Zoysia sinica), “Diamond” zoysiagrass (Zoysia matrella), “J026” zoysiagrass (Zoysia japonica), and “M001” zoysiagrass (Zoysia macrostachya) were grown in plastic pots and exposed to 1/2 Hoagland nutrient solution amended with different amounts of NaCl for 120 days. At the end of the experiment, growth traits and ion contents were determined. The results showed that the salt-tolerance of four zoysiagrasses ranked as “M001” > “Diamond” > “J026” > “S001” according to percent green leaf canopy area (GLCA) after 120 days of salinity treatment. Although dry leaf weight, leaf length/width, and shoot height were significantly decreased by salinity treatments for all turfgrasses, the salt-tolerant species had a smaller drop. Besides, ions secretion capacity and Na+ concentration in leaf and root increased, but K+ concentration together with leaf and root K+/Na+ ratios decreased with the increasing concentration of the salinity. However, the salt-tolerant species exhibited strong K+ absorption and transportation ability and a high salt secretion capacity. The results indicated that growth traits and ions regulation were related to variability in tolerance of diverse zoysiagrasses to salt stress.Salt stress affects plant physiology, development, and growth. This research investigated varied salinity levels on growth traits and ions accumulation of four zoysiagrasses and aimed to identify phenotypic traits associated with variability in salinity tolerance. In this study, “S001” zoysiagrass ( Some zoysiagrasses exhibit strong resistance to salinity [Zoysia matrella, Zoysia japonica, and Zoysia macrostachya [Salt stress is considered as one of the environmental stresses affecting plant growth and development in some areas ,2,3. Salsalinity ,12, whicostachya . These t+ concentration, and osmotic potential were reduced under salt stress [+ and Cl‒ concentrations, osmotic adjustment substances such as free proline, sugar and betaine, salt secretion capacity, and anti-oxidation enzymes activity such as peroxidase, catalase, and superoxide dismutase increased [So far, many researchers have studied the physiological and growth responses of warm-season turfgrasses under salt stress. Leaf water content, Kt stress ,15,16, wncreased ,18,19. Increased ,21. Somencreased ,23. ThesWe carried out the experiment to study the responses of four zoysiagrasses to long-term salinity treatment, and the major objectives were to determine the growth traits and ions accumulation of four zoysiagrasses in the growing season with NaCl concentration ranging from 0 to 400 mM and to identify salt tolerance-related phenotypic traits.22.1This study employed four zoysiagrasses, including “S001,” “Diamond,” “J026,” and “M001.” “S001,” the non-halophyte, was collected at the Jiaozhou Bay of Shandong Province, while the other three turfgrasses were halophytes, among which “Diamond” was a commercial turfgrass, and “J026” and “M001” were collected at Qingdao of Shandong Province and Yancheng of Jiangsu Province, respectively. On April 1st, 2019, stolons of four turfgrasses were collected from the biological garden of Yancheng Teachers University (33°21ʹN latitude and 120°09ʹE longitude) and were cut into stems (10–15 cm in length) with 6 sections. Then, the stems were planted in a plastic pot (15 cm in height and 10 cm in diameter) filled with river sand. Each zoysiagrass was planted into 15 pots, including 3 pots used as controls and 12 pots used as treatments. These pots were suspended in turnover boxes filled with 50 L  of l/2 Hoagland nutrient solution through the foam board. The turnover boxes were placed in the greenhouse and aerated with an air pump every day, the nutrient solution was replaced, and the plants were clipped once every week. The water level in turnover boxes was checked every two days, and water was supplied to make sure that the pot bottom always contacted the liquid level. Meanwhile, the pH of the nutrient solution was kept at 5.5–6.0.2.2Salt stress was conducted by the addition of NaCl to the l/2 Hoagland nutrient solution from June 1st, 2019, until the desired salinities of 100, 200, 300, and 400 mM were obtained, with 0 mM NaCl as control. To avoid the osmotic shock, salinity was incrementally increased by 50 mM every day. Salt stress lasted for 120 days. This study was repeated at the same period in 2020.2.3+ and Na+ levels using the flame photometer .To determine salt secretion capacity, salt crystals on the leaves’ surface were washed with deionized water on a sunny day after one week of salinity treatment. One week later, we cut ten mature leaves, put them at once into the centrifuged tube containing 5 mL of deionized water, and shook for 30 s to completely dissolve all leaf surface salt crystals into water. Later, we dumped the eluent to the other centrifuge tube for determining KAt the end of the experiment, ten leaves from every pot were cut and washed with deionized water, water on leaf surface was wiped and the fresh weight (FW) of leaf was measured at once. Afterwards, the leaves were transferred to Petri dishes containing deionized water and kept in dark for 4 h, so as to restore the turgidity. Subsequently, we eliminated excessive water on leaf surface to measure the turgid weight (TW), and the leaves were then dried at a temperature of 105°C for 30 min and then at 80°C for 48 h to measure their dry weight (DW). Subsequently, relative water content (RWC) was calculated according to the formula below.3 solution for ion extraction. Subsequently, the content of Na+ and K+ were measured using the flame photometer mentioned earlier.Percent green leaf canopy area (GLCA) of each pot was visually estimated. Shoot height, dry leaf weight, leaf length, and leaf width were determined with ten replications per pot. Then, both leaves and roots were ground into fine powders in the context of liquid nitrogen, and digested by 0.5% of HNO2.4p ≤ 0.05.The experiment was performed under complete randomization condition, and each salinity treatment was repeated three times. Differences in data between 2019 and 2020 were not statistically significant, and as a result, data collected at the sampling dates in these two years were combined and the means were used for statistical analysis. The Excel software  was used to draw histograms. The one-way ANOVA was utilized for data analysis by employing the SAS software . Meanwhile, the Duncan’s multiple range test was performed to compare the averages, and the significance level was set as 33.1The GLCA percent of four turfgrasses showed the significant difference under salt stress . Specifi3.2The shoot height of four turfgrasses slightly increased at the salinity level of 100 mM, but it reduced gradually as the salinity concentration increased to other levels . Such re3.3After 120 days of salinity treatment, the leaf water content was not affected at the 100 mM salinity level in all turfgrasses except for “S001” with 5.23% decrease. At the 200 mM salinity level, leaf water content significantly declined in all turfgrasses except for “M001” with largely unaffected leaf water content. The leaf water contents of “J026,” “Diamond,” and “M001” significantly decreased at 300 and 400 mM salinity levels; typically, under 400 mM NaCl treatment, the leaf water content of the turfgrasses “J026,” “Diamond,” and “M001” decreased by 14.91, 20.70, and 17.18%, respectively, compared with control .3.4Leaf length was significantly reduced in “S001” under 100 mM salinity treatment, but there was no significant change in the remaining three species. Besides, the leaf length of all turfgrasses significantly reduced at 200 mM salinity concentration compared with control, except for “Diamond.” Moreover, the leaf length of “J026,” “Diamond,” and “M001” significantly decreased at 300 and 400 mM salinity levels, and the leaf length decreased by 61.63, 45.32, and 30.87%, respectively, at 400 mM salinity level compared with control. The results showed that “M001” had a greater growth capacity at higher salinity concentration . As a ke3.5+ and Na+ ions increased significantly in all turfgrasses with the increase in salinity concentration. Moreover, “M001” exhibited the strongest secretion capacity, in which the K+ and Na+ ions secretion efficiency increased by 97.15–350.53% and 899.15–4692.22%, respectively, at the 100–400 mM salinity levels relative to control. Meanwhile, the Na+/K+ ratio in “M001” was the highest at each salinity level compared with those in the other three turfgrasses  was the salt-sensitive species. In our research, “Diamond” zoysiagrass was the moderate salt-tolerant species, which supported the previous research.Based on the percent leaf firing data, Marcum Kenneth et al.  and QianCynodon dactylon [Agropyron cristatum [RWC is an important physiological index to evaluate the salt tolerance of plant. According to previous research, the RWC of dactylon  and Agroristatum  decreaseTriticum aestivum leaves were reduced in width and length after exposure to salinity stress. For the present study, “M001” showed the highest salt tolerance but still had the greatest shoot height among all turfgrasses under high salinity treatment. Besides, its length, width, and dry leaf weight suffered the least reduction. Based on such results, the leaf growth traits and shoot height were the vital parameters affecting the tolerance to salinity.As suggested by Dudeck and Peacock , Lee et + and Cl−, thus reducing the salt stress-induced damage. Moreover, the salt tolerance is reported to be significantly and positively correlated with the salt secretion ability of zoysiagrasses [+ and Na+ ions, among which “M001” was the most efficient in secreting ions.It was reported by Marcum and Murdoch  that salagrasses ,31. In t+ and Na+ uptake or K+/Na+ ratio were related to the salt tolerance in some plant species [+ uptake high, Na+ absorption low, and thereby the leaf K+/Na+ ratio high [+ uptake and low Na+ accumulation in root and leaf at high salinity levels, which conformed to other studies [+ uptake and avoid excess Na+ accumulation to enhance the salt tolerance of “M001.” Potassium stands for one of the critical nutrient elements necessary during the growth and development of plants. Na+ almost has the same ionic radius as K+, which results in the competitive absorption of K+ with Na+ within cell membrane in the presence of salt stress [+/Na+ ratios are correlated with the salt tolerance of plant. As demonstrated by Guo et al. [Paspalum vaginatum), the salt-tolerant species, showed greater root and leaf K+/Na+ ratios. In this work, “M001” had greater root and leaf K+/Na+ ratios than that of the other three turfgrasses. Therefore, the capacity of K+ uptake and root-to-leaf Na+ transportation inhibition was suggested to be of vital significance to the salt tolerance in “M001.”Salt stress affects ion absorption and transportation in plants . K+ and  species ,33. For tio high . As reve studies ,25,34. St stress . In addio et al. , seashor+ absorption and transportation, high salt secretion capacity, high GLCA percent and shoot height, and low leaf growth reduction, all of which might contribute to the strong salt tolerance of these turfgrasses.Taken together, the “M001,” “Diamond,” and “J026” can adapt their growth to salt stress for 120 days at every levels, and their GLCA percent still remained at above 70% under 400 mM NaCl treatment, demonstrating that they had stronger salt-tolerant capacity. However, the “S001” died on the 20th day under 300 mM NaCl treatment and on the 14th day at the 400 mM salinity level. According to the GLCA percent, after 120 days of salinity treatment, the salt tolerance of four turfgrasses followed the order of “M001” > “Diamond” > “J026” > “S001.” The growth traits and physiological indexes, such as leaf length and width, leaf weight, and RWC were reduced to varying degrees under salinity treatment. Compared with S001, the salt-tolerant zoysiagrasses in this experiment exhibited strong capacity for K


Seedling stage plant biomass is usually used as an auxiliary trait to study plant growth and development or stress adversities. However, few molecular markers and candidate genes of seedling biomass-related traits were found in cotton.Gossypium arboreum accessions, and investigated 11 seedling biomass-related traits including the fresh weight, dry weight, water content, and root shoot ratio. A genome-wide association study (GWAS) utilizing 142,5003 high-quality SNPs identified 83 significant associations and 69 putative candidate genes. Furthermore, the transcriptome profile of the candidate genes emphasized higher expression of Ga03G1298, Ga09G2054, Ga10G1342, Ga11G0096, and Ga11G2490 in four representative cotton accessions. The relative expression levels of those five genes were further verified by qRT-PCR.Here, we collected 215 The significant SNPs, candidate genes identified in this study are expected to lay a foundation for studying the molecular mechanism for early biomass development and related traits in Asian cotton.The online version contains supplementary material available at 10.1186/s12870-022-03443-w. Vegetative growth in crop plants is generally associated with the above and below-ground biomass. The vigorous vegetative growth has been used as an indicator for stress resistance and high yield due to the potential source to sink mobilization of reserves stored in vegetative plant parts, viz., stem, leaves, and roots –4. ThereCotton contributes hefty shares as raw material for the textile industry. Significant variation of biomass-related traits like fresh weight, dry weight, water content, and root shoot ratio, generally influenced by environmental factors, is present among modern cultivars and wild relatives , 13. TheGossypium arboreum, which is the ancestors of the modern cultivated allotetraploid cotton should be an ideal model for basic research in cotton [Recent advances in sequencing technology have led to extensive Genome-wide association studies (GWAS) in crop plants , 23–26. n cotton .G. arboreum accessions by utilizing high-quality resequencing and GWAS. Moreover, we further screened the candidate genes of GWAS by transcriptome data. The yielded information regarding SNPs and putative candidate genes identified could effectively enrich the excellent genetic resources of complex traits like biomass in cotton.The present study aimed to systematically investigate the genetic regulation of early plant biomass in the germplasm of G. arboreum accessions  and SNP Chr02_7231541 (−log P = 6.33), were reached the significant level. All the fresh weight traits including SFW, RFW, and TFW have detected fewer numbers of SNPs, and their -lop P values were also relatively low with no SNP reaching the threshold (−log P > 6.15) (Table SP = 5.65 and 5.61 respectively) and all the fresh traits have detected more SNP numbers on Chr04. Based on the threshold (−log P > 6.15), a total of 83 significant SNPs were detected  of the four G. arboreum accessions, previously have been sequenced by our group (unpublished study).In order to further analyze the GWAS candidate genes, we selected four representative materials for candidate gene expression analysis and qRT-PCR verification. Those four Asian cotton accessions were “AHQJZM”, “DPL971”, “WMSLDMH” and “DGXH”. The “WMSLDMH” and “DGXH” accessions have higher fresh weight, and water content than “AHQJZM” and “DPL971”, especially for the root samples Table S. And theGa10G1342 gene was highly expressed in all the three periods, and interestingly, it was higher expressed in “AHQJZM” and “DPL971” than “WMSLDMH” and “DGXH” on the 12d after sowing. Another gene, Ga11G2490 also showed the same rule, with higher expression levels in the root tissues of “AHQJZM” and “DPL971” both on the 8d and 12d after sowing. However, the Ga11G0096 gene showed the opposite expression pattern, with higher expression levels in “WMSLDMH” and “DGXH” than “AHQJZM” and “DPL971”, especially for the periods of 2d and 8d after sowing. Ga03G1298 has a lower expression on the 2d, and it was increased on the 8d and showed different expression levels among the four cotton accessions on the 12d after sowing. Ga09G2054 showed higher expression on the 2d, and the FPKM value of “AHQJZM” and “DPL971” was higher than the other two accessions. While on the 8d after sowing, little difference can be detected among the four accessions, and it showed an obvious expression change on the 12d after sowing.We utilized the important transcriptomic profiles and compared the different expression levels of GWAS candidate genes in the four cotton accessions Table S. Three pcottonFGD.org [Ga03G1298 have higher TPM expression levels in the leaf samples, whereas Ga09G2054 and Ga10G1342 have higher TPM expression levels in the root samples.The TPM expression data of the GWAS candidate genes for the Asian cotton standard line cv Shixiya1 have also been downloaded from nFGD.org . Forty gGa03G1298, Ga09G2054, Ga10G1342, Ga11G0096, and Ga11G2490) were selected to test and verify their relative expression level in the four representative Asian cotton accessions. Root and leaf samples of 12 das after sowing were collected and used in this study. The qRT-PCR result showed that the Ga03G1298 gene showed higher expression in the leaf samples than in the root samples. Yet, the other four genes especially for Ga11G0096 had a higher relative expression on the root samples, which were consistent with their FPKM/TPM expression result. In addition, we also found the Ga10G1342 gene has detected higher relative expression levels in root samples of “AHQJZM” and “DPL971” than “DGXH” and “WMSLDMH”.As shown in Fig. Early development of plants is critical for providing a strong base for further development. Seedling stage biomass has been extensively studied in plants with its potential relation to the stress environments , 20, 23.The progress of molecular markers identification of biomass and its related traits in cotton is relatively slow. At present, only a few studies have reported molecular markers or QTLs associated with seedling biomass-related traits in cotton. Tang et al. (2017)  used croGa03G1298, Ga09G2054, Ga10G1342, Ga11G0096, and Ga11G2490) that may be most closely related to the seedling biomass related traits. Ga03G1298 was identified for the RSR_FW trait. It could encode the light-harvesting complex-like protein OHP1 and may play a photoprotective role in the thylakoid membrane in response to light stress. In Arabidopsis, a recent study [Ga03G1298 expressed higher in leaves than roots. And we also found the Ga03G1298 was higher expressed in the lower RSR_FW cotton accessions (“AHQJZM” and “DPL971”) on leaf samples, and this may further prove the Ga03G1298 gene is closely related to the RSR_FW trait. The gene name of Ga09G2054 is UBC10. It is a Ubiquitin-conjugating enzyme E2 10. This gene was identified by the RWC trait, however, in our qRT-PCR result, it didn’t show a significant expression difference in root samples among the four representative cotton accessions. Ga10G1342 (RPS15) could encode 40S ribosomal protein S15. A recent study revealed that the ribosomal protein RPS15 exhibited bactericidal activity against Gram-positive Staphylococcus aureus in amphioxus Branchiostoma japonicum [Arabidopsis thaliana, Kathleen et al. (2010) found the ribosomal protein S15a could be classified into two categories (Type I and Type II), and further knock down experiment suggested the RPS15aE isoform (Type II S15a) may act as regulators of translational activity [Ga10G1342 gene was identified in the RDW trait, which means that this gene may be closely associated with the root dry weight in cotton. The FPKM of this gene showed a higher expression level in “AHQJZM” and “DPL971” than “WMSLDMH” and “DGXH” on the period of 12das after sowing. Our qRT-PCR result also showed the same expression pattern in the root, which may further validate the importance of this gene. Ga11G0096 (CYP76A2) was associated with the SWC and TWC trait, which could encode the cytochrome P450 76A2. As early as 1993, Toguri et al. cloned this gene in eggplant seedlings and attributed it to the CYC76 gene family [CYC76 gene has tissue expression specificity. Ga11G2490 is a new gene found in cotton. This gene was identified in the shoot water content trait, which means this gene may also be associated with the water content in cotton seedlings.Based on 1,425,003 high-quality SNPs, we identified 83 significant SNPs, and successfully detected 69 putative candidate genes related to seedling biomass-related traits. After integrating with the transcriptome data in four representative accessions, we finally screened out 5 genes . And 2 weeks later, similar plants for each accession were sampled and were directly cut into two parts of the root and the shoot by scissors  [P > 6.15 was set as the significant threshold. The newly updated G. arboreum genome (download from cottonFGD.org) [The genotype data of the 215 Genetics . SoftwarGenetics , 42. AftGenetics  was chosty SNPs) , and -loFGD.org)  was set The confidence intervals were identified by IGV software , and LD G. arboreum accessions. We extracted the FPKM (Fragments per kilobase of exon model per million mapped fragments) data of GWAS candidate genes in this study for further comparative analysis. Another study is for G. arboreum cultivar Shixiya1 RNA-seq analysis. We downloaded the TPM (Trans per kilobase of exon model per million mapped reads) expression data from the CottonFGD.org website (data accession: PRJNA594268). And we further analyzed the TPM data of different tissues  for our GWAS candidate genes.The transcriptome data comes from two studies. One study (unpublished) was completed by our team, which mainly completed the mRNA sequencing of the roots of “AHQJZM”, “DPL971”, “WMSLDMH” and “DGXH” in three different periods . Each root sample had two replicates, and the plant culture method was similar to our methods of planting 215 For further gene screening analysis, the candidate genes of GWAS were first excluded by the transcriptome data when the gene was not expressed in any tissues. Then, the expression data (FPKM or TPM) of candidate genes were plotted by TBtools v0.67  based on-ΔΔCt method [The root and leaf samples of four cotton accessions  on the 12 das after sowing were collected and stored at − 80 °C until further RNA extraction. Total RNA was extracted for each root sample by using a Plant RNA Purification Kit . The qRT-PCR was performed by 7500 Fast ABI  with TransStart Top Green qPCR SuperMix kit (TransGen Biotech) according to the manufacturer’s instructions. The qRT-PCR reaction system had a final volume of 20 μL, which consisted of 2 μl of cDNA sample, 10 μl of 2 × TransStart Green qPCR SuperMix, 0.4 μl of Passive Reference Dye, 6.8 μl of ddH2O, and 0.8 μl of primers. The reactions were amplified at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 60 °C for 15 s, and 72 °C for 10 s, and then 95 °C for 30 s, 60 °C for 1 min, and 95 °C for 15 s. All reactions were set with three independent biological replications. Primers were designed using Primer5.0 software. The gene β-actin was used as a reference. Primers of selected genes are listed in Table St method .P-value < 0.05 is regarded as significant.The basic descriptive analysis and frequency distribution of the 11 seedling biomass-related traits were performed by GraphPad Prism 8 . The broAdditional file 1: Table S1. Primer sequences of selected genes used for qRT-PCR assay in this study. Table S2. Phenotypes of 11 seedling biomass-related traits in 215 G. arboreum accessions. Table S3. Basic descriptive analysis for seedling biomass-related traits. Table S4. Summary of SNPs that detected in 11 seedling biomass-related traits. Table S5. Significant SNPs identified for the seedling biomass-related traits. Table S6. 69 candidate genes associated with seedling biomass-related traits identified by GWAS. Table S7. FPKM of the GWAS candidate genes in 4 representative cotton accessions. Table S8. TPM of the GWAS candidate genes in Shixiya-1.Additional file 2: Figure S1. The separating process of root and shoot in cotton seedlings. Figure S2. Frequency distribution of 11 seedling biomass-related traits in 215 G. arboreum accessions. Figure S3. Manhattan plot of SFW. Figure S4. Manhattan plot of RFW. Figure S5. Manhattan plot of TFW. Figure S6. Manhattan plot of SDW. Figure S7 Manhattan plot of RDW. Figure S8. Manhattan plot of TDW. Figure S9. Manhattan plot of RSR. Figure S10. Manhattan plot of TWC. Figure S11. Heat map of TPM expression of candidate genes in different tissues of Shixiya-1.


E = 13 ±4 GPa and H = 0.21 ± 0.03 GPa, as revealed bynanoindentation experiments, making them highly resilient and easilytransferable. Finally, the ordering induced by the interface opensmany possibilities for further studies, including those regardingthe supramolecular structure on PDA due to their similarity to 2Dlayered materials.Highly oriented,layered, and mechanically resilient films of polydopamine(PDA) have been synthesized from the air/water interface. The filmsshow a unique layered structure, as shown by scanning and transmissionelectron studies (SEM/TEM) and X-ray diffraction analysis (XRD), whichresemble that of 2D layered materials. The films exhibit a compositiontypical of PDA-based materials, as evidenced by X-ray photoelectronspectroscopy (XPS); moreover, the samples present the distinctiveresonance modes of PDA-based nanomaterials in Raman and infrared spectroscopy(FTIR) experiments. The presence of highly ordinated 3–4 protomoleculestacking, taking place at the air/water interface, with a unique eumelanin-likesupramolecular arrangement is presented. Moreover, the films showsuperior mechanical resilience with  Despitetheir applicability and the rather exceptional properties of PDA,a few studies have shown the use of PDA membranes as wound dressingtools32 and as a calcination template forthe oxygen reduction reaction.33 However,the structure of these PDA layers is typically amorphous and doesnot show any self- or supramolecular organization, despite the similaritiesbetween PDA with the eumelanin protomolecule stacking configuration.35 Nevertheless, the stacking formation should, in principle, allowfor the presence of a pseudo-2D organization in PDA membranes, withlittle or no long-range ordering.The majority of the applicationsof PDA and studies attempt tounderstand the physicochemical characteristics of PDA layers grownon solid supports or directly address amorphous PDA particulates obtainedduring polymerization.E) of 13 ± 4 GPa and hardness (H) of 0.21 ±0.03 GPa, which allows for the easily transferability of the filmto other substrates and even relative large free-standing architectures.Additionally, we propose a mechanism for the formation of these layersusing a combination of the previously described eumelanin protomoleculestacking and the air/water interface.In this work, we describethe synthesis and characterization ofsuch PDA membranes obtained from dopamine polymerization in Tris bufferand their self-assembly in pseudolayered structures. We investigatetheir structure, optical properties, and microscopic features as wellas their mechanical response after transferring to commercially availablesilicon substrates. The results here presented show a previously unreported,highly oriented layered structure with clear similarities to graphite/grapheneoxide materials. We also evaluate the superior mechanical responseby nanoindentation, with a Young’s modulus (2–1). The small stirring barwas put in the Petri dish and rotated at 300 rpm. Finally, the vesselwas covered with a glass lid. For thick samples, a piece of siliconwas immersed in the Petri dish, with a corner covered by Kapton tape.A conventional lime soda glass microscope slide was also placed inthe bottom of the dish for control. The polymerization process wasconducted for 24 h with a nonsealed cover lid (a larger Petri dish)under flowing air from the fume hood. Then the lid was taken out toscoop the thin layers floating on the solution’s surface byusing commercially available Si(001) substrates with native SiOx andmica glasses. The rest of the reaction was left under the hood untilthe solution dried, producing the thick layers on the exposed areasof the Si and microscope slide substrates.The synthesis ofPDA membranes was performed in a Petri dish (14 cm in diameter) containingTris buffer solution  followed by the additionof dopamine , working in 45 kV and 40 mA (Cu source). The out-of-planeordering of the samples was examined by performing GiXRD scans atdifferent Ψ angles (0–45°). Scanning electron microscopy(SEM) images were collected in a JEOL 7001TTLS (JEOL) working at 1kV, without sputtering cover layers. Atomic force micrographs (AFM)were collected in an ICON (Bruker) microscope working in tapping mode.High-resolution transmission electron images (HR-TEM) were collectedin an ARM-200f (JEOL) microscope, working at 80 kV and equipped withan EDX detector. Commercially available bare copper grids (100 mesh)with ∼204 μm hole width (Ted Pella) were used to manuallyscoop the PDA membranes. Samples were left to dry overnight in ambientconditions. Infrared spectroscopy (FT-IR) was performed by using anFT/IR-4700 (Jasco). Raman studies were performed by using a 633 nmlaser, in backscattering geometry in an NT-MDT (Renishaw). Finally,nanoindentation experiments were performed by using a TI-950 (Hysitron)triboindenter equipped with a Berkovich tip. Load and displacementcurves were analyzed according to the Oliver and Pharr method37 and methodology described elsewhere.38We characterized samplesby X-ray diffraction (XRD) and grazing incident X-ray diffraction(GiXRD) using an MRD-X’pert3Figure S1. Thethick sample (i) refers to a silicon piece placed at the bottom ofthe solution, which collected several layers of PDA after the evaporationof the solution. The thin sample (ii) refers to the scooped membranefrom the surface of the dish after 24 h of synthesis. The synthesisvessel is presented in Figure S2a). Furtherinspection using selected area electron diffraction (SAED) patternsshow no in-plane discernible organization, and HR-TEM micrographsshow no distinguishable structures in the nanometers range. AdditionalSTEM images and SAED patterns are shown in Figure S2b. The elemental distribution and quantification were performedby EDX; the calculation was made by analyzing five independent measurementsin different regions of the sample . The pondered results show mainly C(80.9%), O(3.6%%), and N(15.4%)signals, clearly localized on the membrane section of the sample.Electron energy loss spectroscopy (EELS), aiming to identify the thicknessof the layers (zero loss), shows an overage thickness of ∼55nm per membrane layer . At thispoint, it is suspected that the observed layered structures belongto a sort of PDA supramolecular structure or a layered ordering ofthe PDA membrane, which has not been observed before.As described in the Figure S4). The diffractogram shows aset of peaks at ∼10.7° (d = 8.3 Å),∼21.6° (d = 4.1 Å), ∼32.4°(d = 2.8 Å), and ∼41° (d = 2.2 Å), with reducing in intensity at higher 2Θ angles.Grain sizes calculated by the Scherrer equation are ∼10.7°(69.5 nm), ∼21.6° (37.6 nm), and ∼32.4° (12.9nm) after geometrical adjustments.39 Thevalues are smaller than the observed membranes and contrast with thelack of visible grain boundaries on SEM and TEM images. As it is welldocumented in the literature, PDA is an amorphous polymer that doesnot show any distinctive crystallographic features. Therefore, thepresence of these peaks needs to arise from other structural order.It is striking the resemblance of the observed XRD peaks and theirinterplanar distances with those of well-known carbon materials, suchas graphene oxide (GO), ∼10° and ∼40°,40 reduced graphene oxide (rGO), ∼20°,41 and other carbon materials.42 Despite their similarities, it is clear that there is nosynthesis pathway between dopamine and Tris buffer, capable of resultingin 2D-graphene-based materials; thus, the observed structure mustbe a PDA analogue of a 2D-carbon material. A useful method to determineif the 2D-like domains are perpendicularly aligned with the substrate,or randomly oriented within the film, is the 2Θ scan at differentΨ angles as shown in the Figure S5 for other components and Table S1 for quantification). The high-resolutionN 1s region collected for the thin sample shows the presence of twodistinctive nitrogen species centered at 400 eV (3.05% at conc) and401.9 eV (1.11% at conc), which can be assigned to R2–NHand R–NH2, respectively, moieties present in PDAmaterials.43 One of the most well-establishedtechniques for investigating both van der Waals and carbon materialsis Raman spectroscopy.44 It is a referentfor both 2D and pseudo-2D materials as it reflects allows the in-detailinvestigation of their structure. –1) and G (1500–1630cm–1) peaks, typical for carbonaceous materialsand commonly observed in PDA materials;45 moreover, there is a small but clear signature for the second-orderprocess by the apparition of the G′, alsoknown as the 2D (2600–3000 cm–1) peak.46 A comparison between the ID/IG ratio for thick and thin samplesshows a ratio = 0.83 and 0.88, respectively, which are surprisinglylow. Such low values are typically found in pyrolyzed or graphitizedPDA,49 which is surprising since there is no chemical routefor graphitization in our preparation. Nevertheless, applying thegeneralized equation for crystalline size (La),50 we obtain a La = 42.5 nm. This value, although slightly inferior tothe ones measured by XRD, should contain only information about thepseudo-graphitic (π–π) components of the layersinstead of purely graphitic ones. More importantly, the thin sampleshows a group of unreported minor signals, not commonly observed fora PDA-based material. Some of the vibrations can be understood bytheir resemblance to the experimental and theoretical results foreumelanin, specifically the 940–1045 cm–1 vibration, attributed to the C–H, O–H, and O–Hdeformations, and the 1100–1242 cm–1 vibration,attributed to the stretching of C–C, C–OH, C–H,and N–H bound present in amide, phenolic, and pyrrole groups.51 The lower frequencies at 700 and 420 cm–1 are difficult to assign to any specific organic compound;however, these two bands are often observed in defective and twistedcarbons, which have been proposed as an indication of the less presenceof sp2 in carbonaceous samples, especially the ∼400cm–1 band53 previously observedin the literature.54 Additionally, theband at 700 cm–1 is attributed to the sp2 amorphous phase activated by cooperative disorder in the sample,55 which is a feature that melanin shares withpartially disordered carbon materials.34 Furthermore, these studies have also shown some overlapping withthe bands attributed to eumelanin and those of carbon materials, whichcomplicates the distinction between these contributions.Chemical studies performed by XPS on the thin sample are shownin –1 with a peak at 3369 cm–1 is assigned to stretchingmodes of ν(N–H) and ν(O–H) from amino andhydroxyl moieties. The peaks at 2969, 2922, and 2847 cm–1 correspond to stretching C–C modes from aliphatic CH2 groups present in polydopamine. The peak at 1726 cm–1 is due to the presence of carbonyl ν(C=O) from quinones.The signals in the range between 1591 and 1340 cm–1 result from stretching modes of νring(C=C)and νring(C=N), 1460 cm–1 νring(C=C), and 1354 cm–1 νring(CNC) from aromatic amines and the indolering, clearly proving the PDA layer on the support. Those data arein agreement with previously published spectra of PDA layers.56 However, it is impossible to conclude more detailedstructural properties of polydopamine by using the FTIR technique.FTIRstudies aiming to address the chemical structures presentin the samples are shown in 57 show a promising panorama for further studies.Nevertheless, the distinctive structural differences (XRD/SEM/TEM)of the membranes here presented, when compared with other PDA films,58 The authors reportedan E = 2.3 ± 0.84 GPa, which increased to >14GPa upon calcination (600 °C). The films were prepared in a somehowsimilar method; however, pristine membranes were subjected to a heattreatment (300 °C) before mechanical analysis, which resultedin cracks and shrinkage of the membranes. More recent studies haveshown free-standing membranes of PDA synthesized by electrochemicalmethods.52 The mechanical properties ofthese films were investigated by micro-Brillouin light spectroscopy.59 The authors reported a much higher Young’smodulus of ∼12 GPa, which allowed the easy transfer of thefilms.One important aspect to studyis the superior mechanical strength shown by the PDA thin films. Thisis first intuitively observed in the rather large suspended area onthe TEM grid 1c. PreviRq = 2 nm for the thin sample. We performed a series ofindentation tests on the thick PDA sample surface . Nevertheless, the PDA structureremains elusive, and it is still under debate in the literature.The physicochemicalproperties of PDA show significant similarity to those of naturallyoccurring eumelanin. Thus, the conversion of dopamine to PDA is explainedby applying the Raper–Manson mechanism, which is frequentlyused to explain the formation of eumelanin.66 First, the protomolecule stacking has been proposed and investigatedextensively to show the stacking of 3–4 layers consisting offive to seven monomers of indole-5,6-quinone. The interplanar distanceof this stacking has been extensively studied from naturally occurringand synthetic sources, and it is reported as 3.4–4.7 Å,73 which properly correspond to the second register peak in d = 4.1 Å), and are represented in 67 observed for natural eumelanin,which is typically explained by the partially destacking of the population.34 More interestingly, this value also suits theexperimental height of the 3–4 protomolecule stacking (7.4Å)70 and is somehow close to the ∼10.7°(d = 8.3 Å) peak observed in the samples, whichcould suggest a superior ordering and periodic organization of 3–4stacked protomolecules in the film, as presented in 74—more specifically the nanoparticles accompanying the membranes(d = 2.8 Å)75 and ∼41° (d = 2.2Å)76 interplanar distances have beenalso recently observed and attributed to the in-plane periodicityand semicrystalline structure at the mesoscale. These interpretationsgive some understanding to the periodicities observed; however, itis clear that the textured growth of the film is strongly relatedto the air/water interface, as shown in 57 However, the grains size of the2D-like membranes and the lack of visible grain boundaries in bothSEM and TEM experiments suggest a possible mixture of grains withamorphous PDA parts, while retaining the observed 2D-layered structure.Based on the similarity of PDA to eumelanin, there are a few optionsfor the PDA structure observed in this study.embranes1c, in wh74 without any preferential texture,which contradicts the texture observed here. An explanation couldbe that they are formed by using the preordered stacked moleculesat the air/water interface as a “seed” . However, these aspectsneed to be studied in detail in the future as well as the optimizationand control over the membrane/film thickness, optical absorption,and grain size. Further studies are in progress to understand theseaspects and the open questions proposed by this study.Nevertheless, a few questions are still open forresearch, especiallysince the nanoclusters of PDA have been reported to be highly polycrystalline,a “seed” 3c. This 480 profiting from the easy transferability of the layers and ratherunique structure. The 2D PDA layer could show different optical constantswhen compared with traditional PDA due to confinement. Moreover, thecontinuous interest of 2D stacked metamaterials and heterostructures,82 in which layers from different materials are sandwiched together,could benefit from the inclusion of PDA 2D layers. Also, PDA nanometriccoatings have shown enhanced chemical stability toward corrosion83 and more recently enhancement of photocatalyticactivity when in conjunction with semiconductors,9 which could be enhanced by current van der Waals 2D materials.Additionally, here we open the possibility for easy integration inmany known architectures in fields such as biomedical applicationsin wound dressing and antibacterial surfaces of PDA.7 Furthermore, the mechanical strength of the membranes iscomparable to that of other resilient PDA membranes reported in theliterature, allowing many applications on free-standing architectures,such as resonators with a potentially high-quality factor.59 Finally, our study and films show a set of unique structural andoptical properties, which open the possibility for many new studiesand experiments in PDA membranes and films.We have prepared free-standing and easilytransferable PDA filmsby a simple method that exploits the air/water interface. The filmsshowed a previously unknown macromolecular arrangement of PDA resemblingthat of eumelanin clusters. More interestingly, the membranes showa unique layered structure and organization, which resembles thatof 2D materials, making them an excellent candidate for further researchand integration in several fields. Here, we outline some of the possibleapplications of our materials. One of the clear candidates is chromaticcontrol and coating technology,


NBCe1 is highly selective to HCO3− and does not transport other anions; the molecular mechanism of anion selectivity is presently unclear. We previously reported that replacing Asp555 with a Glu (D555E) in NBCe1 induces increased selectivity to other anions, including Cl−. This finding is unexpected because all SLC4A transporters contain either Asp or Glu at the corresponding position and maintain a high selectivity to HCO3−. In this study, we tested whether the Cl− transport in D555E is mediated by an interaction between residues in the ion binding site. Human NBCe1 and mutant transporters were expressed in Xenopus oocytes, and their ability to transport Cl− was assessed by two-electrode voltage clamp. The results show that the Cl− transport is induced by a charge interaction between Glu555 and Lys558. The bond length between the two residues is within the distance for a salt bridge, and the ionic strength experiments confirm an interaction. This finding indicates that the HCO3− selectivity in NBCe1 is established by avoiding a specific charge interaction in the ion binding site, rather than maintaining such an interaction.The Na/HCO NBCe1 is highly selective to HCO3− and does not transport other anions, including Cl− [3 transport mediated by NBCe1 and its family proteins, collectively the Na+-coupled bicarbonate transporters, in humans. NBCe1 exists as multiple variants, due to different N- and C-terminal sequences, and each variant differs in tissue expression, intrinsic functional properties, and regulation [+:HCO3− stoichiometry of 1:3 in renal proximal tubules and 1:2 in other cells, as well as in heterologous expression systems. Overall, NBCe1 plays an important role in the physiology and pathophysiology of many different organs, such as kidneys, heart, brain, eyes, enamel organs, and intestines [NBCe1 is a membrane protein that mediates electrogenic Nany cells . NBCe1 wny cells , where iding Cl− . The gengulation . The tratestines ,8,9,10.+ and CO32− (HCO3−) move. The ion accessibility pathway is formed by TMs 1, 3, 5, 8, 10, 12, and a short loop connecting TM13 and TM14, and the diameter along the pathway varies, ranging from >12 Å in the entrance region to ~2 Å diameter in the middle of the protein. Many amino acid residues lining the pathway were previously recognized as residues critical for transport function by mutagenesis studies [+-driven Cl/HCO3− exchanger NDCBE [3 changer AE1 [+, Cl−, HCO3−, and CO32− transport via SLC4A bicarbonate transporters.The cryoEM structure of NBCe1  has prov studies ,16,17. Ter NDCBE  and Cl/Hnger AE1 , NBCe1 s555, Lys558, Lys559, and Lys562, all of which are in TM 5, and site S1 has residues from multiple TMs and loops. We have previously reported that substituting Asp555 with a Glu (D555E) causes the transporter to be permissive to other anions, including Cl−, NO3−, SCN−, I−, and Br− [3−; thus, it produces an outward current (INBC) when HCO3− is available but a Cl− current (ICl) when HCO3− is unavailable. This modified selectivity should be related to a geometrical difference in the carboxyl side chain of Asp vs. Glu, due to an additional carbon backbone. On the other hand, all members of Na+-coupled bicarbonate transporters contain either Asp or Glu at the corresponding site, implicating that the geometrical difference in the carboxyl side chain is not the only cause for altered anion selectivity and an additional mechanism should be involved. Elucidating that mechanism will help us understand how bicarbonate transporters selectively transport HCO3−, while excluding other anions.Recently, Zhekova et al.  performe and Br− . D555E mXenopus oocytes and subjected to recordings of ClI, INBC, and intracellular pH (pHi). The results show that ICl is induced by a charge interaction between residues in site 2. The two residues involved in the interaction are not simultaneously present in any member of the SLC4A bicarbonate transporters; thus, the HCO3− selectivity is maintained by avoiding a charge interaction in the ion binding site, located at the entrance of the ion accessibility pathway. We also find that Na+ is required for HCO3− access to the transporter, consistent with a conventional concept of a Na+ prerequisite for substrate movement in secondary active transporters.In this study, we investigated the effect of charge interactions between residues in site 2 on anion selectivity. Candidate residues were changed by TM5 replacement and site-directed mutagenesis, and the mutant transporters were expressed in ICl, produced by D555E, we expressed human NBCe1 and mutant D555E in oocytes and applied them with 71 mM Cl− during superfusion of Cl−-free solution. ICl, in response to bath Cl−, but produced an outward INBC upon exposure to 5% CO2, 25 mM HCO3−, consistent with its electrogenic Na/HCO3 cotransport activity. In contrast, D555E produced an outward ICl, in response to Cl−  is summarized in ICl produced by D555E was reduced in the presence of CO2/HCO3− . In other experiments, we then determined I–V relationships for ICl and INBC to compare the current responses at different voltages in NBCe1 vs. D555E. As shown in − , reflecting Cl− influx. However, in CO2/HCO3− solution . The two I–V curves were parallel to each other in the outward direction, as they are INBC. The curves crossed at a negative potential (approximately −80 mV), probably due to Cl− efflux via D555E in the inward direction.To record e to Cl− B. ICl was report  that D55solution E, NBCe1 − transport by D555E is less favorable than HCO3− transport, when both ions are present in the bath. To determine whether this feature depends on Na+, we performed two sets of experiments. In the first set of experiments, we recorded ICl in Na+-free CO2/HCO3− solution and tested whether ICl could be reduced under this condition. Representative recordings of ICl, produced by NBCe1 and D555E, are shown in ICl in the absence of CO2/HCO3− and, more importantly, in the Na+-free CO2/HCO3− solution. The current amplitudes were similar in both solutions, indicating that HCO3− has negligible effect on ICl under the Na+-free condition. ICl between groups in these two solutions from other oocytes (n = 5 NBCe1 and 10 D555E). No significant difference was observed within groups. In the second set of experiments, we induced ICl in Na+-free solution and tested whether the induced ICl could remain after CO2/HCO3− application under the continued Na+-free condition. As shown in ICl, D555E produced ICl under the Na+-free condition, regardless of bath CO2/HCO3−. A slight decrease after CO2/HCO3− application is probably due to Cl− mismatch between solutions. Consistent with this result, comparison of mean ICl (n = 5/group), before and after CO2/HCO3− application, resulted in no significant difference  as HCO3− is continuously transported into the oocyte by NBCe1 and associates with intracellular H+. Applying CO2/HCO3− also elicited an outward INBC (arrowhead), consistent with an influx of a net negative charge, due to 1 Na+ and 2 HCO3− (or 1 CO32−). i and INBC, produced by the TM5-replaced chimeric transporter, subjected to the same experimental protocol. The chimeric transporter had a slower pHi recovery rate (dpH/dt) and smaller INBC in CO2/HCO3− solution than NBCe1. Consistent with this observation, mean dpH/dt and INBC from 5 oocytes per group were significantly decreased in the chimeric transporter , calculated from the slope of the ICl–V relationship . GCl of the chimeric transporter was negligible.D555E is charge-conserved but has different geometry of the carboxyl group in the side chain due to an additional carbon backbone. This led us to postulate that Gluge clamp A,B. In oydration A. The pHICl in the chimeric transporter indicates that Glu555 is not the sole residue for ICl and additional residues are involved. Those residues should be in TM5 because other TMs were unchanged in the chimeric transporter. Asp555 is a residue in the anion binding site S2 that includes Lys558, Lys559, and Lys562 . I–V relationships for the chimeric transporter without mutation. As expected, no significant difference was observed in the I–V curves before and after Cl− application (red line in the figure). In contrast, replacing Glu558 with a Lys (E558K) increased the slope in the outward direction upon Cl− application (562 with a Lys (D562K) had no effect  increased the slope in the outward direction upon Cl− application . We also compared mean INBC between groups to evaluate the effect of Lys mutations on Na/HCO3 cotransport and found a decrease in INBC by the mutations .The identification of Lys.1 mol/L A,C, cons05 mol/L B but had.1 mol/L D. The rentration E. The re555 and the amino group of Lys558 using the structure editing function with Dunbrack rotamer library [555 and the amino group of Lys558 was 5.63 Å (555 and Lys558 was 3.79 Å (559 and Lys562 were higher than 4.0 Å (data not shown). Thus, the bond length was consistent with a weak electrostatic interaction between Glu555 and Lys558, but neither Lys559 nor Lys562. The importance of the Glu555/Lys558 interaction for ICl was further examined by replacing Glu555 and Lys558 residues with other amino acids and comparing their ability to produce ICl  or Lys558 with an Asp (E–E) near completely abolished ICl. In contrast, replacing Lys558 with a positively charged Arg (E–R) retained measurable ICl. One-way ANOVA, with Sidak post-test, revealed a significant change in ICl for N–K, Q–K and E–E pairs compared to E–K and E–R pairs . Water-injected control showed no current.To further determine the above salt bridge interaction, we analyzed the bond length between the carboxyl group in the side chain of Glu library  built ins 5.63 Å A, highers 3.79 Å B. The leduce ICl C. Replac555 and other charged residues in the entrance anion binding site S2 on Cl− selectivity and made the following observations. (i) Replacing Asp555 in NBCe1 with a charge-conserved Glu induces a permissiveness to Cl− that is normally not a substrate. This replacement does not alter HCO3− selectivity as INBC is favorably produced when both HCO3− and Cl− are present. (ii) Under the Na+-free condition, D555E produces ICl even if HCO3− is available in the bath. The reason is that the anion binding site is not occupied with HCO3− in this condition; as a result, Cl− is accessible to the site. Thus, Na+ is required for HCO3− to access its binding site. (iii) The ICl induced by D555E is due to a charge interaction between Glu555 and Lys558. Other Lys residues in site S2 have negligible effects on Cl− transport. Glu555 and Lys558 are not simultaneously present in any member of the SLC4A bicarbonate transporters, indicating that the high HCO3− selectivity in these transporters is maintained by avoiding a charge interaction between the two residues. This molecular feature is interesting as it is generally understood that electrostatic interactions contribute to protein structure and create a suitable environment for protein function such as enzyme catalysis, protein-ligand binding, thermal stability, and macromolecular assemblies [In this study, we examined the effects of Asp/Glusemblies ,24,25. I555, Lys558, and Lys562 in NBCe1, all of which constitute site S2 located near the entrance of the ion accessibility pathway. Site S2 also contains Lys559, a DIDS-interacting residue [− selectivity because it is conserved in all SLC4A transporters. The SLC4A transporters contain either Asp555 or Glu555 but maintain a high selectivity to HCO3−; thus, a residue capable of interacting with these residues should not be conserved. The CryoEM of NBCe1 [555 and Lys558 are located to the protein center, while Lys559 and Lys562 are positioned further away from the center. The bond length between Asp555 and Lys558 is higher than the maximum length required for a salt bridge to take place, but Glu555 substitution has decreased the length , but D555E produces a larger NO3− current than ICl. It is, thus, likely that site S2 is molded to sterically distinguish HCO3− or CO32− from other polyatomic anions in a trigonal planar arrangement. The charge interaction between Glu555 and Lys558 in D555E modifies this steric arrangement in a way that other structurally similar ionic compounds, including NO3−, are allowed. The modified steric arrangement also allows Cl− to access the site but, given its monatomic molecule and competition with HCO3− or CO32−, we think that a Cl− leak occurs at one of the three coordinating residues for peripheral oxygen atoms of HCO3− or CO32−. This interpretation is consistent with the MD simulations that Lys558 and Lys559 are the closest coordinating residues of CO32−, determined from ion density maps and contact frequency analysis.The amino acid residues in the chimeric transporter we examined correspond to Asp residue , but we of NBCe1  shows the length A,B. We h555–Lys558 pair produces ICl in the absence of CO2/HCO3− and INBC in the presence of CO2/HCO3−; that is, the presence or absence of ICl reflects whether the anion binding site is occupied with HCO3− or CO32−. ICl is produced in Na+-free CO2/HCO3−; thus, the binding site is not occupied in the absence of Na+, indicative of Na+ precondition prior to anion binding. Based on this interpretation, a model of the ion binding process can be made. In NBCe1, ion transport begins with a recruitment of Na+ to its binding site. The Na+ binding then allows HCO3− or CO32− to access its anion binding site and as a result both ions are bound to the transporter. The steric arrangement of Asp555, Lys558, and Lys559 in site S2 is critical for distinguishing HCO3− or CO32− from other anions. The same ion recruitment process also takes place in a mutant transporter containing the Glu555–Lys558 pair, such as D555E. However, the charge interaction between the two residues modifies the steric arrangement of residues in S2, such that other anions, such as Cl−, are permissive; as a result, Cl− is accessible to the anion binding site. Our model proposes that Na+ binding is a necessary first step prior to anion binding and, thus, should be independent of external HCO3− or CO32− levels. In this sense, it is interesting to note that the apparent affinity of NBCe1 for Na+ is independent of external HCO3− concentrations [555 facilitates Na+ recruitment from the extracellular fluid surrounding the transporter. One might argue that Na+ should overcome an electrostatic repulsion from the positively charged Lys residues before reaching its binding site. Decreased INBC by E5558K and E558K/D562K , comparable to ICl in response to Cl−. However, we did not observe IHCO3 under the Na+-free condition. The lack of IHCO3 under the Na+-free condition reflects that HCO3− movement via D555E is solely mediated by electrogenic Na/HCO3 transport that generates INBC. The important finding is that ICl is significantly inhibited by electrogenic Na/HCO3 transport , implicating a salt bridge between the two residues. It will be interesting to examine whether this mutation can cause Cl− leak. Additionally, depending upon NBCe1 variants, intracellular Cl− can regulate the transporter activity [− leak in NBCe1 is beneficial for cellular HCO3− homeostasis and epithelial electrolyte secretion.Lastly, our study leads us to a discussion about a pathological implication of Cldination ,29,30,31s et al. ,33 have y glands . Anothery glands . Our anaactivity . Thus, t32− or HCO3− recruitment to the binding site and ion binding sequence. Future studies will be of the molecular mechanism underlying ion selectivity and translocation in other Na/HCO3 transporters.In summary, by analyzing the TM5 chimeric transporter and relevant point mutants, we identified a charge interaction in site S2 as a key factor for anion selectivity and provided new insights into COD555E made on human NBCe1-A  was described previously . The TM5Xenopus laevis oocytes, at stages V-VI, were purchased from Ecocyte Bioscience . For cRNA synthesis, plasmids containing NBCe1 or mutant transporter DNAs were linearized and transcribed using the mMessage/mMachine transcription kit . Transcribed RNAs (15–25 ng in 46 nL) were injected per oocyte. Equal amounts of RNAs were used when multiple samples were compared. Controls were water injection. Oocytes were maintained for 3–4 days at 18 °C before use.2, 1 MgCl2, and 10 HEPES, pH 7.4) and impaled with two borosilicate glass electrodes filled with 3 M KCl. The tip resistance was 0.5–2 MΩ. After stabilization of the resting membrane potential, the oocyte was clamped at –60 or 0 mV using the voltage-clamp amplifier OC-725C . For recording ICl, an oocyte was superfused with Cl−-free solution, which replaced all NaCl in ND96 solution with Na/gluconate, and then with 71 mM Cl− solution, which replaced 25 mM NaCl with the same amounts of Na/gluconate. A small amount of Cl− (<3 mM) was included to minimize junction potential. For recording INBC, 25 mM NaCl was replaced with the same amounts of NaHCO3 equilibrated with 5% CO2. Recording ICl in CO2/HCO3− solution was achieved after INBC reached steady-state. Na+-free solutions were made by substituting Na+ with N-methyl-D-glucamine NMDG. Current-voltage (I–V) relationships were determined by a staircase voltage command between −120 to +60 mV, with 20 mV increments for 100 ms duration. The voltage command was applied immediately after a current reached steady state. Voltage signals were sampled by Digidata 1322A  and data were acquired using pClamp 10 (Molecular Devices). Signals were filtered using an 8-pole low pass Bessel filter, with a cutoff frequency of 0.1–1 Hz. Recordings were made at room temperature.An oocyte was placed in the recording chamber containing ND96 solution . The rate of pH change (dpH/dt) was determined by drawing a line during the first 4 min of recovery from CO2-induced acidification.Oocyte pHd before . Brieflyi is the molar concentration of ion i (mol/L), and Zi is the charge number of that ion.For assessment of salt bridges, an oocyte expressing the mutant transporters was clamped at 0 mV and superfused with 96 mM Na/gluconate (plus 5 mM mannitol) or 197 mM mannitol, plus a small amount of chloride (<3 mM), until base line currents became stable. Then, a series of test solutions containing 1, 10, 20, 40, and 96 mM of NaCl were applied. NaCl in each test solution replaced the equivalent amount of mannitol or Na/gluconate. Each test solution was bracketed with NaCl-free solution to maintain steady-state baseline between test solutions. The ionic strength (I) was determined using the equation:555 and amino group of nearby Lys residues was identified when the distance between them was <4 Å. For D555E or the TM5-replaced chimeric transporter, amino acid changes were analyzed using the structure editing function with Dunbrack rotamer library [Analysis of the binding site S2 was performed with CryoEM structure of the human NBCe1 (accession code: 6CAA) from the RCSB Protein Data Bank using the molecular visualization program UCSF ChimeraX 1.1 . A hydro library  built inICl or INBC among multiple mutants; and (iv) two-way ANOVA for comparison between ICl vs. INBC among multiple mutants. The p value of less than 0.05 was considered significant. Data were analyzed using Prism 7  and Microsoft Office Excel add-in Analysis ToolPak .Data were reported as mean ± standard error. The level of significance was determined using (i) unpaired, two-tailed Student t-test for comparison between NBCe1 and D555E or chimeric protein; (ii) paired, one-tailed test for comparison of single transporters in two different solutions; (iii) one-way ANOVA for comparison of


Background: Emery–Dreifuss muscular dystrophy (EDMD) is a rare disease characterized by early joint contractures, slowly progressive muscular dystrophy, and cardiac involvement, which includes arrhythmia, dilated cardiomyopathy, hypertrophic cardiomyopathy, heart failure, and sudden death.Methods: Clinical data of the proband and family members were collected. The next-generation sequencing technology was used to analyze the pathogenic variants and copy number variations. Polymerase chain reaction was used to sequence the breakpoints of gene locus rearrangements.Results: Here, we report two siblings with EDMD in a family. The proband, a 17-year-old boy, manifested a dilated right heart, bradycardia, mild muscle weakness, and joint contractures. His younger brother only showed a mild bowing limitation with elevated creatine kinase. Next-generation sequencing revealed the complete deletion of EMD and a rearrangement in FLNA (exon29_48dup) in these two patients. The EMD deletion and partial FLNA duplication were accompanied by a 5 bp overlap (GTCCC) on the background of the FLNA-EMD inversion. These findings support the pathogenic mechanism of microhomology-mediated nonhomologous end joining.Conclusion: We report two siblings with complete EMD deletion and FLNA duplication in a family. A microhomology-mediated nonhomologous end joining event involving EMD and FLNA acts as the underlying mechanism. EMD, LMNA, SYNE1, SYNE2, FHL1, and TMEM43  duplication in an unconsanguineous family, supporting the pathogenic mechanism of microhomology-mediated nonhomologous end joining.Emery–Dreifuss muscular dystrophy (EDMD) is a rare inherited disease that is characterized by early joint contractures, slow-progressive muscular dystrophy, and cardiac involvement , includid TMEM43 . In addiuse EDMD . Among tt common . EMD, loThe research protocol (2015[916]) was reviewed and approved by the Ethics Committee of Peking University First Hospital. Written informed consent, which also included consent for the publication of medical data, was obtained from the patients and their parents.Medical records of the patients were reviewed. Clinical and laboratory data, including age of onset, onset of recognizable symptoms, motor development, contractures, creatine kinase (CK) levels, electrocardiography (ECG), Holter, ultrasound cardiography (UCG), cardiac magnetic resonance imaging (MRI), limb muscle MRI, and muscle biopsy findings, were collected.http://www.hgmd.cf.ac.uk/ac/index.php), LOVD (LOVD: http://www.dmd.nl/), ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/), 1000 Genomes (http://www.1000genomes.org/), ExAC (ExAC: http://exac.broadinstitute.org/), and gnomAD (gnomAD: http://gnomad.broadinstitute.org/) databases, were used to identify previously reported mutations and discover potential novel mutations. Predictions for the pathogenicity of missense mutations were obtained using PolyPhen-2, SIFT, and Mutation Taster. The predicted splicing mutations were tested through the Human Splicing Finder. The pathogenicity of candidate variants was classified according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) guidelines  were successively used to detect genetic variations in the proband. Candidate variants in the probands were validated by Sanger sequencing in the family. Disease association databases and population databases, including the HGMD . Ratios near 0 indicated deletions in male patients . In contrast, ratios near 1.5 indicated the presence of heterozygous duplications in female patients , and ratios higher than 1.75 indicated duplications in male patients . Polymerase chain reaction (PCR) was used to confirm the deletion of EMD in male patients. Statistical results were illustrated using GraphPad Prism 8 software .Next-generation sequencing (NGS) data were also analyzed to detect copy number variations (CNVs) by comparing the number of sequence reads between the patient and control samples. First, the number of reads in each amplicon was counted for the patient and control samples. For the purposes of this comparison, sequencing data from at least three control samples (genomic sequencing had been performed for patients diagnosed with other diseases) were used as the reference. Subsequently, the patient/control ratios were calculated by dividing the number of patient reads in each amplicon with the average of the total reads regarding this amplicon from control samples. For the genes, including EMD deletion and to sequence the breakpoints of the CNVs. Primers were designed separately for exons 1–6 of EMD using Primer 5.0 . The two primers designed for exon 1 were Primer F 5′-ACCGCGAGACCTTTTGCTC-3′ and Primer R 3′-GCGAGGCTCTCACCAGAGAA-5′. The two primers designed for exon 2 were Primer F 5′-CGCACGGGCCTGTAGTAGGT-3′ and Primer R 3′-GACGAAGTCGGGTGAAGGTG-5′. The two primers designed for exon 3 were Primer F 5′-CGGCCAGGATCAACTCGTAG-3′ and Primer R 3′-TCCTAAGGCTGCTGGAGTGG-5′. The two primers designed for exon 4 were Primer F 5′-ACCTTCACCCGACTTCGTCA-3′ and Primer R 3′-TCCTGAATGGCTCTGGGTGT-5′. The two primers designed for exon 5 were Primer F 5′-GAGCCATTCAGGAGGGTGTG-3′ and Primer R 3′-GCTGAAACAGGGCGGTAGTG-5′. The two primers designed for exon 6 were Primer F 5′-TGTGGGTTCCTGGCCTCTAA-3′ and Primer R 3′- AAAAATGCGGACCCAACAAA-5′. For the breakpoints, two primers were designed for a location in exon 2 of FLNA  and exon 29 of FLNA . PCR amplifications were performed using GoldStar Taq DNA Polymerase in an Applied Biosystems Veriti Thermal Cycler . Each PCR amplification was repeated for 35 cycles . Before sequencing, the PCR products were analyzed by electrophoresis on a 2% agarose gel.PCR and Sanger sequencing were performed to verify +, while that of the distal arms was V. The muscle strength grade of the proximal lower limbs was V−, with a distal dorsal flexion power of IV and a plantar flexion power of V−. Bilateral knee tendon reflex could not be induced. To confirm the diagnosis, laboratory tests were performed. The level of CK was 500–2,500 U/L . Skeletal muscle biopsy indicated muscular dystrophic changes   and array CGH. Finally, we used the NGS data of WES combined with PCR to help verify the region of deletion. The depth of coverage was used to detect the CNV by comparing its copy number ratio between the patient and the control. The ratio was 0 in the region of EMD, suggesting the deletion of the entire EMD gene  starts from exon 29, we proposed that one breakpoint in FLNA was located in intron 28 of FLNA. Besides, FLNA exon 2 was unaffected. Therefore, another breakpoint in FLNA may occur in intron 1 of FLNA to the linkage region of FLNA-EMD. A model including the inversion of FLNA-EMD is shown  between FLNA intron 28 and FLNA intron 1 on the background of FLNA-EMD inversion.A muscular disease gene panel that was performed in a local hospital detected no pathogenic variation in the proband. We reanalyzed the NGS data. We did not find any pathogenic variation but found that the depth of coverage was low in the whole region of ons 1–6)  in the pEMD gene . On the g region . The whodeletion . Therefodeletion , we propis shown  to explaEMD deletion and partial FLNA duplication. He was followed up and given rehabilitation training regularly. The mother was confirmed as a carrier.The family pedigree is shown in The proband had symptomatic right heart disease accompanied by mild muscle weakness. Due to his normal primary motor milestones, his mild elbow joint contracture and rigid spine may not have received enough attention. After a detailed physical examination, he was found to have mild muscle weakness, humeroperoneal muscle atrophy, mild elbow joint contracture, and a rigid spine. Based on his muscle weakness, high CK level, and pathological muscular dystrophic changes, the diagnosis of muscular dystrophy could be confirmed. Combining both cardiac and muscle involvement, skeletal muscle cardiomyopathy was confirmed. Given the severe cardiac involvement, humeroperoneal distributed muscle weakness, and special locations of the contractures, he was suspected to have EDMD. Finally, this diagnosis was confirmed by genetic analysis.EMD, located in Xq28, encodes the nuclear protein emerin. The complete deletion of EMD in the proband caused an X-linked EDMD phenotype. The cardiac phenotype often emerges within the end of the second decade along the third decade of life in classical X-linked EDMD . Given this consideration, we predicted that FLNA is not a causative gene in this patient, despite the FLNA rearrangement.Filamin-A is encoded by malities . In heteolvement . This 17EMD and FLNA, with a 5 bp overlap (GTCCC), acts as the underlying mechanism. Microhomology-mediated end joining is an error-prone repair mechanism for DNA double-strand breaks. It needs longer microhomology (more than 2 bp) and is independent of Ku70/Ku80, which is distinct from classic nonhomologous end joining   . The fir repeats . Two othts (FLNA . They arEMD deletion and partial FLNA duplication. A microhomology-mediated nonhomologous end joining event involving EMD and FLNA acts as the underlying mechanism.In conclusion, we reported two cases of EDMD with a complete


After treatment with hydrolysates at room temperature, a much higher total Fe content of 781.99 mg/kg was observed compared to other treatment conditions. The release of amino acids increased with temperature and was 520.54 mg/g for the hydrolysate-treated sample versus 413.12 mg/g for the control seal meat sample treated in buffer. Overall, this study provides useful data on the potential use of seal meat as a food product with high nutritive value and seal meat hydrolysates with antioxidant activity as preservatives to control oxidation in food.Seal meat is of high nutritive value but is not highly exploited for human food due to ethical issues, undesirable flavors, and loss of nutrients during the processing/cooking step. In this work, commercially available processed seal meat was treated with its hydrolysates as preservatives with the aim of improving nutrient bioavailability. The contents of the nutrients were analyzed after digestion using a simulated dynamic digestion model, and the effects of different processing conditions, i.e., low-temperature processing and storage (25 °C) and high-temperature cooking (100 °C), of seal meat were investigated. Hydrolysates with antioxidant activity decreased the amounts of the less desirable Fe Pagophilus groenlandicus) is a common seal species found in the Atlantic Ocean, particularly in waters that border Canada [The harp seal , magnesium (Mg), and zinc (Zn) [2+) is the form that is readily absorbed in the human gastrointestinal tract [2+ and Fe3+ obtained from seal meat after digestion. The conventional preservation methods for fresh seal meat cuts include freezing, vacuum packaging, and canning, and long-term storage can cause the gradual oxidation of Fe2+ and nutrient loss. The processing of seal meat at high temperatures (cooking) can lead to the oxidation of the preferred Fe2+ form to the less desirable Fe3+ form [As food, seal meat is commercially processed into different cuts, such as loins, flippers, and trims, and is consumed in a number of countries, including Canada, Greenland, Japan, Norway, Iceland, and Siberia . Althouginc (Zn) . Taking inc (Zn) . Becausee3+ form , which sReactive oxygen species (ROS) and free radicals are usually created during food processing, storage, and cooking. Antioxidant reagents, such as antioxidant hydrolysates, can react with ROS to extend the shelf life of food products. The antioxidant properties were analyzed for “Hydrolysates 25”, “Hydrolysates 100”, “Buffer 25”, and “Buffer 100”, as well as “Buffer” and “Hydrolysates”. DPPH free radical scavenging activities, as shown in It is also worth noting that processing at an elevated temperature (100 °C) affected the antioxidant properties of hydrolysates and buffer solutions compared to processing at 25 °C. The antioxidant properties of protein hydrolysates and peptides depend not only on their amino acid compositions but also on their structural features ,22, whicAfter treatments with the hydrolysates or buffer at 25 °C or 100 °C, seal meats were collected, freeze-dried, and digested via the dynamic rat digestion model, and the liquid digests after stomach and intestine digestions were collected, as described in For the amino acid analysis, only the intestinal digests were considered, as amino acids are mainly absorbed in this portion of the GIT . Figure The amino acid composition results of the four samples showed similar trends. Mineral analysis was performed for the (i) gastric digests and (ii) intestinal digests, but only the results of the intestinal digests are discussed in detail in this section, because the majority of minerals are absorbed in the small intestine in the human body . AccordiThe contents of various minerals in the gastric and intestinal digests are shown in 2+ and Fe3+ ions before and after digestion in the simulated gastric and intestinal tract were measured, and the results are presented in 2+ ions in green and Fe3+ ions in orange). In contrast to the results regarding the Fe mineral content in 2+, than their control counterparts treated with buffer. The antioxidant properties of the hydrolysates that were used to treat seal meat probably prevented or reduced the oxidation of Fe2+ to Fe3+, explaining the lower contents of Fe3+ in sample (a) compared to (c). 3+ was found, which was probably due to the denaturation of antioxidant hydrolysates at elevated temperatures or the high-temperature-induced oxidation of Fe2+ to Fe3+. Fe2+ is of special interest for people suffering from iron deficiency, as it can be readily absorbed by the human body in the gastrointestinal tract [The concentrations (mg/g) of Feal tract . Thus, sPagophilus groenlandicus) loins were obtained from SeaDNA Canada Inc., Québec, Canada. Antioxidant hydrolysates from seal meat were prepared using enzymatic hydrolysis in phosphate buffer (pH 7.5) following an established protocol [4), 1,10-phenanthroline, sodium phosphate, hydroxylammonium chloride, and hydrogen peroxide (H2O2) were purchased from Sigma-Aldrich Co. .Fresh adult harp seal meat . The treatment of seal meat in either hydrolysates or buffer solution was performed at 25 °C (representing low-temperature processing or room temperature storage conditions) and 100 °C (representing cooking conditions at high temperature) for 30 min under continuous stirring. Afterwards, the meat residues were separated from the mixture using a 0.22 µm vacuum filtration membrane. The meats treated with hydrolysates at 25 °C and 100 °C and buffer solution at 25 °C and 100 °C were labeled “Meat Hydrolysates 25”, “Meat Hydrolysates 100”, “Meat Buffer 25”, and “Meat Buffer 100”. The corresponding solutions collected after separation were labeled “Hydrolysates 25”, “Hydrolysates 100”, “Buffer 25”, and “Buffer 100”. The original “Buffer” and “Hydrolysates” solutions were utilized as controls. All samples were frozen at −40 °C and then freeze-dried to obtain a powder using a Labconco freeze-dryer before subsequent analysis. The workflow is summarized in Fresh seal meat was washed and blended into minced meat before being added to the hydrolysates or the buffer solution  , followed by the addition of H2O2. The absorbance was measured at 536 nm using a spectrophotometer . Both methods were performed following the protocol described in a previous study [The antioxidant activity of solutions collected after separation, i.e., “Hydrolysates 25”, “Hydrolysates 100”, “Buffer 25”, and “Buffer 100”, were measured via (i) DPPH free radical scavenging activity and (ii) hydroxyl radical scavenging activity, as shown in us study .The digestion of the freeze-dried forms of seal meats treated with hydrolysates at 25 °C and 100 °C and buffer solution at 25 °C and 100 °C, i.e., “Meat Hydrolysates 25”, “Meat Hydrolysates 100”, “Meat Buffer 25”, and “Meat Buffer 100” (as shown in The composition and contents of amino acids in gastric and intestinal digests of seal meat described in The composition and amounts of minerals in gastric and intestinal digests of seal meat described in 2+ and Fe3+ ions in gastric and intestinal digests of seal meat described in 2+ contents in the supernatants were measured using the 1,10-phenanthroline method. The total Fe ion content was measured using the same method after converting all Fe3+ to Fe2+ via hydroxylammonium chloride. The Fe3+ content was back-calculated.The contents of FeTriplicate measurements were performed for antioxidant activity and Fe ion determination.2+ ions due to the oxidation process during long-term storage and high-temperature processing was reduced due to the antioxidant properties of the hydrolysates used to treat the seal meat. Additionally, the effect of low and high temperature in combination with hydrolysate treatment showed that the digestibility and nutritional intake depended on the processing conditions. Generally, higher contents of all studied nutrients were achieved after the treatment of seal meat with hydrolysates, for which higher amounts of amino acids were found for treatment at 100 °C, whereas for mineral intake, treatment at 25 °C was shown to be the preferable processing temperature. In summary, the treatment of seal meat with seal meat hydrolysates can be considered a promising bioprocessing method to improve the biological availability and nutrient values of seal meat.Seal meat can be considered a good source of high-quality protein, since it contains important amino acids and minerals, especially Fe ions. In this work, seal meat was treated with seal meat hydrolysates as a preservative agent, and there was an improvement in the release and availability of beneficial amino acids and minerals. In addition, the loss of desirable Fe


We aimed to assess the level of knowledge, attitude and practice regarding breast cancer screening among Sudanese female workers at a secondary-level hospital.this is a cross-sectional study carried out at the largest governmental hospital of Ad-Damazin City  in 2018. It surveyed female healthcare providers “group A” compared to the non-medical female staff at the same hospital “group B” to assess their awareness, beliefs and behavior concerning Breast Cancer Screening (BCS). Chi-squared and Student t-tests were used for analysis with a significant p value of <0.05.participants were 110, included 78 (70.9%), (“group A”) and 32 (29.1%) (“group B”) women. Good overall knowledge score (47.4%) vs (43.8%), for “group A” and “group B”, respectively, p=0.000. Positive attitude was scored by 63 (80.8%) vs. 23 (71.9%) participants in “group A” and “group B” respectively, p= 0.305. Obvious denial trend regarding susceptibility to this disease was noted in both groups. BCS practices were seriously unsatisfactory in both groups. As “group A” vs “group B” regarding breast self-examination, n=13 (16.7%) vs n=10 (31.3%); clinical breast examination n=4 (5.1%) vs n=4 (12.5%) and mammography was not performed by any woman in both groups.the modest knowledge and poor BCS practices of our study groups strongly recommends appropriate official and educational actions. Breast Cancer (BC) has the highest incidence and is the first cause of death from cancer in women worldwide. Despite extensive Breast Cancer Screening (BCS) programs in the western countries, BC still causes significant morbidity. Currently, the estimated number of newly diagnosed BC cases worldwide is about 2.1 million ,4. AwareSudan as a large developing African country is struggling to provide essential preventive health services in low resource settings. The few available records showed that Sudan is experiencing a rapidly increasing BC prevalence and high incidence characterized by late and complicated stages at presentation, and a high frequency related to a several risk factors . AdditioDesign: a descriptive, hospital-based cross-sectional study.Study setting: study was conducted during the period from November 2016 to January 2018 at Ad-Damazin Teaching Hospital . This is the main governmental state hospital serving over 1100000 papulation in Ad-Damazin City, capital of the Blue Nile State in the South-Eastern Sudan. Hence, it can be defined as a secondary-care institution [Participants: the study population were adult female university graduates and postgraduates working at that hospital. The medical personnel comprised doctors, pharmacists, nurses, radiology technologists, laboratory scientist and technologists, dieticians. The non-medical participants were the accountants, statisticians and other office employees.Exclusion criteria: women who suffered from BC or any other cancer; women in a holiday and women been absent for any reason during data collection period were not included in this study.Variables: included in 97 items within four sections: Section 1: sociodemographic data included age; marital status; occupation; level of education; work experience; health insurance status and having close person suffered from breast cancer (9 items). Section 2: knowledge (21 items) divided into predisposing factors; clinical manifestations and BCS methods . Section 3:attitude assessment based upon the revised Champion's Health Belief Model Scale (CHBMS) six concepts and 63 items processes [rocesses ,12. The rocesses -13. SectData collection technique: through direct interview of the participants by the principle researcher.Data collection tool: was a predesigned, pretested and a self-administered questionnaire. Its validation was via a draft questionnaire designed after meticulous search in the literature. Its contents were face validated and reviewed with expert consultants. A pilot test was carried out in 20 participants  working at Sudanese Chinese Friendship Hospital at Ad-Damazin City. The pre-test data was analysed and the final questionnaire was produced after elimination and modification of some items.Questionnaire reliability: Alpha Cronbach´s test was performed to assess the internal consistency and reliability of the questionnaire items. A high value of the test coefficient (0.939) was obtained. Response rate 94.8 % . The average time to answer this questionnaire was 12-16 minutes.Questionnaire format, scoring and grading system:Knowledge: the questions were close-ended of the “Yes”, “No” or “I don´t know” format. For each correct answer a candidate was awarded one mark and a zero mark for wrong, “I don´t know”, blank or a more than one checked answers. A full mark was 21, graded as “Poor” (0-7 (≤ 33%)); “Moderate” (8-14 (34-65%)) and a “Good” (15-21 (≥ 66%)) level of knowledge.Attitude: assessment format was three-point Likert scale  was used to a cover narrow range of attitude. For every positive attitude “I agree”; the participant was awarded one mark, for negative attitude “I´m neutral” or “I don´t agree” answer the participant was awarded zero mark. A full attitude score was 63 points, graded as “Negative” = 0-31 (< 50%) and “Positive” ?32 (?50%) level of attitude.Practice: assessment format was “Done” and “Not done”. For every best practice screening methods the participant was awarded one mark for “Done” response, and for “Not done” response, zero mark was awarded. Highest practice score was three marks, where score of 0-1 graded as unsatisfactory and 2-3 as satisfactory level of practice. To identify the medical education effect, the participants were divided into medical personnel “group A”, and non-medical participants “group B” for comparison purposes.Potential bias management: selection bias: as a cross sectional study the potential bias due to convenience sampling was eliminated by the high response rate (94.8 %) of the participants. Information bias: The information bias was addressed by using a the valid and reliable Champion's Health Belief Model Scale.Confounding: to avoid confounding causal comparison was avoided in this study.Sampling: addressing selection bias a nonprobability (convenience) sampling of all the females working at Ad-Damazin Teaching Hospital and satisfying the inclusion criteria was approved.Statistical analysis: data completeness assessment, cleaning, entry, then analysis via Statistical Package for Social Sciences  was accomplished. Regarding knowledge, attitude and practice among the two groups´ percentage and mean scores of each piece of data calculated and analysed, excluding missing data. Categorical variables were noted in numbers and percentages. Continuous variables were expressed as mean ± standard deviation. Statistical methods used were Chi-squared tests for association, independent t-test to compare the both groups for each mean of independent variables, the p value of significance was < 0.05.Ethical issues: ethical approval for this study was obtained from the Dean Faculty of Medicine, Blue Nile University ethical committee and the General Secretary for Ministry of Health. The participants were informed and consented for the study. Participants` confidential information was guaranteed through unnamed questionnaire. This study assessed the level of BCS among Sudanese female workers at a secondary healthcare hospital.Socio-demographic: the study included 110  participants. The majority of the participants in both groups, were young (22-39 years); married; nurses and technicians. Most of them had less than five years´ experience and have health insurance. A minority of them knew some person who had cancer before. Details of their socio-demographic data are shown in  vs 3.19 ± (2.7), p=0.001; clinical manifestations were 5.88 ± (2.5) vs 4.62 ± (2.5), p=0.019; early detection methods were 1.91 ± (1.1) vs 1.34 ± (1.2), p=0.020. The highest overall knowledge grade was “moderate” (60.8% for “group A” and 43.6% for “group B”), p=0.000.Attitude: positive attitude score was detected in the majority of both groups, as demonstrated in (rated in .Practice: satisfactory BCS practices regarding BSE were reported in 13 (16.7%) participants in “group A” and in ten (31.3%) participants in “group B”, p=0.045. Satisfactory CBE was reported by four (5.1%) participants in “group A” and by four (12.5%) participants in “group B”, p=0.039 where mammography was not performed by any participant, more details shown in  revealed that “group A” was significantly better than “group B”. The highest score grade of overall knowledge in “group A” participants was moderate, while that of “group B” was poor. The poor knowledge outcome detected in our “group B” go on the same line with earlier surveys carried out regionally in Nigeria and Yemen [nd Yemen ,21. We bAttitude: assessment of attitude in both groups revealed their almost equal positive trends towards certain elements of attitude regarding breast cancer screening  and Magnetic Resonance Imaging (MRI) machines. The reason for this defect is unknown and reflects a defective public and medical authority strategy towards BCS in our country. A moderate (unclear and unsatisfactory) level of confidence to perform breast self-examination was also detected by our survey. The overall attitude in score  presentePractice: concerning early BCS, knowledge and attitude-directed practice had already been emphasized [et al. and Bener et al. [phasized ,24. On tphasized . Concernr et al. ,27. Thisr et al. . This faOur study finding that healthcare workers are not doing any better regarding BCS practice, regardless of the knowledge and attitude detected levels . Inadequet al.[et al.. An addeet al.,38. Our Our study limitations were that it was a single center; hospital-based (rather than community-based). Assessment of practice was so brief and in a form of close-ended questionnaire, rather than actual observation due to the practical sensitivity of the issue. Moreover, undergraduate and community-based ladies were outside the scope of this study. The results of our study open the door widely for further studies to detect reasons behind the negative attitude towards BC susceptibility; lack of mammography machines in most of our hospitals; the lower confidence level to perform breast self-examination by our participants and the inferiority of the medical group compared to non-medical group in performing breast self-examination.Concerning BCS, our study of medical and non-medical staff showed obvious denial features towards susceptibility to a very well-known threatening disease. Furthermore, the study highlighted the alarming poor practice accompanying moderate knowledge and positive behavior. These findings noticeably necessitate further generalization via national studies and in-depth investigation of the possible local socio-cultural and educational causes. Doctors and rest of health care providers should highly be focused as leaders of the health care services leading other woman along the road to successful BCS practice.Advanced cases of breast cancer are seen in Africa;Breast cancer screening helps in early detection of breast cancer.The level of knowledge, attitude and behavior regarding breast cancer screening among healthcare providers in a big city in the Sudan is seriously low;The knowledge, attitude and behavior level regarding breast cancer screening among those healthcare workers is not better than the ordinary  workers.