:2000 dilution), and caspase-3 (1:1000 dilution) and specific mouse antibodies against β-actin . We used the β-actin antibody as a loading control to check equal loading of protein on each lane. We detected immunoreactive CHOP and BiP using enhanced chemiluminescence (ECL) Western Blotting Detection Reagent  after the isolated cellular proteins were complexed with horseradish-peroxidase-conjugated anti-rabbit immunoglobulin G (IgG). For the detection of GAPDH and Siah-1, cell fractions were prepared using radio-immunoprecipitation assay (RIPA) buffer containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 200 µM phenylmethylsulphonyl fluoride (PMSF), 100 µM sodium orthovanadate, and proteinase inhibitor cocktail solution. Quantitative analyses were conducted using Image J , Bethesda, MD), as previously reported i and ROS production. Karthikeyan et al. [In this study, we demonstrated that ER stress induces apoptotic cell death in retinal Müller cells, through experiments for the detection of DNA fragmentation, cleaved caspase-3, and CHOP expression, which are key markers of apoptosis. Previous reports have suggested that nuclear accumulation of GAPDH plays a role in apoptotic Müller cell death under high-glucose conditions  and is in et al.  demonstrn et al. . Severaln et al. ,40. The The pharmacological actions of PMFs are directly related to their chemical structure and depend on the number and positions of hydroxyl groups and methoxy groups ,42. For In conclusion, Müller cells have unique characteristics, such as maintenance of BRB integrity and stem cell potential, that make them suitable targets for the prevention and treatment of DR. In this study, we demonstrated that nobiletin prevents ER-stress-induced Müller cell death by inhibiting the translocation of GAPDH to the nucleus. Therefore, therapies targeted toward preventing GAPDH nuclear localization in retinal Müller cells should help in arresting the development and also the progression of DR. We need further research to clarify weather ER stress actually induces Müller cell death through GAPDH nuclear translocation in animal models. We will examine the preventive action on Müller cell death in diabetic retina by nobiletin. In addition, we will examine nobiletin-augmented PEDF expression in Müller cells, which may lead to the protection of BRB integrity through improvement in the VEGF/PEDF ratio. Müller cells have been shown to become mired in dysfunction in the early stages of DR; therefore, we consider that adequate modulation of Müller cell responses by intake of nobiletin may lead to a promising prevention therapy for DR. Prospects of this research include conjugating the nobiletin molecule with a nanocarrier for effective and sustainable delivery to the degenerating retina.


Escherichia coli (AIEC) has been linked to the pathogenesis of Crohn’s Disease. AIEC’s characteristics, as well as its interaction with the gut immune system and its role in intestinal epithelial barrier dysfunction, have been extensively studied. Nevertheless, the currently available techniques to investigate the cross-talk between this pathogen and intestinal epithelial cells (IECs) are based on the infection of immortalized cell lines. Despite their many advantages, cell lines cannot reproduce the conditions in tissues, nor do they reflect interindividual variability or gut location-specific traits. In that sense, the use of human primary cultures, either healthy or diseased, offers a system that can overcome all of these limitations. Here, we developed a new infection model by using freshly isolated human IECs. For the first time, we generated and infected monolayer cultures derived from human colonic organoids to study the mechanisms and effects of AIEC adherence and invasion on primary human epithelial cells. To establish the optimal conditions for AIEC invasion studies in human primary organoid-derived epithelial monolayers, we designed an infection-kinetics study to assess the infection dynamics at different time points, as well as with two multiplicities of infection (MOI). Overall, this method provides a model for the study of host response to AIEC infections, as well as for the understanding of the molecular mechanisms involved in adhesion, invasion and intracellular replication. Therefore, it represents a promising tool for elucidating the cross-talk between AIEC and the intestinal epithelium in healthy and diseased tissues.Over the last decades, Adherent-Invasive  Escherichia coli (E. coli) strains are widely known inhabitants of the healthy human gut microbiota, being one of the first colonizers as well as among the most prevalent microorganisms in the intestines  pathotype, have acquired a virulent nature. Despite the lack of typical enteropathogenic E. coli virulent factors in AIEC isolates, these are able not only to adhere to and invade intestinal epithelial cells (IECs), but also to replicate within macrophages without inducing cell death, thus evading protective host immune responses  and may constitute more than the 50% of the total number of bacteria both in early and chronic ileal lesions , 9. AIECty in CD . AIEC mety in CD , 21 as wty in CD . Adhesioty in CD , 24. On ty in CD . All thety in CD  and intety in CD . AIEC arty in CD –30, and ty in CD . Overallty in CD .in vivo environment offer the advantage of providing a more physiological experimental setting due to their mimesis of the tissue of origin, phenotype and structure. Hence, infecting human colonocytes derived from patient biopsies might represent a promising strategy for studying the intestinal epithelium response to AIEC, as well as new pathogenicity mechanisms associated with this pathobiont. Such an approach could lead to the discovery of new disease biomarkers and new therapeutic targets. To our knowledge, there are few publicly available reports that analyze the interaction between enteric pathogens and human isolated IECs : intestinal samples of healthy sigmoid colon with no evidence of macroscopic inflammatory lesions were obtained from subjects undergoing surgery for left-sided colorectal cancer (CRC) or routine endoscopy for CRC screening. For surgical pieces, a segment of healthy mucosa was collected at least 10 cm from the margin of the affected area. Biopsy samples showed no evidence of neoplastic lesions. However, biopsies were not specifically assessed for signs of microscopic inflammation.Surgical or biopsy samples were immediately used for generating EpOCs. Cell lines: Intestine-407 – I407 –  cell line.E. coli K12 C600 [a prototypical derived laboratory strain which has been extensively used for molecular microbiology and bacterial physiology studies since its isolation in 1954 .Heat inactivated – at 56°C for 30 minutes – fetal bovine serum – FBS – South American .Washing medium (WM) : -80°C stored bottles were thawed overnight (ON) on ice. 500 µl aliquots were prepared and frozen at -20°C for later use. Once thawed, aliquots were stored at 4°C for no longer than one week.Cell Recovery solution .Dissociation medium (Wnt3a-conditioned medium + Y (STEM+Y medium) .Differentiation medium (DIFF medium) . WC: 170,000 U/L trypsin and 200mg/L EDTA.EMEM Complete Medium (Liquid Luria-Bertani (LB) Broth .Minimal media .Ringer Solution .LB Agar .Chloroform .RNeasy Kit .High Capacity cDNA Reverse Transcription kit .RNAse Inhibitor .TaqMan™ Fast Universal PCR Master Mix (2X), no AmpErase™ UNG .Nuclease Free Water .MYC (Mm00487804_m1), MKI67 (Mm01278617_m1), AXIN2 (Hs00610344_m1), TJP3 (Hs00274276_m1), TFF3 (Hs00902278_m1), MUC2 (Hs03005094_m1), LGR5 (Hs00173664_m1), FYN (Hs00176628_m1), CDCA7 (Hs00230589_m1), ZG16 (Hs00380609_m1), TLR3 (Hs01551078_m1), TLR4 (Hs00152939_m1), CCL20 (Hs01011368_m1), CXCL1 (Hs00605382_gH), CXCL2 (Hs00601975_m1), ANPEP (Hs00952642_m1), FABP2 (Hs01573164_g1), AQP8 (Hs00154124_m1), CA1 (Hs01100176_m1), CHGA (Hs00154441_m1), CEACAM7 (Hs03988977_m1), OCLN (Hs00170162_m1), PHGDH (Hs01106330_m1), CYP1B1 (Hs00164383_m1),  and ACTB  with VIC dye.Pre-designed TaqMan Assays : Paraformaldehyde aqueous solution – PFA – . WC: 20 mM.Bovine serum albumin – BSA – . WC: 1%.Primary antibodies: mouse anti-EpCAM , rabbit anti-E-Cadherin , mouse anti-KI67 , rabbit anti-MUC2 , mouse anti-VILLIN  all diluted in 1% BSA.Secondary antibodies: anti-mouse Cy3  and anti-rabbit Alexa 488  all diluted in 1% BSA.4′,6-diamidino-2-phenylindole (DAPI) .Alexa Fluor™ 555 Phalloidin .Mounting medium: glycerol (Sigma. Ref. G5516-500). WC: 80%.Dulbecco phosphate-buffered saline – DPBS – .Triton X-100 .2O.Distilled HCellTox™ Green Cytotoxicity Assay .Digitonin . WC: 100 µg/ml.1.5 ml tubes .1.5 ml tubes RNAse free .Falcon 15ml Sterile Disposable Conical Centrifuge Tubes .Falcon 50ml Sterile Disposable Conical Centrifuge Tubes .Filtered pipette tips – 10 µl, 20 µl, 200 µl, 1000 µl – .Serological pipettes: 5, 10, 25 ml and 50 ml .Scalpels .Microscope slides .® Glasstic Slide 10 With Counting Grids .KOVABD Emerald 5 ml syringes .® 3 21Gx1’’ 0.8mmx25mm .BD MicrolanceMicroAmp™ Optical Adhesive Film .48-well plates .24-well plates .µ-Slide 8 Well ibiTreat: #1.5 polymer coverslip, tissue culture treated, sterilized .T25 and T75 tissue culture flasks .120x120mm Petri dishes .Microplate 96 well qPCR FAST THERMAL CYCLING .Vortex mixer.C Microvolume UV-Vis Spectrophotometer Precision Scale.Thermo Scientific™ NanoDrop™ OneVeriti 96-well Thermal Cycler (Applied Biosystems).Benchtop shaker (BOECO Mini-Rocker Shaker MR-1).Benchtop refrigerated centrifuge .Inverted microscope (Olympus X51 Inverted Microscope).Fluorescence Inverted Microscope Nikon S Ti.2).Cell incubator .Leica TCS_SP5 scanning spectral confocal microscope  equipped with an DMI 6000 inverted fluorescence microscope, blue diode (405nm), Argon (488nm), diode pumped solid state (561nm) lasers and a Apochromat 63X oil immersion objective (NA 1.4).Zeiss LSM880 laser scanning spectral confocal microscope  equipped with an Axio Observer 7 inverted microscope, blue diode (405nm), Argon (488nm), diode pumped solid state (561nm) and HeNe (633nm) lasers and a Plan Apochromat 63X oil (NA 1.4) immersion objective lenses.Micropipettes and Pipettor.Tube racks.Refrigerated racks.Aluminum foil.Cell-counter.Forceps.Scissors.Spectrophotometer Cuvettes.https://imagej.net/Fiji).Image processing software .Data software analysis Graphpad Prism 5  before being infected by In this section we will accurately describe the optimized protocol for ODM generation from EpOCs, ODM differentiation and AIEC infection of d-ODMs to evaluate AIEC’s invasive capacity in differentiated primary epithelial cells.EpOCs were generated as previously described , 45. BriΔ CRITICAL. Based on our experience, every EpOCs drop contains around 40,000-100,000 cells. Thus, depending on the final number of single cells needed for the invasion assay, a determined number of EpOCs drops will be used at the starting point.To generate ODMs from EpOCs, the protocol was as follows:(1) Matrigel drops containing EpOCs were washed with cold DPBS and collected in Cell Recovery solution (300 µl/well) at 4°C for 40 minutes. Every 5-10 minutes, cell suspensions were gently inverted upside-down.g for 4 minutes at 4°C.(2) 4-5 ml of washing medium (WM) ((3) Supernatant was discarded and the pellet was resuspended in Dissociation Medium ((4) After organoid release, cells were mechanically disaggregated using a 5 ml syringe with a 21G needle until the cells were totally dissociated  Cells were centrifuged at 800(6) The remaining pellet was resuspended in 1-2 ml of WM for manual cell counting:a. Cells (10 µl) were diluted 1:1 with Trypan blue Solution.b. 10 µl of the cell suspension was loaded into a Glasstic Slide 10 With Counting Grids and the cell number was estimated according to the manufacturer’s recommendations. The mortality rate  was usually below 10%.5 cells/well/250 µl.(7) Single cells were again centrifuged, and the pellet was resuspended in the required volume of STEM+Y medium (2 ((8) Cells were seeded on Matrigel pre-coated 48-well plates and incubated for 24h at 37°C 5% CO2 for an additional 48h.After incubation, ODMs were induced to differentiation. To this end, STEM + Y medium was discarded and ODMs were washed with DPBS and Advanced DMEM/F12 medium (300 µl/well) at RT to remove dead cells. DIFF medium (250 µl/well)  reached 100% confluence 1-2 days after differentiation (Δ FOR SYSTEM SET UP ONLY. The methodology described in this section was only utilized during optimization and until the protocol we established was entrenched (2O). Target genes were amplified and quantified using ACTB as the endogenous control. PCR reaction was run in the ABI PRISM 7500 Fast RT-PCR System using the following program: a holding stage for 20 seconds at 95°C and a cycling stage for 3 minutes at 95°C and 30 seconds at 60°C during 40 cycles. Target gene expression values relative to ACTB were expressed as arbitrary units (AU) following this formula:Both ODMs and d-ODMs were harvested in Trizol for RNA extraction  After two DPBS washes, the cell monolayer was fixed with 2% PFA (1:1 4% PFA + DPBS) for 5 minutes at RT and then with 4% PFA for 10 minutes at RT.st fast; 2nd and 3rd 5 minutes at RT.(2) Cells were washed three times with DPBS: 1Δ CRITICAL. STOP POINT – Cells were stored at 4°C covered in DPBS (300 µl) or were immediately used for staining.(3) 250 µl of 20mM Glycine was added for 10 minutes at RT to reduce background staining.(4) DPBS washes were conducted as described in step (2).(5) For permeabilization, 250 µl of 0.25% Triton X100 were added for 20 minutes at RT.(6) Cells were then washed 3 additional times – 5 minutes each – with DPBS.(7) To block non-specific binding, 250 µl of 1% BSA was applied and incubated at RT for 30-45 minutes.(8) Primary antibodies (150-200 µl/well) – EpCAM, E-Cadherin, MUC2, Villin or KI67 – were added at the specified dilutions (in 1% BSA) and incubated ON at 4°C.Materials section) in 1% BSA for 1h of incubation at RT. Cells were washed 3x with DPBS at RT as described in step 6.(9) After 3 DPBS washes (as in step 6), cells were incubated with 150-200 µl/well of the secondary antibodies – Anti-mouse Cy3 and Anti-rabbit 488 – at the specified dilutions (see (10) For DNA counterstaining, DAPI (250 µl/well) was added and incubated at RT for 10 minutes. Washes were repeated as in step 6.(11) Finally, 200 µl/well of mounting medium (80% Glycerol in DPBS) were added. Samples were stored at 4°C for subsequent fluorescent microscope observation.Δ CRITICAL. After adding the secondary antibodies, cells were kept in the dark.Δ CRITICAL. For short-term storage, stained cells were kept at 4°C or at -20°C for up to 6 months.E. coli K12 strains were cultured in 1.5 ml of LB Broth and incubated for 12-18 hours at 37°C without shaking :The assay was performed at Multiplicity of Infection (MOI) 10, as described previously , 47. InfΔ FOR SYSTEM SET UP ONLY. This model of infection was only performed until establishment of the d-ODM-based gentamicin protection assay.5 EpOCs-derived single cells/well in 48-well plates based on previous experience (data not published), although this may need to be adjusted by each lab as culture conditions can vary slightly. To monitor the number of cells present in the plate at 100% confluency, experiments were performed seeding the above number of cells/well and counting cells present in d-ODM prior to infection. This step proved decisive in order to adjust the needed inoculum of bacteria and achieve the desired MOI. Briefly, d-ODMs were washed with DPBS to remove non-attached cells. Trypsin-EDTA (150 µl) was added to the culture for 10-15 minutes at 37°C 5% CO2. Detached cells were collected and resuspended in Advanced DMEM/F12 + 10% FBS. These last two steps were repeated until complete cell-detachment was achieved. Cells were centrifuged at 800g for 4min and at 4°C and resuspended in 200 µl of Advanced DMEM/F12 + 10% FBS for cell counting as explained in a previous section (see the ODM generation section).To infect cells with a determined MOI, it is crucial to know the exact number of cells seeded as a monolayer at the time of infection. In our particular case, we seeded 2x10Δ CRITICAL. It is important to not exceed the 10-15 minutes incubation with Trypsin-EDTA in order to prevent cell death.5 cells/well prior to infection  were multiplied 20- or 100-times to determine the bacterial colony forming units (cfu)/ml required for reaching each MOI value. In our case, 3,6x106 or 18 x106E. coli cfu/ml were needed.Thus, d-ODM counted-cells   ON bacterial cell suspensions (500 µl) were diluted 1:1 with LB medium and 1 ml was transferred to a cuvette.(2) The OD was measured with a spectrophotometer at a wavelength (λ) of 600 nm.(3) OD adjustment was achieved in accordance with the following formula:iV; Initial Volume (required volume for the ON culture)fOD; Final OD (0.1 in this case)fV; Final Volume (1 ml)mOD; Measured OD(4) The calculated iV and DIFF-MM  and negative (non-invasive) control, respectively. Infection was performed using d-ODMs generated from 7 different subjects  and fresh DIFF-MM was added (500 µl/well). Then, the corresponding volume of OD 0.1 bacterial suspension Table 1 Δ CRITICAL. The Triton X-100 step should not take longer than 30 minutes in order to avoid bacterial cell death.-1 and 10-2, as well as the non-diluted samples, were plated (25 µl) in LB agar plates  was calculated considering the amount of bacteria initially inoculated to d-ODMs:6 for MOI 20 or 18 x106 for MOI 100.†: in this context, 3.6x10As previously described by Darfeuille-Michaud et al., who studied AIEC infection by using immortalized cell lines , we consAnticipated Results section).Notice that even though the techniques detailed herein are not mandatory, they were performed to obtain a deeper understanding of the results obtained from the AIEC infection of d-ODM (see Immunofluorescence Staining section). DAPI (250 µl/well) was then added and the protocol continued as described in steps 10 and 11. The assay was performed with cells obtained from 3 different subjects  and MOI 100) seeded in µ-Slide 8 Well ibiTreat chambers, were processed for fluorescent cyto-staining. This procedure was identical to that used for ODM/d-ODM characterization until step (7) of the Immunofluorescence Staining section. After incubation with the blocking solution, 150-200 µl/well of Phalloidin diluted 1:40 in 1% BSA was added for staining of the actin filaments. After 1-hour incubation at RT, cells were washed 3x with DPBS at RT as in step 6  previously diluted according to the manufacturer’s instructions (1:500 in Assay Buffer). After ≥15 minutes of incubation at 37°C in the dark, cultures were observed using a fluorescence microscope. A positive control of cell death was included by adding 100 µg/ml of digitonin in the uninfected d-ODM for 1 hour.Quantitative data are expressed as the standard error of the mean (SEM). A paired t-test was performed to examine statistically different expression patterns between 2 groups, and a 2-way ANOVA test to examine statistical significance in multiple group data sets, followed by a Tukey test correction for multiple testing. A p-value of <0.05 was considered statistically significant. Data were analyzed using Graphpad Prism 8 (version 8.2.1).The intestinal crypt is organized so that the stem-cell compartment resides at the bottom, thereby protected from the luminal content, while the differentiated and surface epithelium is more directly in contact with the microbiota and its metabolites. In order to develop a model that would more closely resemble the type of upper crypt epithelium that is more susceptible to bacterial interactions and based on previous results from our lab , 50, we 5 single cells/well. On day 1, cells created clusters that alternated with empty areas, while on day 3, the monolayers reached 100% confluence, the requirement for AIEC infection  for the AIEC invasion assay. Based on previous experiments by our lab, we seeded 2x10lls/well , 50.AXIN2, MYC and MKI67, , were significantly higher in ODMs compared to d-ODMs. On the other hand, transcriptional levels of the differentiation markers TFF3 and MUC2, showed an up-regulation, despite not statistically significant, in d-ODMs compared to ODMs. Similarly, TJP3, representative marker of epithelial cell junctions, was significantly up-regulated in d-ODM. Other markers included in the analysis  showed an invasion index (INV-I%) below the established background (<0.1%). Moreover, LF82 showed a time-dependent increment of the INV-I%, and thus of the invasion capacity and/or intracellular multiplication in the AIEC-reference strain. Nevertheless, this capability was significantly higher compared to the K12 strain, both at 6 and 7 hours after infection for MOI 20 (via the CellTox Green assay (data not shown).To the date, the characteristics and pathogenicity of AIECs have been studied so far by employing immortalized cell lines . Here, wBy staining the eukaryotic actin filaments Figure 4In summary, we demonstrated the capacity of AIECs to invade the epithelial cells of d-ODMs. Thus, we present here a method that can be applied in multiple AIEC-IEC cross-talk studies, not only to discover new AIEC pathogenic mechanisms and host implicated molecules, but also, and more relevantly, to establish a possible starting point for further clinically oriented applications.In the following section we will highlight which, in our opinion, are the most noteworthy advantages and disadvantages that this protocol presents. By doing so, we can focus on its practicality and try to overcome its limitations.Working with samples isolated from their natural surroundings (the human intestine in this case), preserves the cytoarchitecture and most of the intercellular connections and interactions. Moreover, it also provides the option to consider the interindividual variability that exists between different subjects.Working with ODMs and d-ODMs offers accessibility to the IECs-apical side, contrary to 3D-organoid structures which may be required for infectious models.We also demonstrate its great reproducibility, a highly relevant feature when one considers the differences between individuals and their responses to microbes.Given the fact that ODMs and d-ODMs can be generated from potentially any individual, including patients suffering from IBD, this method offers the possibility of testing personalized treatment approaches.Time-consuming. EpOCs and ODM cultures are time intensive. Nonetheless, once the system is set up, organoid-derived single cells can be more rapidly obtained, shortening the time required for the entire procedure.Costly. EpOCs and ODM cultures could remain unaffordable for some research groups due to the high costliness of most of the reagents that are required.Access to patient specimens is required.Sample-to-sample variability might lead to differences in the number of cells obtained from every EpOCs drop. This might be an important limiting aspect that should be considered when applying the method described here.ex vivo primary cultures (as we mentioned in the previous section) is that these might offer a more physiological view of the host’s response to AIEC infections. However, unpredictable biological variability could hinder the obtainment of the necessary cell concentration at the starting point. In that context, establishing an accurate and standardized protocol is crucial to facilitating reproducibility and enabling results comparisons. In our case, reproducibility was assessed first, by testing the gene and protein expression levels of the 2D cultures derived from the different donors. Moreover, AIEC infections were carried out in duplicate, exposing those EpOCs-derived d-ODMs from seven different individuals to E. coli. This validation approach is of great importance in host-pathogen interaction studies, considering the real differences in infection susceptibility among individuals and the divergence in host responses to a pathogen  achieve the optimal differentiated phenotype of the monolayer cultures, and (2) to properly adjust the number of exposed bacterial cells to the d-ODMs (MOI), which can greatly affect the results.While a more extensive characterization of the d-ODM at protein level would add robustness to our culture system, our results suggest that ODMs and d-ODMs preserve the characteristics of the intestinal epithelium E. coli). Over time, increasing amounts of invasive bacteria were detected, with higher values evident when smaller amounts of bacteria (MOI 20) were added to the culture at the starting point. Based on this finding, we concluded that adding more bacteria does not directly correlate with higher invasion values. Similar results were obtained by Boudeau et al. in 1999 with Hep-2 cells  under grant agreement number FPI BES-2016-076642. ID is funded by the Horizon 2020 Framework Programme (EU for Research and Innovation H2020), Grant 720905. QB-R was supported by the Programa d’Ajuts per a Investigadors en Formació de la Universitat de Girona – IFUdG 2019/21. AS is funded by the Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD) and Grant RTI2018- 096946-B-I00 from the Ministerio de Ciencia, Innovación y Universidades, Spain. MM-M is funded by the Spanish Ministry of Economy and Competitiveness through project SAF2017-82261-P, which is co-funded by the European Regional Development Fund.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.


Tamarindus indica, and isolated, identified, and characterized 50 fungal strains from the leaf litter. The fungal diversity encompassed three phyla, namely Mucoromycota, Ascomycota, and Basidiomycota, and 23 genera. Using various media, we further investigated their functional potential. Most of the fungal strains produced siderophores and presented proteolytic activities. The majority were also able to decompose cellulose and xylan, but only a few were able to solubilize inorganic phosphate. Regarding oxalate metabolism, several strains were able to produce calcium oxalate crystals while others decomposed calcium oxalate. These results challenge the current view of the OCP by indicating that fungi are both oxalate producers and degraders. Moreover, they strengthen the importance of the role of fungi in C, N, Ca, and Fe cycles.The oxalate-carbonate pathway (OCP) is a biogeochemical process linking oxalate oxidation and carbonate precipitation. Currently, this pathway is described as a tripartite association involving oxalogenic plants, oxalogenic fungi, and oxalotrophic bacteria. While the OCP has recently received increasing interest given its potential for capturing carbon in soils, there are still many unknowns, especially regarding the taxonomic and functional diversity of the fungi involved in this pathway. To fill this gap, we described an active OCP site in Madagascar, under the influence of the oxalogenic tree  Microorganisms are essential drivers of plant material degradation and recycling in terrestrial ecosystems , and as aK1 = 1.27; paK2 = 4.27) forms its conjugate base, namely oxalate, which can bind to calcium cations. The resulting Caox crystals can be found in leaf, wood, and root tissues, as well as in the surrounding soil [3) under environmental conditions, this can lead to the formation of a CaCO3 phase. The OCP has been observed mainly in tropical ecosystems, and this process has been reported to occur in soil [The OCP is a biogeochemical process involving plant, fungal, and bacterial partners . In thising soil ,18. Simiing soil ,20. Thesing soil , leading in soil  and in t in soil .i) isolating and phylogenetically identifying fungi associated with an active OCP, (ii) characterizing the functional potential of these fungal strains, especially regarding their contributions to C, N, P, Ca and Fe cycles, and (iii) clarifying the role of fungi in the functioning of the OCP.The OCP has recently received a lot of interest for its potential role in the capture and fixation of carbon in a mineral form in soils ,25,26, bTamarindus indica in November 2014, in the Kirindy forest, Madagascar . Mean annual temperature and mean annual precipitation were 24.8 °C and 785 mm, respectively. We collected the litter in 50 mL sterile Falcon tubes, using sterile tweezers to prevent biological contamination. We only sampled the upper litter in order to avoid contamination from soil organisms. The sampled litter was stored at 4 °C until laboratory analysis. Additionally, we dug and sampled four soil profiles, three at the base of the T. indica trunk and another at 15 m away from the tree. The latter was considered as not under the influence of other oxalogenic plants. Each profile was 80 cm deep. For each profile, we sampled 50 g of soil from 5 cm layers, at four depths: 0–5 cm, 10–15 cm, 20–25 cm, and 40–45 cm.We sampled litter of 2O with a MetrohmTM pH-meter, using 10 g of 2 mm sieved soil mixed with 25 mL of deionized water and agitated for 16 h [We measured soil pH Hfor 16 h . Litter for 16 h .2PO4 0.1%; MgSO4∙7H2O 0.02%; KCl 0.02%; NaNO3 0.2%; yeast extract 0.02% and agar 1.3% (w/v) [0 to 10−5. We plated fifty µL of each dilution in Petri dishes also containing LCA medium with 2 antibiotics (chloramphenicol 0.01% and streptomycin 0.01%). For each dilution, we made 5 replicates. All Petri dishes were incubated at 22 °C in dark conditions. We checked the plates daily for fungal growth, and we transferred individual mycelia to new LCA plates containing the same two antibiotics.To cover a broader range of microenvironments, we used two approaches for fungal isolation. First, we sampled various litter fragments under sterile conditions and washed them 3 times with sterile water by vortexing for 5 min. Subsequently, we directly inoculated the litter fragments in Petri dishes containing lignocellulose agar (LCA) medium with 2 antibiotics (chloramphenicol 0.01% and streptomycin 0.01%). LCA medium contains glucose 0.1%; KH3% (w/v) . The pH 3% (w/v) . Second,−1), pH adjusted to 7, and with the fungicide cycloheximide 100 mg·L−1. For each dilution, 5 replicates were made, and all plates were incubated at 22 °C in the dark. The presence of halos around the colonies indicated oxalotrophic activity of the colony. We estimated the bacterial concentration by counting the number of colony-forming units (CFU) per gram of litter and using only one dilution of the suspensions .To quantify the proportion of culturable oxalotrophic bacteria, we plated the microbial suspensions and the dilutions described above on a modified bi-layered AB Schlegel medium  with calWe extracted DNA from the mycelium of each fungal isolate using the ZR Fungal/Bacterial DNA MiniPrep kit  and following the manufacturer’s instructions. Subsequently, we targeted the 28S large subunit (LSU) rRNA gene as a fungal taxonomic marker. We used the primer pair LROR_F 5′-CCGCTGAACTTAAGCATATCAATA-3′ and LR5-F 5′-CGATCGATTTGCACGTCAGA-3′ to amplify a fragment of the LSU rRNA gene , with thFor each 28S rRNA gene sequence, we performed a BLASTN search  with botn = 3 replicates) was plotted over time, and a linear growth phase was identified. We estimated hyphal extension rate by linear regression of the linear growth phase [−1 as sole source of carbon; K2HPO4 1.0 g·L−1; (NH4)2SO4 1.0 g·L−1; MgSO4∙7H2O 0.5 g·L−1; NaCl 0.5 g·L−1; agar 15 g·L−1; and pH was adjusted to 5 [−1 as the sole source of carbon; K2HPO4 1.0 g·L−1; (NH4)2SO4 1.0 g·L−1; MgSO4∙7H2O 0.5 g·L−1; NaCl 0.5 g·L−1; agar 15 g·L−1; and pH was adjusted to 5 [−1) agar (15 g·L−1) medium supplemented with 5 g·L−1 CaCO3 [−1) with 5 g·L−1 CaCO3 was also tested, but since not all the strains were able to grow on it, it was not included in the final analysis. We also investigated the dissolution of Caox by inoculating strains on a bi-layered AB Schlegel medium [−1 Caox. Oxalate degradation by a fungal strain was indicated by the presence of a halo around the mycelium. We searched for the production of siderophores by the fungal strains by inoculating them on a chrome azurol S (CAS) agar medium prepared following the method of Tarnawski et al. [−1, Migros) [3(PO4)2 5 g·L−1; MgCl2∙6H2O 5 g·L−1; MgSO4∙7H2O 0.25 g·L−1; KCl 0.2 g·L−1; (NH4)2SO4 0.1 g·L−1; glucose 10 g·L−1; agar 15 g·L−1; pH 7. All the media were autoclaved at 121 °C for 20 min.To describe the functional potential of the fifty isolated strains, we used ten selective media. First, we used the LCA medium (described above) as a minimal medium to measure fungal radial growth. We observed the fungal colonies daily and measured the diameter of the colonies until they reached the periphery of the plates. For each strain, the diameter of the colony  . Finally Migros) , composen = 3 Petri dishes of each medium with a single fungal plug. Subsequently, we sealed them with Parafilm M and incubated them at 30 °C in dark conditions before analysis. In total, we analyzed 10 media × 50 strains × 3 replicates = 1500 Petri dishes.For each strain, we inoculated −1) agar (15 g·L−1) medium supplemented with 5 g·L−1 CaCO3, we evaluated the production of Caox crystals for each fungal strain with a Leica DMR optical microscope and a ×400 magnification, following the procedure described in [Using a malt (12 g·Lribed in . Becauseggplot2 package [D statistic [caper package (phylo.d function with 10⁵ permutations). A value of D = 0 indicates phylogenetically conserved traits as expected under a Brownian motion model, whereas D = 1 indicates randomly distributed binary traits. For the continuous trait, we used Pagel’s λ metric [phytools package (phylosig function) [We performed statistical analyses with R version 4.0.2  and visu package . The exitatistic , computeλ metric  with theunction) . Here, λ3). Concerning the first element, T. indica is known to be an oxalogenic plant in which calcium oxalate crystals have been observed in secondary xylem [7 bacterial CFU.g−1 litter, including 1.76 107 oxalotrophic CFU.g−1 litter , was able to degrade RBBR that was used as a proxy for ligninolytic activity, and this strain also degraded xylan  to 1709.7 µm h−1 . No significant differences were observed between the distribution of hyphal extension rates within the different detected functions . For all these traits, we tested for the presence of a phylogenetic signal , Caox crystal production , Caox degradation , siderophore production  and hyphal extension rate .Each fungal strain was further characterized by measuring eight binary and one continuous trait, using various selective media . All theed xylan . Caox pred xylan , encompac signal . A signiT. indica as an oxalogenic tree driving an active OCP. This species is relatively widespread in Madagascar [We reported here the first evidence of an active OCP in Madagascar. To date, the OCP has been observed in numerous tropical and semiarid environments, with different tree species, and across various geographical areas, such as Israel , the USAdagascar , but mordagascar , suggestIn the studied litter samples, the concentration of culturable oxalotrophic bacteria appeared to be two times higher than the one observed in OCP soil from Bolivia . While cFusarium, Trichoderma and Penicillium [Ceratobasidium genus, was also reported as an abundant member in decomposing leaf litter [While the main goal of the present study was not to obtain a taxonomic overview of the fungal diversity associated with the OCP litter, we provide the first glimpse of this diversity via a culture-dependent approach. The described fifty strains represent 23 genera encompassing three phyla. With 42 strains, Ascomycota was the most abundant phylum in the dataset, which was also the case for studies investigating fungal litter diversity via metabarcoding approaches ,67. Amonicillium . Lastly,f litter , this fuf litter  to betten = 27) were identified as both CMC and xylan degraders, suggesting that these fungi can decompose the two most abundant plant carbohydrates , potentially with synergistic metabolisms. For CMC degradation, the trait was not conserved at the genus level, with variation among strains of some genera, such as Absidia, Pseudoseptoria, Aspergillus and Penicillium. Regarding xylan degradation, a weak but significant phylogenetic signal was observed , indicating that this trait was not randomly distributed. The potential ligninolytic activity was observed for the only isolated Basidiomycota, Ceratobasidium sp. (strain 35). Interestingly, this genus has already been reported to be associated with leaf litter, and this presence was correlated with laccase and peroxidase activities [To investigate the functional diversity of each fungal strain, we used various selective media, which are important tools for fungal ecology . Becausetivities , confirmProteolysis is known to be a widespread trait among soil microorganisms , and ourAspergillus strains isolated here, four of them were able to produce Caox crystals, confirming previous reports about members of this genus [Absidia and Penicillium genera were identified as Caox producers, which was also previously reported [Ceratobasidium and Ciliochorella being able to produce Caox crystals. Regarding the fungi able to degrade oxalate, we found one strain of Fusarium and Trichoderma, two genera for which this capacity has already been reported [Absidia, Bartalinia, Lichtheimia, Nigrosabulum, Ochroconis, Pseudocoleophoma, Purpureocillium and Sirastachys, and thus, we extended the knowledge on the diversity of this functional group [Regarding oxalate metabolism, we identified fifteen strains involved in Caox degradation and nine involved in Caox crystal production. Therefore, our results provide evidence that fungi associated with the OCP are not only involved in oxalate production but also in oxalate degradation, changing the current paradigm of the OCP in which oxalate degradation is mainly the result of bacterial activity. It has been shown in vitro that some white-rot fungi (Basidiomycota) can produce and dissolve calcium-oxalate crystals . Here weis genus . Similarreported ,78. Howereported . Interesal group . FurtherTamarindus indica tree. In addition to the capacity to decompose cellulose and hemicellulose, the characterization of fifty strains revealed the importance of fungi in the OCP for siderophore production, proteolysis, Caox crystal production but also surprisingly for oxalate degradation. These results provide new elements to better understand the role of fungi in the OCP. This study provides the first insights into the taxonomic and functional diversity of the litter fungi associated with an active OCP in Madagascar under a


Mycobacterium avium-complex (MAC) is an infectious granulomatous disease which is associated with hypercalcemia especially in immunocompromised patients. We present an unusual case of MAC infection in an immunocompetent patient presenting as hypercalcemia.A 76-year-old immunocompetent male was admitted for hypercalcemia of 12.6 mg/dl found on outpatient evaluation for fatigue. PTH level was low 8 pmol/L, Vitamin D 25hydroxy was 29 ng/ml, 1,25 dihydroxy vitamin D 2 vitamin D) levels was low at 11 pg/ml, PTH related peptide was 1.1 pmol/L. Hypercalcemia resolved with intravenous hydration and bisphosphonate administration. CT chest identified a nodule with central cavity in the right upper lobe. Pathology from percutaneous biopsy of thenodule demonstrated granulomatous inflammation. AFB culture came positive for MAC. Patient was treated with Azithromycin, Rifabutin and Ethambutol for twelve months.Granulomatous diseases like MAC cause hypercalcemia via activation of macrophages which express extrarenal 1- alpha -hydroxylase. It converts vitamin D to its active form 1,25(OH)2 vitamin D causing its excess, leading to hypercalcemia. Interestingly, in our patient calcium level was elevated with appropriately low PTH but 1,25(OH)2 vitamin D level was also low. There are few reported cases of hypercalcemia in granulomatous disease with normal levels of 1,25(OH)2 vitamin D levels, and our case is the first one to have MAC associated hypercalcemia with low 1,25(OH)2 vitamin D levels, suggesting an alternative mechanism for hypercalcemia in these patients Mycobacterium avium-complex (MAC) infection is a granulomatous disease that can cause hypercalcemia and has been increasingly reported in the literature over the years, especially in immunocompromised patients A 76-year-old male patient with a past medical history of chronic kidney disease stage 4, atrial fibrillation, and coronary artery disease was admitted for hypercalcemia of 12.6 mg/dl found on outpatient laboratory evaluation for fatigue. The levels remained elevated at 12.1 mg/dl on repeat testing at 48 h despite a dose of Pamidronate. This prompted the admission for further evaluation. He was on apixaban, allopurinol, amlodipine, donepezil, epoetin, esomeprazole, hydralazine, metoprolol, and simvastatin before admission. Of note, the patient used to be on calcitriol in the past but this was discontinued at least a month before admission and was unlikely to be related to the new hypercalcemia. He had complaints of chronic non-productive cough, fatigue, night sweats, and unintentional weight loss of 80 pounds within one year.On physical examination, the patient was cachectic, afebrile, normotensive, and had dry mucosal membranes. Laboratory findings during admission were significant for a hemoglobin of 10.2 g/dl on the complete blood count. The metabolic panel was pertinent for elevated creatinine of 3.47 mg/dl increased from his baseline of 2.9 mg/dl, GFR of 17 ml/min, calcium level of 11.6 mg/dl, albumin of 2.7 mg/dl, and a mildly elevated alkaline phosphatase level of 73 U/L. Further workup revealed ionized calcium of 1.39 mmol/L, 25 hydroxy-vitamin D of 29 ng/ml, 1, 25 (OH) 2 vitamin D levels low at 11 pg/ml, PTH level low at 8 pmol/L, and PTH related peptide (PTHrP) unremarkable at 1.1 pmol/L.Imaging modalities included a chest x-ray that showed new nodular opacities in the right upper and lower lobes along with another one in the left mid-lung. Subsequently, computed tomography of the chest (CT) showed multiple, bilateral ill-defined nodular opacities that were in a clustered configuration with some areas of branching. A 1.3 × 0.9 cm nodule with a central cavity in the right upper lobe and another 1.2 cm pleural-based nodule in the right posterior lobe were noted. There were multiple nonspecific sub-pleural ground-glass opacities and small bilateral pleural effusions . The cluThe patient underwent a CT-guided biopsy of the right upper lobe cavitating nodule. In the meantime, he also underwent a Positron Emission Tomography (PET) scan that showed multifocal hypermetabolic pulmonary nodules within both lungs, more on the right, and consistent with inflammation . Gram stEven though an uncommon clinical entity, MAC-associated hypercalcemia has been described extensively in HIV, lymphoma, and other immunocompromised patients but it is a rare phenomenon in immunocompetent patients Hypercalcemia has been reported in granulomatous infectious diseases other than MAC, like tuberculosis, leprosy, fungal infections such as cryptococcosis, candidiasis, and coccidioidomycosis Another interesting aspect of the current case is that our patient had hypercalcemia in the presence of low 1, 25(OH) 2 vitamin D. There are a few reported cases of hypercalcemia in patients with granulomatous disease where 1, 25(OH) 2 vitamin D levels are not elevated Our patient had already received pamidronate outpatient and responded well to aggressive hydration which led to the resolution of hypercalcemia inpatient. In other case reports of MAC-associated hypercalcemia, patients responded to steroids Hypercalcemia is a common entity in both outpatient and inpatient settings, but an association with infectious causes such as MAC is very rarely considered. Although the exact mechanism for hypercalcemia in MAC with low 1, 25(OH) 2 vitamin D levels is still unknown, we present this case to increase awareness of this rare presentation. These patients should undergo appropriate diagnostic evaluation and be treated appropriately to prevent further complications.Tulika Chatterjee: Writing – original draft, Contributed in writing the manuscript, Revised the manuscript. Yeshaswini PS Reddy: Contributed in writing the manuscript. Manasa Kandula: Ssupervised and edited the manuscript, Revised the manuscript.


Plasmodium falciparum gametocytes. Primaquine is metabolized by a highly polymorphic cytochrome P450 2D6 (CYP2D6) enzyme. Mutations in the gene encoding this enzyme may lead to impaired primaquine activity. This study assessed if 0.25 mg/kg single-dose primaquine is safe and sufficient to reduce transmission of gametocytes in individuals with no, reduced, or increased CYP2D6 enzyme activity.Primaquine is a pro-drug and its active metabolite is potent against mature P. falciparum malaria were enrolled in the study. The enrolled patients were treated with a standard artemether-lumefantrine regimen plus 0.25 mg/kg single-dose primaquine and followed up for 28 days for clinical and laboratory assessment. Primaquine was administered with the first dose of artemether-lumefantrine. Safety assessment involved direct questioning and recording of the nature and incidence of clinical signs and symptoms, and measurement of haemoglobin (Hb) concentration. Blood samples collected from 100 patients were used for assessment of post-treatment infectiousness on day 7 using mosquito membrane feeding assays. Molecular methods were used to determine CYP2D6 and glucose-6-phosphate dehydrogenase (G6PD) status. The primary outcome was the safety of 0.25 mg/kg single-dose primaquine based on CYP2D6 status.Between June 2019 and January 2020 children aged 1–10 years, attending at Yombo dispensary, Bagamoyo district, with confirmed microcopy-determined uncomplicated t = 0.012, p = 0.990). The day 3 mean absolute Hb concentration reduction in G6PD deficient, G6PD normal and heterozygous female was 1.82 g/dL (95% CI 1.32–2.32), 1.48 g/dL (95% CI 1.30–1.67) and 1.47 g/dL (95% CI 0.76–2.18), respectively . Sixteen percent (16/98) of the patients each infected at least one mosquito on day 7, and of these, 10.0% (2/20) and 17.9% (14/78) had reduced and normal CYP2D6 enzyme activity, respectively .In total, 157 children [median age 6.4 (Interquartile range 4.0–8.2) years] were recruited, of whom 21.0% (33/157) and 12.7% (20/157) had reduced CYP2D6 and deficient G6PD activity, respectively. Day 3 mean absolute Hb concentration reduction was 1.50 g/dL [95% confidence interval (CI) 1.10–1.90] and 1.51 g/dL (95% CI 1.31–1.71) in reduced and normal CYP2D6 patients, respectively (P. falciparum gametocytes regardless of CYP2D6 or G6PD status.Single-dose 0.25 mg/kg primaquine was safe and sufficient for reducing transmission of Trial registration Study registration number: NCT03352843.The online version contains supplementary material available at 10.1186/s12936-022-04100-1. Plasmodium falciparum gametocytes in many malaria-endemic settings and this has contributed significantly to the global decline of malaria transmission [P. falciparum gametocytes, and, therefore, not sufficient to prevent transmission [The scale-up of malaria control efforts particularly using insecticide-treated bed nets and artemisinin-based combination therapy (ACT) has led to the global decline of malaria transmission, consequently renewing the interest in the possibility of eliminating the infection –3. Artemsmission , 4. HoweP. falciparum resistance against artemisinin in Southeast Asia [Anopheles resistance against pyrethroid in Africa [The emergency of ast Asia –7, and An Africa –10, is an Africa .Plasmodium vivax and Plasmodium ovale infection [P. falciparum gametocytes, a parasite stage responsible for onward transmission of the infection [P. falciparum gametocytes [Primaquine is an 8-aminoquinoline pro-drug indicated for radical cure of nfection –14. It infection , 15, 16.nfection . It may etocytes –23, and etocytes . A failuetocytes –23.P. vivax and P. ovale hypnozoites, and P. falciparum transmission [P. falciparum gametocytes probably at a lower concentration than that needed to kill and clear them from the circulation [P. falciparum gametocytes in patients with uncomplicated malaria based on CYP2D6 alleles common in African Tanzanian.Primaquine gametocytocidal effect and haematological toxicity are thought to depend on cytochrome P450 isoenzyme 2D6 (CYP2D6) conversion of a pro-drug into active phenolic metabolites –29. But smission . The nulsmission , 35. Redsmission , hence rsmission . Howeverculation . Of noteculation –40, howeculation , 31, whiculation , 33. Incculation . This stThe study was carried out at Yombo primary health facility, Bagamoyo district, Tanzania, between June 2019 and February 2020. The health facility is located southwest, about 20 km from Bagamoyo town. It serves approximately 7000 people and can carry out routine malaria microscopy and rapid diagnostic tests . The inhPlasmodium falciparum and Anopheles arabiensis are the predominant malaria parasite species and vector, respectively [Malaria transmission in the Bagamoyo district occurs throughout the year with peaks corresponding to the long and short rainy seasons from May to July and November to December, respectively. ectively . Malariaectively . Artemetectively , and priectively . Insectiectively . Previouectively .P. falciparum gametocytes. Patients with microscopically confirmed uncomplicated P. falciparum mono-infection were enrolled in the trial, treated with standard artemether-lumefantrine regimen plus 0.25 mg/kg single-dose primaquine, and then followed-up until day-28 after treatment initiation for clinical and parasitological assessment. Individuals with reduced CYP2D6 activity were defined as those with CYP2D6*17 and CYP*29 alleles, whereas those with no activity were those with CYP2D6*4 and CYP2D6*5, and normal individuals were those with CYP2D6*1, while those with CYP2D6*2 were defined as having increased activity [There is no rapid diagnostic test at the moment that could be used to evaluate and group participants based on their CYP2D6 status at the enrolment. Thus this study was a single-arm clinical trial to assess the safety and efficacy of 0.25 mg/kg single-dose primaquine added to standard artemether-lumefantrine treatment in individuals with CYP2D6 reduced or no activity compared to those with normal or increased activity for clearance and reducing transmission of activity , 46. AddP. falciparum mono-infection, parasitaemia level of 2000–200,000/µL, ability to swallow oral medication, ability and willingness to abide by the study protocol and the stipulated follow-up visits, and a written informed consent from a parent/guardian and assent from children aged ≥ 7 years. Children aged below 1 year were not included in the study because primaquine is contraindicated in this group, and children up to the age of 10 years were included to increase the recruitment rate. Exclusion criteria were evidence of severe malaria or danger signs, known allergy to trial medicines, reported anti-malarial intake ≤ 2 weeks, haemoglobin < 5 g/dL, blood transfusion within last 90 days, febrile condition other than malaria, and known underlying chronic or severe disease .Patients presenting at the health facility with suspected acute uncomplicated malaria were screened for eligibility. Inclusion criteria were age from 1 to 10 years, weight ≥ 10 kg, body temperature ≥ 37.5 °C or history of fever in the last 24 h, microscopy confirmed At the health facility patients and their parents/guardians were informed in detail about the trial, purpose, the risks and benefits of participating in the trial, and their rights, and encouraged them to participate in case they are diagnosed with malaria. Thereafter, patients were evaluated clinically, and those with signs and symptoms of malaria were directed to the laboratory for parasitological evaluation.P. falciparum malaria infection, and met all the inclusion and none of the exclusion criteria their parents/guardians were requested to allow their children to participate in the trial. The parents/guardians were then administered with the consent form. The consent form was self-administered to the literate parents/guardians and for illiterate one’s the trial clinician explained to them. All the procedures were done in the presence of a witness to ensure that there is no coercion. Parents/guardians were given time to ask questions to their satisfaction. All consented parents/guardians signed the form either by a written signature for literate or a thumbprint together with a written signature of their witnesses for the illiterate.For the children who were found to have uncomplicated P. falciparum malaria [Enrolled patients were treated using a standard 3-day course of artemether-lumefantrine  according to Tanzanian national treatment guidelines for uncomplicated  malaria . The art malaria . Accurac malaria , 25. AllPatients were withdrawn from the study in case of vomiting the study drug > 3 times, withdrawal of consent, the onset of a serious febrile illness, intake of any drug with anti-malarial properties outside the study protocol, or any protocol violation. Patients who missed scheduled follow-up visits and did not show up on the successive days despite efforts to trace them at their residences were considered lost to follow-up and were consequently censored from the analysis on the last day they were seen. Participants who returned before the last day of follow-up were not considered as lost to follow-up. Patients with symptoms/signs of severe disease (including repetitive vomiting of study drug) were managed according to the Tanzania national guidelines and followed up until recovery, but were censored from the analysis on the day of withdrawal . PatientClinical and laboratory assessments were performed on days 0, 1, 2, 3, 7, 14, 21, and 28 or any day of recurrent illness as follows:The assessment involved taking a history of clinical symptoms, possible adverse events (AEs), concomitant drug consumption, and clinical examination including measurement of axillary temperature. Fever was defined as a body temperature ≥ 37.5 °C. All the clinical and laboratory data were recorded in a case record form (CRF).Safety assessment for AE and serious adverse events (SAE) was performed using the Primaquine Roll Out Monitoring Pharmacovigilance Tool (PROMPT) . An AE wThe safety assessment involved direct questioning and recording the nature and incidence of AE and/or serious adverse events (SAE). Signs and symptoms such as fatigue, weakness, dizziness, headache, palpitations or allergic drug reactions (rash), diarrhoea, abdominal pain, dizziness, itching, fever, and haemoglobin level  were recorded and determined to whether they were related to the trial medicine. Patients with AE were kept under observation, and those requiring treatments were treated accordingly. Patients with SAE were admitted and if necessary referred to the Bagamoyo district hospital.All the patients received an information card with instructions on how to identify signs and symptoms of commonly reported AEs including monitoring of urine colour. In addition, patients were given clear containers and asked to provide urine samples on day 0 before the first drug dose and days 1, 2, and or 3. The urine colour estimation for haemoglobinuria was gauged against the Hillmen colour chart with a colour score ranging from 1 to 10 . A colouLaboratory assessment involved collection of finger-prick blood samples that were used to measure haemoglobin concentration, prepare the thick film for microscopy to assess presence and density of asexual parasitaemia and gametocytaemia, thin-film for asexual parasite species determination, and also blotted on filter paper (Perkin Elmer 266) for molecular analysis of CYP2D6 and G6PD status. On day 7, 3 mL of venous blood were collected in vacutainers from 100 patients randomly selected using research randomizer software   and usedHaemoglobin concentration was measured using a portable spectrophotometer, HemoCue Hb 201 + , with a precision of ± 0.3 g/dL. The HemoCue was calibrated every morning using a control cuvette at 16.0 ± 0.3 g/dL according to the manufacturer’s instructions. Anaemia was classified as haemoglobin level < 11 g/dL (mild), < 8 g/dL (moderate) and < 5 g/dL (severe).Thin blood smears were prepared only at enrolment, whereas thick blood smears were prepared at all sampling time points. At the enrolment, two pairs of thin and thick smears were collected, and the first pair was quickly stained using 10% Giemsa for 15 min to accelerate the enrolment of patients, whereas the second pair was stained slowly using 3% Giemsa for 1 h. Thick smears collected during follow-up were stained slowly using 3% Giemsa for 1 h. Asexual and sexual parasites on thick smears were counted against 200 white blood cells (WBC). This number was then multiplied by 40, assuming 8000 leukocytes per microlitre of blood, to gain an approximate parasite density per microlitre of blood. A blood slide was considered negative if no parasite was seen after examining 100 fields. Two, independent microscopists read all the microscopy slides. In case they disagreed on the presence of parasitemia or if the density differed by more than 25%, a third independent reading was performed. In the case of positive versus negative results, a third independent reading was used to confirm the reading of the first two readers.The genomic DNA was extracted from 100 μL of dried blood spots on filter papers using Quick-DNA™ Miniprep plus Kit Catalog Nos. D4068 and D4069 .To detect CYP2D6 point mutations, the extracted DNA was amplified using mass array VeriDose CYP2D6 CNV Panel . The CYPThe G6PD analysis was performed through amplification of the extracted genomic DNA using a single-round PCR followed by gel electrophoresis. Amplicons were loaded on agarose gel containing GelRed™ (Biotium) and then visualized under ultraviolet- transillumination. The molecular weight of the DNA fragments was sized using Gene Ruler TM 100 bp molecular weight marker. Amplicons with an expected band size of 1061 bp for Exon 3–5 were sequenced using the Sanger sequencer. The interested two most common polymorphisms associated with G6PD deficiency in Africa i.e., A376G (rs1050829 A > G/T > C) and G202A (rs1050828 G > A/C > T) were found in Exon 3–5 after analysis of sequences data by Geneious prime software. The outcomes were classified as follows; For males A was defined as wild-type/normal and A− as hemizygous/deficient G6PD status, whereas for females A−A− was defined as homozygous/deficient, AA− and BA− as heterozygous/intermediate and AA and BA as wild-type/normal G6PD status .P. falciparum block 3 of merozoite surface protein (MSP) 2, block 2 of MSP 1, and region II (RII) of glutamate-rich protein using a nested PCR method [To differentiate recrudescence from new infection, the extracted DNA from patients classified during the 28 days follow-up period as late clinical failure or late parasitological failure was genotyped using a stepwise genotyping of R method . BrieflyAnopheles gambiae sensu stricto mosquitoes through an artificial membrane. These mosquitoes were starved for a minimum of 5 h before the experiments. The membrane feeder setup was maintained at 37 °C using a temperature-controlled water bath and was run for 15 min. Only fully fed mosquitoes were transferred into a holding cage in the insectary and kept on 10% glucose, at 28 °C for eight days for oocyst maturation, whereas the partially fed and unfed ones were destroyed. A minimum of 25 mosquitoes that survived to this period was dissected in 0.5% mercurochrome, and their midguts were examined using microscopy for oocyst prevalence and density. The proportion of infected mosquitoes and the density of oocysts in infected mosquitoes were determined. An infected mosquito was defined as any mosquito with oocysts detected after dissection. Membrane feeding experiments were performed at the IHI, Bagamoyo, Tanzania.Infectivity was assessed using mosquito membrane feeding experiments. Blood samples collected on day 7 from a subset of 100 randomly selected participants were used in the experiment to assess the biological evidence of gametocyte sterilization –58. BrieP. falciparum gametocytes in patients with uncomplicated P. falciparum malaria based on CYP2D6 status defined as mean maximal absolute Hb (g/dL) fall during follow-up (day 0–28). Secondary outcomes included: follow-up day of haemoglobin nadir defined as mean day of follow-up (day 0–28) per group with lowest Hb measurement; maximal percentage fall in haemoglobin defined as the percentage relative change of maximal haemoglobin drop during follow-up (day 0–28) from baseline; percentage of participants with anaemia during the days 0–28 of follow-up; percentage of patients receiving blood transfusion during days 0–28; proportion of patients with urine colour change score ≥ 5 using Hillmen Urine Colour Chart during days 0–28; incidence of serious adverse events by sign, symptom, laboratory parameter during days 0–28; proportion of participants with gastrointestinal symptoms within day 0–7 after taking study drug; proportion of patients with G6PD deficiency; proportion of patients who were infectious to mosquitoes on day 7 determined by direct membrane feeding assay; and proportion of patients with PCR-adjusted adequate clinical and parasitological response (ACPR) by day 28. All the evaluated study outcomes were compared between CYP2D6 or G6PD status. The primary outcome was the safety of 0.25 mg/kg single-dose primaquine for reducing transmission of The study was conducted following Good Clinical Practices (GCP), the Declaration of Helsinki, and applicable regulatory requirements in Tanzania. Ethical clearances to conduct the trial were obtained from the Muhimbili University of Health and Allied Sciences Institutional Ethics Review Board and the National Health Research Ethics Review Committee of the National Institute for Medical Research. Permission to import and use primaquine was sought from the Tanzania Medicines and Medical Devices Authority. An independent data and safety monitoring board (DSMB) was established, visited the trial site, and performed the interim analysis of safety data. Written informed consent was obtained from parents/guardians of the participants whereas children aged ≥ 7 years also provided assent before their enrollment. The trial is registered at clinicaltrials.gov with the number NCT03352843.The study was powered to measure the efficacy of primaquine (0.25 mg/kg primaquine dose) in maximum reduction of gametocytaemia from day one through day 28. A sample size of 155 evaluable subjects was chosen to estimate a prevalence of gametocytes carriage measured by QT-NASBA on day 7 of 10% among the individuals with normal/increased activity compared to 35% in reduced activity at a power of 80% . All theIn this trial, a similar prevalence of CYP2D6 genotypes was assumed to be as described by Wennerholm et al. ; 63% norNo sample size calculation was performed for the number of patients required for membrane feeding experiments but based on other studies –23 100 pData were double entered in an electronic database, cleaned, and analysed using R version 3.2.3  software. The data were analysed based on intention to treat and per-protocol analyses.t-test. In addition, a one-sample t-test was used for comparisons of the mean maximal fall Hb with that reported in Uganda by using 0.4 mg/kg single-dose [Chi-square or Fisher exact test. Data were censored at the time of withdrawal for patients lost to follow-up, withdrawal of consent, and PCR determined reinfection or uncertain PCR outcome. A p < 0.05 was defined as statistically significant.The primary safety end-point, the mean maximal haemoglobin concentration (Hb) reduction during 28 days of follow-up was assessed and compared between the CYP450 2D6 status using independent two paired samples gle-dose . ProportP. falciparum infection. Of the infected patients, 157 were enrolled in the study after fulfilling all the inclusion and none of the exclusion criteria  had microscopy-confirmed % had micThe pre-treatment characteristics of the enrolled patients are presented in Table At the enrolment, 69.4% (109/157) of the patients had a fever. Following the initiation of treatment, fever clearance was rapid with only 22.9% (36/157), 3.2% (5/157), and 0.6% (1/157) of the patients remaining febrile on days 1, 2, and 3, respectively. Microscopy-determined asexual parasites decreased to 81.5% (128/157) on day 1 and 13.4% (21/157) on day 2, after the initiation of the treatment, and none of the patients had parasitaemia on day 3. Only one (0.64%) patient had microscopy-determined gametocytes at the enrolment, and these gametocytes were cleared on day 2 after the initiation of treatment. Another patient had gametocytes detected on day 1 after the initiation of treatment, but was not detected again on day 2 and onward.Fisher’s test = 0.736, p = 0.513). In addition, there was no statistically significant difference in the infectiousness between children under five years of age (17.6% (6/34) and those aged 5 years and above (15.6% (10/64) .One hundred patients were randomly selected to provide blood samples for mosquito membrane feeding experiments on day 7 after the initiation of treatment, and of these, 98 (98.0%) provided the blood sample. Sixteen out of 98 (16.3%) patients were infectious to at least one mosquito on day 7. Of the infectious patients, 10.0% (2/20) and 17.9% (14/78) had reduced and normal CYP2D6 enzyme activity, respectively, and the difference between the groups was not statistically significant  patients had late clinical and parasitological failure, 6.9% (10/143) had a late parasitological failure, and 6.9% (10/143) had PCR adjusted recrudescence. The day 28 artemether-lumefantrine per-protocol PCR-adjusted adequate clinical and parasitological response (ACPR) was 93.0% (133/143), , whereas, 25.0% (5/20), 20.7% (24/116) and 23.8% (5/21) of them were G6PD deficient, normal and heterozygous females, respectively .Furthermore, during follow-up, acute haemolysis defined as haemoglobin reduction of ≥ 25% occurred in 21.7% (34/157) of the patients. Of the patients with acute haemolysis, 18.2% (6/33) and 22.6% (28/124) had reduced and normal CYP2D6 activity, respectively .On the other hand, 45.0% (9/20) of G6PD deficient patients had reduced CYP2D6 enzyme activity. Out of 11 participants with G6PD deficiency and normal CYP2D6 enzyme activity, 5 (45.5%) had acute haemolysis, whereas none (0/9) of participants with G6PD deficiency and reduced CYP2D6 had acute haemolysis . Likewise, the prevalence of anaemia at the enrolment was 55.0% (11/20), 53.4% (62/116), and 57.1% (12/21) in G6PD deficient, G6PD normal, and heterozygous patients, and was not significantly different between the status . During follow-up, the prevalence of anaemia increased significantly from 54.1% (85/157) at the enrolment to 89.8% (141/157) on day 3  and 82.8% (130/157) on day 7 . Prevalence of anaemia was not significantly different between CYP2D6 status on days 3  and day 7 , but was significantly different between G6PD status with higher prevalence occurring in G6PD normal patients on day 3 (93.1% (108/116), trend x2 = 6.136, p = 0.047) and in G6PD deficient patients (100% (20/20), trend x2 = 7.994, p = 0.018) on day 7.At the enrolment 39.4% (13/33) and 58.1% (72/124) of patients with reduced and normal CYP2D6 enzyme activities had anaemia, but the prevalence of anaemia was not significantly different between the status (About 5.7% (9/157) of the patients were both reduced CYP2D6 and G6PD deficient. The haematological changes in patients with both reduced CYP2D6 and G6PD deficiency compared to other CYP2D6 and G6PD status are presented in Table x2 = 1.29, p = 0.257) or G6PD  status was not statistically significant. Likewise, all other AEs were not statistically significantly different between the CYP2D6 or G6PD status.Other adverse events (AEs) occurred in 65.6% (103/157) of the patients. In total 124 AEs were reported during follow-up and were all mild and self-limiting, Table P. falciparum gametocytes [P. falciparum gametocytes, to probably be influenced by the polymorphic nature of the gene encoding this enzyme [Primaquine is the only approved anti-malarial drug potent against mature etocytes , 59, 60.s enzyme , 36, 61.s enzyme , 63. This enzyme , 25, 64 s enzyme , 65, 66.s enzyme , 25, 67.s enzyme –70. In aP. falciparum gametocytes. Following mosquito membrane feeding assay, 16.3% of the patients were infectious to at least one mosquito on day 7. However, there was no statistically significant difference in the prevalence of infectiousness between CYP2D6 status. In addition, there was no statistically significant differences in the rate of infectiousness between the underfives and those aged above five years. Contrarily, a previous study has reported a higher prevalence of gametocytes carriage in CYP2D6 poor/reduced activity [Anopheles mosquitoes. Importantly, sterilization of P. falciparum gametocytes occurs at a lower primaquine concentration and earlier than it is required for them to get killed and cleared from the circulation [P. falciparum gametocytes transmission; second, other factors such as poor intestinal absorption especially during the acute clinical malaria infection may be responsible for the failure in reducing the transmission; and third, the CYP2D6 reduced activity has a more significant role in P. vivax hypnozoites clearance rather than P. falciparum gametocytes sterilization.Reduced CYP2D6 activity is thought to reduce primaquine efficacy probably by impairing the production of phenolic metabolites that determines the drug’s efficacy , 61. Theculation , therefoP. falciparum parasite clearance is a hallmark of artemisinins [P. falciparum K13 gene.Rapid fever and asexual misinins . In thismisinins , 73, othmisinins –76, and misinins –79 usingP. falciparum gametocytes was not assessed.Despite its strength, the study had limitations including, at the enrolment patients were not tested and then grouped based on the CYP450 2D6 status as there is no rapid test for the condition, thus it was not possible to recruit an equal number of participants based on their CYP2D6 status, and therefore, it was not possible to have a double-blinded randomized clinical trial with artemether-lumefantrine alone versus artemether-lumefantrine plus SLD primaquine. However, the attrition rate of 20% was used to ensure that the required proportions of patients with CYP2D6 normal/increased and reduced/null are recruited. Another limitation is that the sample size calculation was based on previous studies that assessed PQ efficacy for reducing gametocytes transmission using QT-NASBA, but during the implementation, the efficacy was assessed using mosquito membrane feeding assays only, and the study was not powered to assess efficacy using membrane feeding assay as the primary outcome. Furthermore, the available primaquine concentration in the plasma of treated patients was not measured, therefore, its relationship with sterilization of P. falciparum gametocytes, as assessed by mosquito membrane feeding assays, regardless of the CYP2D6 or G6PD status, and the observed adverse events were mild and self-limiting.The SLD primaquine was safe and sufficient for reducing transmission of Additional file 1. Dataset.