Little is known about whether sodium intake is associated with the clinical effects of SGLT2 inhibitors (SGLT2is); however, SGLT2is may increase urinary sodium excretion. Thus, we investigated the impact of daily sodium intake on the estimated glomerular filtration rate (eGFR) via an SGLT2i, tofogliflozin (TOFO), in patients with type 2 diabetes (T2D).Individual‐level data on 775 T2D patients in TOFO Phase 3 trials were analysed. Adjusted changes in variables during 52 weeks of TOFO therapy were compared according to basal daily salt intake (DSI), which was measured based on estimated daily urinary sodium excretion using the Tanaka formula. Multivariable analysis was used to investigate the impact of basal DSI on changes in eGFR at Weeks 4 and 52.MDRD and median DSI were 58.5 years, 8.0%, 25.6 kg/m2, 83.9 mL/min/1.73 m2 and 9.3 g/d, respectively. In all participants, eGFRMDRD sharply dipped during Week 4, and gradually increased by Week 52, showing a significant increase overall from baseline to Week 52. Multivariable analysis showed that basal DSI and HbA1c levels were independently correlated with eGFRMDRD changes at Weeks 4 and 52. Additionally, lower baseline HbA1c and DSI levels were significantly correlated with a greater increase in eGFRMDRD at Week 52.Sixty‐six percent of participants were men; mean age, HbA1c, body mass index, eGFRMDRD via TOFO. Thus, an appropriate dietary approach to therapy should be considered before treatment of T2D patients with an SGLT2i.Dietary salt intake, in addition to glycaemic control, correlates with changed eGFR In diabetes, increased expression and activity of SGLT22A pooled analysis was conducted on two Phase 3 studies (Table S1) of administration of TOFO to patients with T2D. Various doses of TOFO, either as monotherapy or as an adjuvant antidiabetic agent, were compared. The CSG004JP study  and the CSG005JP study  were both 52‐week, randomized, controlled, open‐label, Phase 3 studies.MDRD) was estimated using the Modification of the Diet in Renal Disease (MDRD) formula for the Japanese populationCKD‐EPI was estimated using the Chronic Kidney Disease Epidemiology Collaboration (CKD‐EPI) formula for the Japanese population.CRE + CYS from the Japanese equationThe following laboratory variables were measured at baseline: HbA1c, fasting plasma glucose (FPG), sodium, potassium, uric acid, brain natriuretic peptide (BNP), urine creatinine, urine sodium, urine potassium, urine albumin‐to‐creatinine ratio (ACR), creatinine and cystatin C. The eGFR of creatinine  (mmol/d) = 7.59 × (Kspot [Spot urinary potassium]/Crspot × PrUCr24h)0.431, PrUCr24h = 14.89 × weight (kg) + 16.14 × height (cm) − 2.04 × age (years) – 2244.45. Estimated daily salt intake (DSI) (g/d) = 24‐hour urinary sodium (mmol/d)/17.24‐hour urinary sodium (Na) (mmol/d) = 21.98 × (Naspot [Spot urinary sodium]/Crspot [Spot urinary creatinine] × PrUCr24h [predicted 24‐hour urinary creatinine])MDRD, BNP and ACR were analysed using an analysis of covariance (ANCOVA) model, with the baseline values age, sex and eGFRMDRD as covariates to determine changes across quartiles. In the evaluation of adjusted assessments of cystatin C, eGFRCKD‐EPI and eGFRCRE + CYS, changes were analysed using the ANCOVA model, with baseline values, age and sex as covariates. The adjusted assessment of creatinine was also analysed, using the ANCOVA model with baseline values, age and sex as covariates across quartiles of DSI.To assess the effects of basal DSI on changes in metabolic variables during the use of TOFO, participants were divided into four groups according to quartiles of basal DSI . Adjusted assessments of HbA1c, FPG, sodium, potassium, body weight, SBP, DBP, eGFRMDRD were assessed in participants receiving, and in those not receiving, renin‐angiotensin system (RAS) inhibition drugs (ARB and/or ACE inhibitor [ACEI]) as concomitant antihypertensive therapy and in participants with or without hyperfiltration. Participants were divided into two groups according to whether they received RAS inhibition drugs and into another two groups according to basal eGFRMDRD .Changes in eGFRMDRD at Weeks 4 and 52 and baseline variables were analysed by Pearson's product‐moment correlation coefficients. Correlations between changes in eGFRMDRD and blood pressure at Weeks 4 and 52 and baseline DSI were investigated in the entire participant population and in those using or not using RAS inhibition drugs as concomitant antihypertensive therapy. Correlations between changes in eGFRMDRD and blood pressure and body weight at Week 4 were also analysed in the entire participant population and in each quartile group according to DSI. Finally, correlations between changes in eGFRMDRD and changes in HbA1c from Week 4 to Week 52 were investigated.Correlations between change in eGFRMDRD and creatinine level at weeks 4 and 52, 14 variables at baseline  were initially identified based on clinical considerations, although eGFRMDRD and creatinine were adjusted for each other. Moreover, to identify clinical factors that might independently influence changes in eGFRMDRD from Week 4 to Week 52, 12 variables  were initially identified based on clinical considerations. Finally, to identify clinical factors that might independently influence eGFRMDRD changes from Week 52 to Week 54, 13 variables  were initially identified based on clinical considerations.To identify baseline clinical factors that might independently influence changes in eGFRt test. In this study, all HbA1c values are presented using the National Glycohemoglobin Standardization Program units. The (two‐sided) significance level for each test was 0.05.For each group, participants' demographics were summarized with appropriate descriptive statistics . Additionally, differences in between‐group assessments were analysed using ANOVA and chi‐square test. Differences from baseline, from Week 4 to Week 52, and from Week 52 to Week 54 were analysed using the one sample 3MDRD and median estimated DSI were 58.5 years, 8.1%, 25.6 kg/m2, 83.9 mL/min/1.73 m2, and 9.3 g/d, respectively , gradually increased from Week 4 to Week 52 (+6.1 mL/min/1.73 m2) . Two weeks after termination of TOFO therapy, eGFRMDRD further increased from Week 52. We also observed an inverse time course in serum creatinine levels compared to eGFRMDRD .This pooled analysis included 775 participants (66% men) who were receiving TOFO Table S1). Mean age, HbA1c, BMI, eGFR. Mean agMDRD, eGFRCKD‐EPI, eGFRCRE + CYS) and the proportion of participants with albuminuria (ACR = >30 mg/g Cre) were higher, based on increased basal DSI, while glycemic status and blood pressure were consistent among all quartiles. Changes in variables, glycaemic status, body weight and blood pressure, at both Weeks 4 and 52 were consistent across quartiles (Table S2). Moreover, differences in BNP level and urine ACR were insignificant across quartiles at Week 52. On the contrary, at Week 4, the reductions in eGFRMDRD and eGFRCKD‐EPI were higher, based on increased basal DSI. At Week 52, the increase in eGFRMDRD and eGFRCKD‐EPI tended to be smaller, according to the increased basal DSI, while the reduction in eGFRCRE + CYS and increase in cystatin C were greater, based on increased basal DSI.Participants were divided into four groups according to quartiles of estimated basal DSI Table . Age wasMDRD (Table S3). There were significant differences in the change in eGFRMDRD at Week 4 according to dosage of TOFO, use of concomitant antihypertensive drugs including ARB and RAS inhibition drugs, and ACR category (<30 mg/g Cre or ≥ 30) (Table S4). At Week 52, age, baseline HbA1c, fasting plasma glucose, cystatin C and urinary sodium excretion, and DSI were correlated with change in eGFRMDRD (Table S3). At Week 52 there were significant differences in the change in eGFRMDRD, according to the use of concomitant antihypertensive drugs, including the use of ARB (Table S4).At Week 4, baseline HbA1c, fasting plasma glucose, SBP, BNP, urinary sodium and potassium excretion, and DSI were significantly correlated with the change in eGFRMDRD at Week 4 was not correlated with change in DBP  in the entire participant group, while change in eGFRMDRD at Week 4 was significantly correlated with change in SBP  and with change in body weight . There was not a significant correlation between change in eGFRMDRD from Week 4 to Week 52 and change in HbA1c .Change in eGFRMDRD at Week 4 , suggesting that the lower levels of HbA1c soon after TOFO treatment might have contributed to that recovery. Further, change in eGFRMDRD from Week 4 to Week 52 was not correlated with change in HbA1c. These results suggested that, not only the correction of salt intake‐induced glomerular relative‐hyperfiltration, but also the well‐controlled hyperglycaemia before and soon after TOFO administration, might contribute to the recovery of and increase in eGFRMDRD after the initial dip. Prolonged hyperglycaemia directly induces mesangial expansion and injury,MDRD from the initial decrease might be attributed to, not only attenuation of glomerular relative‐hyperfiltration, but also the baseline renal status induced by a sustained well‐controlled glycaemic status.We also observed a gradual increase in eGFRMDRD during TOFO treatment, according to whether participants used RAS inhibition drugs . There were significant differences in the change in eGFRMDRD at both Week 4 and Week 52, according to use of RAS inhibition drugs. Results of multivariable ananysis indicated that the use of RAS inhibition drugs as concomitant antihypertensive medication might be negatively correlated with change in eGFRMDRD at Weeks 4 and 52. Therefore, the use of RAS inhibition drugs as concomitant antihypertensive medication might contribute to the effects of TOFO treatment on eGFRMDRD levels, independent of basal DSI. The preferential effect of SGLT2 inhibition on afferent renal arteriolar resistance was reported to be important in reducing intraglomerular pressure,MDRD, followed by decreased intraglomerular pressure. On the contrary, our findings also supported the association of basal DSI with change in eGFRMDRD at Weeks 4 and 52, independent of the use of RAS inhibition drugs. However, the monitored period of TOFO treatment was limited and histological confirmation was not performed to evaluate structural changes in the kidney. Therefore, further investigation will be needed to evaluate the effects of RAS inhibition drugs on changes in eGFR as the result of long‐term SGLT2i treatment.We observed changes in eGFRMDRD. SGLT2 is co‐expressed with NHE3, which reabsorbs approximately 30% of filtered sodium. Recent studies provide evidence that SGLT2 may be functionally linked to NHE3, such that SGLT2 inhibition may also inhibit NHE3 in the proximal tubule.MDRD during long‐term TOFO treatment. The interaction between SGLT2 and NHE3, and change in the reabsorption of sodium leading to increased delivery of sodium to the macula densa after SGLT2 inhibition, could be relevant in explaining the effect of SGLT2i on eGFR. Further investigations are required to clarify those interactions and their effect on eGFR as the result of SGLT2i treatment.From our results, we could not explain the association of the Na+/H + ‐exchanger 3(NHE3), which contributed to reabsorption of urinary sodium, with the effects of long‐term TOFO treatment on eGFRMDRD levels had further increased from Week 52, which was consistent with previous reports.MDRD and urine ACR from Week 52 to Week 54 was observed in participants with albuminuria . This result suggested that attenuation of glomerular relative‐hyperfiltration might contribute, in part, to the reduction in urine ACR. The report by Cherney et al. also suggested that long‐term administration of empagliflozin might contribute to functional improvement of the kidney.MDRD during the termination period was not significantly correlated with the initial decrease, which might be caused by the attenuation of glomerular relative‐hyperfiltration . Moreover, DSI was not significantly associated with increased eGFRMDRD during the termination period, based on results of multivariable analysis (Table S7). These findings suggested that increased eGFRMDRD after the termination of TOFO treatment might be caused, not only by the released attenuation of glomerular relative‐hyperfiltration, but possibly by other factors as the result of long‐term use of the medication, such as functional changes in the kidney.Two weeks after termination of treatment with TOFO, eGFRMDRD value at baseline of 83.9 mL/min/1.73m2, 30 of these participants had eGFRMDRD levels ≥120 mL/min/1.73m2 (hyperfiltration). The proportion of participants with hyperfiltration tended to increase in association with increased basal DSI. We further investigated the effects on eGFRMDRD, according to whether participants had hyperfiltration during the study period . Reduction in eGFRMDRD at Week 4 was greater in participants with hyperfiltration than in those without hyperfiltration, while changes in eGFRMDRD from Week 4 to Week 52, from baseline to Week 52, and from Week 52 to Week 54 were identical between participants with and without hyperfiltration. Changes in eGFRMDRD during TOFO treatment were similar between participants with and without hyperfiltration, with the exception of the initial dip in eGFRMDRD. However, further study involving a large number of participants with hyperfiltration is needed to clarify the effects of basal DSI on eGFR as the result of SGLT2i treatment.Although study participants had a mean eGFRMDRD as the result of TOFO treatment. Interestingly, independent of glycaemic status, lower salt intake at baseline was also associated with subsequent increase in eGFRMDRD.. Our findings suggested that basal DSI, in addition to correlation with glycaemic control, correlates with eGFRMDRD as the result of long‐term TOFO treatment in participants with T2D, independent of the use of RAS inhibition drugs as concomitant antihypertensive treatment. Thus, an appropriate dietary approach to therapy should be considered before initiation of treatment with SGLT2is. On the contrary, glomerular relative‐hyperfiltration induced by higher DSI might be attenuated by TOFO treatment, leading to a further reduction in eGFRMDRD levels. More studies are required to clarify whether these differences in results regarding the changes in eGFRMDRD that are influenced by basal DSI may contribute to clinically meaningful reductions in the development and progression of diabetic kidney diseases in patients with T2D. Unfortunately, we could not monitor changes in urinary sodium excretion in order to estimate changes in salt intake during TOFO treatment. Sustained lower‐controlled salt intake may contribute to the further increase in eGFR during SGLT2i treatment, but additional investigations will be needed. SGLT2i inhibits the reabsorption of, not only urine glucose, but also sodium, which may have multifactorial effects on the risk factors for cardiovascular disease and deterioration of kidney function. However, dietary approaches may be needed, particularly in monitoring salt intake, to maximize the renal effects of SGLT2i.Current approaches to preventing kidney complications in patients with diabetes have focused on lowering blood pressure, HbA1c, body weight, albuminuria and cholesterol, which have been shown to reduce the risk of cardiovascular disease and deterioration of kidney function.We recognize that the estimation of DSI in our study is a serious limitation. Although we performed the analyses according to quartiles of basal DSI, SBP and DBP levels at baseline did not differ among the quartiles of DSI, nor did the proportion of concomitant antihypertensive drugs at baseline. Of note, the use of diuretics coincided with increases in basal DSI, although the use of diuretics might be clinically effective in controlling blood pressure in salt‐sensitive hypertensive participants. Our study was based on pooled analyses from two TOFO studies that had been designed to evaluate the safety and efficacy of TOFO as monotherapy or as an add‐on to other oral antidiabetic agents in T2D patients. Thus, the prescribed antihypertensive drugs might not have been selected according to basal DSI. Among study participants, 47.2% received antihypertensive drugs and 8.3% were received diuretics. We found that the proportion of participants with ACR levels ≥30 mg/g Cre was significantly increased, based on increased basal DSI. In Japanese T2D patients with microalbuminuria, the prevalence of hypertension that was salt sensitive was greater than that in patients without microalbuminuria,MDRD level in almost all participants was less than 120 mL/min/1.73m2. We could not measure the actual GFR and variables related to intrarenal haemodynamic function. Histological confirmation was not performed to evaluate structural changes in the kidney. This study was the first to elucidate baseline predictors of changes in eGFRMDRD at Weeks 4 and 52 from the results of multivariable analyses. However, the contribution of these predictors to change in eGFRMDRD according to the multivariable analyses might be small; more studies to determine greater contributions to eGFR as the result of SGLT2i treatment are required. Also, our results were obtained in Japanese participants with T2D, whose DSI might be higher than that in other populations.in vitro molecular actions and pharmacological factors are required to confirm conclusions based on the results of this study.This study has several other limitations. We could not perform a comparison between a placebo and TOFO, and the baseline eGFRMDRD after long‐term treatment with an SGLT2i, TOFO, in patients with T2D. Moreover, both low basal HbA1c and DSI levels were associated with a greater increase in eGFRMDRD at Week 52. The effects of the SGLT2i, TOFO, on eGFR might be associated with the handling of, not only glucose, but also sodium, through SGLT2 in the proximal tubule.In conclusion, basal DSI was independently correlated with changes in eGFRK. K. has been an advisor to and has received honoraria for lectures from Astelas, Novo Nordisk Pharma, Sanwa Kagaku Kenkyusho, Takeda, Taisho Pharmaceutical, MSD, Kowa, Kissei, Sumitomo Dainippon Pharma, Novartis, Mitsubishi Tanabe Pharma, Nippon Boehringer Ingelheim, Daiichi Sankyo, and Sanofi. A. Y. is an employee of Kowa Pharmaceutical. H. S. is an employee of Kowa. Y. S. had no conflicts of interest.All authors participated in writing the manuscript. K. N. and Y. S. wrote the draft of the manuscript and contributed to the discussion. K. K. contributed to the discussion and reviewed/edited the manuscript. A. Y. researched data and reviewed the manuscript. H. S. had full access to all data in the study and takes responsibility for the integrity of the data and the accuracy of the data analyses. K. K. is a director of data collection. H. S. and K. K. are the guarantors of this work.Figure S1. eGFRMDRD changes from week 4 to week 52 and from week 52 to week 54.Figure S2. Time course of creatinine levels.Figure S3. Time course of eGFRMDRD levels according to the participants with or without RAS inhibition drugs (ARB and or ACEI) as concomitant antihypertensive drugs.Figure S4. Correlation between eGFRMDRD and urine ACR changes from week 52 to week 54.Figure S5. Correlation between eGFRMDRD changes from baseline to week 4 and from week 52 to week 54.Figure S6. Time course of eGFRMDRD levels according to participants whose basal eGFRMDRD levels were < 120 mL/min/1.73m2 and = > 120.Click here for additional data file.Table S1. Integrated analysis of two clinical studies.Table S2. Change in variablesat week 4 and week 52 according to quartiles of daily salt intake.Table S3. Baseline predictors for the change in serum creatinine levels at week 4 and week 52.Table S4. Predictors for change in eGFRMDRDfrom week 4 to week 52.Table S5. Predictors ofchange in eGFRMDRDfrom week 52 to week54.Table S6. Correlations between the change in eGFRMDRDat week 4 and week 52 and baseline continuous variables.Table S7. Change in eGFRMDRDat week 4 and week 52 according to baseline categorical variables.Click here for additional data file.


Mycobacterium bovis Bacille Calmette-Guérin (BCG) is not only used as a vaccine against tuberculosis but also protects against leprosy and is used as part of bladder cancer treatment to induce a protective immune response. However, protection by BCG vaccination is not optimal. To improve vaccine efficacy, recombinant BCG expressing heterologous antigens has been put forward to elicit antigen-specific cellular and humoral responses. Cell surface localized or secreted antigens induce better immune responses than their cytosolic counterparts. Optimizing secretion of heterologous proteins or protein fragments holds therefore unexplored potential for improving the efficacy of recombinant BCG vaccine candidates. Secretion of heterologous antigens requires crossing the mycobacterial inner and outer membrane. Mycobacteria have specialized ESX or type VII secretion systems that enable translocation of proteins across both membranes. Probing this secretion system could therefore be a valid approach to surface localize heterologous antigens.M. marinum, albeit with low efficiency. Subsequent random mutagenesis of LipY-OVA and screening for increased secretion resulted in mutants with improved heterologous secretion. Detailed analysis identified two mutations in OVA that improved secretion, i.e. an L280P mutation and a protein-extending frameshift mutation. Finally, deletion of the linker domain of LipY enhanced secretion of LipY-OVA, although this mutation also reduced surface association. Further analysis in wild type LipY showed that the linker domain is required for surface association.We show that ESX-5 substrate LipY, a lipase, can be used as a carrier for heterologous secretion of an ovalbumin fragment (OVA). LipY contains a PE domain and a lipase domain, separated by a linker region. This linker domain is processed upon secretion. Fusion of the PE and linker domains of LipY to OVA enabled ESX-5-dependent secretion of the fusion construct LipY-OVA in We show that the ESX-5 system can be used for heterologous secretion. Furthermore, minor mutations in the substrate can enhance secretion. Especially the C-terminal region seems to be important for this. The linker domain of LipY is involved in surface association. These findings show that non-biased screening approaches aid in optimization of heterologous secretion, which can contribute to heterologous vaccine development.The online version of this article (10.1186/s12934-019-1093-1) contains supplementary material, which is available to authorized users. Mycobacterium bovis Bacille Calmette-Guérin (BCG) is well known for its use as a vaccine against the infectious disease tuberculosis, caused by Mycobacterium tuberculosis. One of the major characteristics of BCG vaccination is the induction of the T-helper cell 1 (Th1)-polarized CD4+ T cell responses that confer cellular immune responses against bacterial infection . Fluorescence was measured using a plate reader , bottom mode. The lipase inhibitor Paraoxon  was used as a control for inhibition of LipY lipase activity.To measure lipase activity, bacteria were grown to an OD2 [To predict the structure of the lipase domain and analyze homology with other proteins aa 158–443 of LipY were analyzed by Phyre2 .wt , containing amino acids 259–357 from ovalbumin, including the major known epitopes, i.e. Major histocompatibility complex (MHC) class I epitope SIINFEKL and MHC class II epitope ISQAVHAAHAEINEAGR , 42. Prewt Fig. a.Fig. 1Ewt, surface proteins were extracted with the mild detergent Genapol X-080. The fusion protein was readily expressed in M. marinum, but only a modest amount was found in the cell surface-enriched and nothing was detected in the supernatant fraction  showed that this modest secretion of LipY-OVAwt was ESX-5 dependent, resembling the secretion of LipYtub. Thus, LipY can serve as a carrier for heterologous secretion of OVA in an ESX-5 dependent manner, although the efficiency is rather low.To study surface localization of LipY-OVAion Fig. b. Analys+ T-cell responses [wt, we introduced random point mutations in the gene coding for LipY-OVAwt by amplification of the DNA fragment with error-prone DNA polymerase. Subsequently, a library of these error-prone constructs were introduced in M. marinum and colonies were screened for increased secretion of LipY-OVA by double filter screening.Secretion, or surface localization, of mycobacterial antigens is required for the induction of specific CD4esponses , 43, 44 wt was not secreted on double filter, mutants 1 (LipY-OVA1), 2 (LipY-OVA2) and 3 (LipY-OVA3) showed secretion. Especially mutant 2 (LipY-OVA2) showed a strong increase in secretion, e.g. twofold higher than LipY-OVA1. Strikingly, both LipY-OVA1 and LipY-OVA3 had independent frame shift mutations in the same codon, resulting in a nineteen amino acid extension after the HA-tag (Table 1 the extension was caused by a 2 basepair (bp) frameshift deletion in the last two codons before the stop codon. In LipY-OVA3 a 1 bp insertion in the penultimate codon caused a frameshift resulting in a nearly identical extension of the HA-tag. The most striking alteration was located in LipY-OVA2, instead of a single basepair change we observed a deletion that removed aa 98–201 of the gene, which spans almost the complete LipY linker domain, leaving only the PE domain in front of OVA.We identified three LipY-OVA clones with increased secretion Fig. a. SequenWT) or the original OVA-encoding domain (OVAWT). Whereas LipYwt-OVA1 and LipYwt-OVA3-showed high secretion levels, similar to LipY-OVA1 and LipY-OVA3, secretion of LipY1-OVAwt and LipY3-OVAwt was not detectable . Double filter analysis showed however that LipY-OVA1(PPP/AAA) was secreted to a similar extent as LipY-OVA1, indicating that enhanced secretion is not caused by the proline triplet  showed secretion and subsequent surface localization of the processed form, resembling the phenotype found for LipY-OVA3 . In contrast, LipY-OVAwt was mainly present in its full length form and was only partially processed   in LipY-OVAace Fig. . To furtM. marinum, the amount of LipY-OVA3 was higher than LipY-OVAwt (twofold), whereas the amount in the pellet fraction was less for this mutant (fivefold). The other two chimeras, LipY-OVA1 and LipY-OVA2 were not increased in the surface-enriched fraction as compared to LipY-OVAwt. As published before [tub was not secreted by BCG in vitro, as was shown by the absence of the processed band (35 kDa) in all fractions.Since application of heterologous secretion for vaccine strategies would ultimately require expression in BCG, the selected LipY-OVA mutant constructs were also introduced in BCG. All these LipY-OVA constructs were expressed at comparable levels  and LipY-OVA3 (4.1 fold increase) have a higher surface labeling as compared to LipY-OVA1 and LipY-OVA2 . This resembles the subcellular fractionation data, in which LipYtub and LipY-OVA3 were present in significant amounts in the genapol supernatant fraction. Unexpectedly, no clear difference was seen between the surface labeling of LipY-OVAwt (5.0 fold increase) and LipY-OVA3, despite the clearly enhanced presence of LipY-OVA3 in the cell surface-enriched fraction. This might be explained by the observation that LipY-OVA3 is mainly present in its processed form. Due to the orientation and/or length of the processed protein, the HA-tag could be less well accessible on the cell surface for antibody labeling.Surface localization does not necessarily equal surface accessibility, since proteins that are detected in denatured conditions in cell surface fractions might be shielded by other capsular components and are therefore not exposed to the exterior of the cell. Therefore, we analyzed surface accessibility of LipY-OVAtub Fig. . The inc2 showed a deletion of aa 98–201, which is almost completely coinciding with the predicted linker domain (aa 100–205). This mutant showed a higher level of secretion, as was shown by both double filter assay  was mimicked in LipYtub (LipYΔ98–201). Expression and secretion of all LipY deletion constructs were analyzed in M. marinum. Deletion of the N-terminal part of the linker domain (LipYΔ100–145) did not result in a stable product and was not observed on double filter nor upon cell fractionation. This finding resembles previous data, which showed that the PE-domain with only the first 25 aa of the linker domain alone is not stable [Δ158–180, LipYΔ158–205 and LipYΔ181–205 also showed smaller bands that correspond to their relative molecular weight after processing after amino acid 149. Surprisingly, processing was also observed for LipYΔ98–201, although this construct lacks the protease cleavage site at amino acid 149 in the linker. This suggests that the PE-domain is still removed, since the length of the smaller band corresponded to the predicted molecular weight of the lipase domain only, which is in line with previous observations [LipY-OVAsay Fig. . These rnum Fig. a. To rett stable . All othrvations .Fig. 6ThΔ158–180 and LipYΔ181–205 were mainly found in the genapol X-080 supernatant, indicating that they were processed and retained at the cell surface. In comparison, LipYΔ158–205 was present in much lower amounts in this fraction. Apparently, partial deletion of the C-terminal part of the linker domain (aa 158–180 or aa 181–205) did not affect cell surface localization, while deletion of the whole C-terminal part (aa 158–205) reduced surface localization.The processed bands of LipYM. marinum the C-terminal part of the linker domain is involved in retention of LipY to the surface. Deletion of the full linker domain reduced surface localization of both LipYΔ98–201 and LipY-OVA2. However, in contrast to LipY-OVA2, culture supernatant levels of LipYΔ98–201 did not increase.Together these results indicate that in lipY gene results in significantly increased lipase activity, which can be reduced again by addition of the lipase inhibitor POX. The lack of activity of LipYΔ100–145 is likely due to the instability of the construct. Surprisingly, also most other deletion mutants did not show any lipase activity exceeding the background lipase activity of M. marinum WT even though production levels of these variants were in fairly similar amounts. Only LipYΔ158–180 still showed lipase activity. This was most clear for LipYΔ181–205, which seems in amounts comparable to intact LipY, but which showed no lipase activity above background levels. This means that, even though partial deletions of the linker domain do not affect surface localization of LipY, lipase activity is severely hampered, especially when the C-terminal part of the linker domain is removed.To examine whether the linker domain is also important in LipY activity, we assessed the lipase activity of the different linker deletion mutants. Lipase activity was measured using a whole cells assay. Lipase activity was almost reduced to zero in a secretion deficient ESX-5 negative background Fig. c. IntrodMycobacterium tuberculosis antigens [The BCG strain is one of the most widely used vaccines worldwide. Because of the immunogenic properties of BCG that induce clear cellular immunity against many intracellular pathogens, several attempts have been made to enhance vaccine efficacy by recombinant expression of mycobacterial and non-mycobacterial antigens . Since santigens , we aimeM. marinum, although secretion efficiencies were reduced as compared to the wild-type protein. By introducing random mutations in LipY-OVA we were able to identify mutants that showed enhanced secretion and surface localization, indicating that this is a viable approach that could be used for other heterologous constructs. These experiments showed two different mutations in the cargo that improved secretion. The first group of mutations had their stop codon removed by small insertions or deletions. Remarkably, both these alterations gave rise to the same frameshift resulting in an extension containing a proline triplet. Proline triplets can in theory be responsible for enhanced secretion, by securing better folding of the protein due to ribosome stalling [By using LipY as a carrier we provided a proof of principle that the ESX-5 system could indeed be used for heterologous secretion in stalling . HoweverL280P induced processing of LipY-OVA in a way similar to the processing seen for LipY. Particularly, this processed form showed increased surface localization. Most likely, this increased surface localization and subsequent processing are the result of enhanced secretion through the ESX-5 system. Prolines are known to induce kinks in peptides [The second mutation improving secretion was L280P in the OVA-domain. In addition to increased secretion, we found that LipY-OVApeptides , which cpeptides . On the 3 was detected in high amounts in the genapol-extracted fraction in denatured form, but not on the surface of intact cells. Nevertheless, LipY-OVA was clearly present on the cell surface, although cell surface accessibility did not significantly differ between mutants.Surface localization of LipY-OVA was further analyzed by immunolabeling of whole cells. Whereas surface localization was well visible after secretion analysis on western blot, the differences on whole cells, analyzed with flow cytometry, were relatively small. This might be due to reduced accessibility of the HA-tag in a folded state on the cell surface and putative shielding by the mycobacterial capsule. This could explain why LipY-OVAM. tuberculosis [The absence of LipY secretion by BCG in culture most likely resembles previous observations in rculosis . This baM. marinum. It is likely that surface localization is linked to processing of the linker domain, since LipY-OVA was retained at the cell surface only after processing of the linker domain, as seen in LipY-OVAL280P. These data correspond well with our previously published model of LipYtub secretion, in which the protein is cleaved after Gly-149, after which the C-terminal part stays associated with the cell surface [tub showed that when the major part of the linker domain (aa 98–201) or its C-terminal part (aa 158–205) was deleted, surface localization was severely reduced.The deletion of the linker domain (aa 98–201) enhanced secretion of LipY-OVA. This compelled us to study the functional role of the LipY linker domain in Δ158–180 and LipYΔ181–205 were only slightly affected, especially LipYΔ181–205 showed strongly reduced lipase activity in whole cells. Previously, it has been shown that the PE domain negatively regulates lipase activity [2 [Although surface localization and processing of LipYactivity , 49, 50.ivity [2 ) indicativity [2 .M. marinum and BCG. This paves the way to improve recombinant expression approaches that already have been taken to improve vaccine efficacy. More importantly, we show a viable and affordable method for optimization of heterologous secretion in mycobacteria by combining error-prone PCR with double filter screening methods. In principle, this method can be adopted to any carrier-cargo sequence to find individual mutations required for enhanced secretion, which in turn may lead to improved performance of recombinant mycobacterial vaccine candidates. If one could combine this screen with Flow Cytometry Cell Sorting many more mutants could be analyzed, although one would miss mutants with improved secretion without increased surface exposure, such as LipY-OVA2.While further research is needed to optimize LipY-mediated heterologous secretion in BCG, this study shows that ESX-5 substrate LipY can act as a carrier for secretion of heterologous proteins both in M. marinum. Subsequent random mutagenesis of the fusion protein LipY-OVA showed that minor changes in the heterologous substrate can enhance secretion. In addition deletion of the linker domain of LipY-OVA enhanced secretion, but reduced surface association. In agreement with the latter finding, the linker domain was also shown to be important for surface localization of intact LipY. Together, this work is the first to methodologically analyze and optimize heterologous secretion in mycobacteria. This could give new momentum to recombinant BCG development, with implications for recombinant vaccines and BCG bladder cancer treatment.Here we have shown that the mycobacterial ESX-5 system can serve as a route for heterologous secretion. By fusing the PE domain and linker domain of ESX-5 substrate LipY to a fragment of OVA, OVA was secreted in an ESX-5 dependent manner in Additional file 1: Table S1. Primers used in this study.Additional file 2: Figure S1. Expression of LipY-OVA swap constructs in M. marinum. LipY-OVA swap mutants showed similar expression levels, as was analyzed by collecting pellet fractions and staining with anti-HA and anti-GroEL2 (cytosolic control).Additional file 3: Figure S2. Double filter assay of LipY-OVAPPP/AAA Double filter assay showing LipY-OVA in which the proline triplet in the extension of LipY-OVA1 was replaced by an alanine triplet. HA-tagged proteins were detected by immunoblotting with anti HA.Additional file 4: Figure S3. Secretion analysis of LipY-OVA in M. bovis BCG. LipYtub and LipY-OVA constructs were expressed in BCG. Whole cell lysate (WCL), genapol pellet (GP), genapol supernatant (GS) and culture filtrate (CF) were collected and analyzed on western blot. Proteins were visualized by anti-HA-tag and anti-GroEL2 (cytosolic control). Proteins in genapol supernatant were only visible after long exposure . Total amount of HA-labeled protein was calculated for each blot separately.


Domains are distinct units within proteins that typically can fold independently into recognizable three-dimensional structures to facilitate their functions. The structural and functional independence of protein domains is reflected by their apparent modularity in the context of multi-domain proteins. In this work, we examined the coupling of evolution of domain sequences co-occurring within multi-domain proteins to see if it proceeds independently, or in a coordinated manner. We used continuous information theory measures to assess the extent of correlated mutations among domains in multi-domain proteins from organisms across the tree of life. In all multi-domain architectures we examined, domains co-occurring within protein sequences had to some degree undergone concerted evolution. This finding challenges the notion of complete modularity and independence of protein domains, providing new perspective on the evolution of protein sequence and function. Domains are basic functional and structural elements of proteins. In addition to sequence mutations, protein evolution is driven by combining existing domains into novel arrangements. The modular nature of domains arises from their ability to adopt well-defined three-dimensional 3D) structures  that oftD structuStructural changes driven by mutations in the primary sequence are one mechanism underlying the acquisition of novel domain functions. , 7, 8 StIn this work, we present an information-theoretic analysis of coevolutionary signals among protein domains in multi-domain arrangements. Based on the functional implications these signals carry, we test the notion of evolutionary and functional independence of domains and examine their adaptability to their primary sequence context. In contrast to the aforementioned studies, we examine coevolution as a global property of a domain pair, and introduce an appropriate continuous measure to quantify its effect. Using this measure, we show that coevolution among protein domains is a much more widespread phenomenon than previously anticipated.We defined protein domains as sequence families recognized in release 31.0 of the Pfam database.  Pfam 31.Domain architecture was established for each URPs sequence. We defined the domain architecture of a protein as a vector of Pfam 31.0 sequence families identified within the protein sequence ordered according to their proximity to the N-terminus. A total of 278,458 distinct domain architectures were recognized. In order to reduce small-sample effects , 26, onlFor each HPA, we compiled a list of all URPs sequences in which it was realized. Sequences of domains found in these proteins aligned to the respective Pfam 31.0 profile HMMs were retrieved from the Pfam family MSAs. Proteins that contained only standard amino acid, insert, and delete state symbols in the alignments of sequences of each of their domains to the respective Pfam 31.0 profile HMMs were identified for all HPAs. The lists of UniProt identifiers of these proteins for individual architectures are available in i contained two domains A and B with respective sequences iA and iB, the concatenated sequence iA‖iB was created. By generating this string for each protein, we created a multi-domain MSA for each HPA.Residue symbols found at positions corresponding to match or delete states (consensus columns) in the alignments of the respective domain sequences to the profile HMMs were extracted from each domain sequence within these proteins. This action corresponds to localizing all but the insert state positions in the respective MSAs, as residues assigned to insert states are, by definition, unaligned, and therefore irrelevant to this study.  SequenceX) of a random variable X taking on values from a finite alphabet f(xi) is the relative frequency of observing xi and 0 log2 0 is defined as zero. The mutual information MI of a pair of random variables X, Y can be estimated asf is the joint frequency of observing xi and yj simultaneously. Since 2 is chosen as the base of the logarithms in Eqs K and L are equal to 21, as all sequences in the MSAs contain only symbols for the standard amino acids and the delete state.Individual columns in a MSA can be viewed as random variables, with residue symbols found in the columns acting as the values of their respective observations. The Shannon entropy H showing the values of mutual information between each pair of columns within a MSA was calculated for multi-domain MSAs corresponding to the 2,063 selected multi-domain HPAs. The general structure of a MIM for a two-domain architecture is shown in D for each domain within each HPA asn is the number of positions i,DX corresponding to domain D. This corresponds to calculating the average values of the dark blue or dark red elements for the MIM shown in We calculated the average entropy N domains. For all such pairs of domains D, E within each architecture , the average value of mutual information m, n are the numbers of positions i,DX, j,EY corresponding to domains D and E, respectively. This corresponds to calculating the average value of matrix elements in pink rectangles in the general MIM shown in There are a total of i.e.,We calculate the normalized average inter-domain mutual information D and E, independent of the internal sequence variability of each domain. There were a total of 5,205 domain pairs for which the value of The value of rtainty.  It repreAB was found in N protein sequences and the corresponding multi-domain MSA contained sequences A1‖B1, A2‖B2, …, NA‖NB, then two sets of sequences, {A1, A2, …, NA} and {B1, B2, …, NB}, were obtained after the original sequences had been split. After this splitting, sequences of individual domains within these sets were then randomly shuffled and rejoined so that the original architecture was reestablished. For example, if protein i with domain architecture AB originally contained a concatenated domain sequence iA‖iB, the shuffling process resulted in the sequence iA‖jB, with j being a different URPs sequence with an AB architecture. This way, any native evolutionary coupling among protein domains was disrupted.To compare the extent of evolutionary crosstalk among protein domains in the selected multi-domain architectures with the crosstalk in sequences that share no evolutionary history, we have designed the following test. First, we split strings consisting of concatenated domain sequences at the domain boundaries. If domain architecture For each HPA, we calculated a second MIM from the perturbed MSA of disrupted sequences in which fragments corresponding to individual domains almost certainly originated from different proteins. For each pair of domains in each architecture, the value of To further examine the influence of background information noise arising among uncorrelated sequences, we studied the effects of random sequence composition fluctuations on the resulting values of There were a total of 5,205 domain pairs for which the values of i.e., the p-value of 0.0. The difference between the distributions shown in p-value effectively zero.It is clear from ank test  on the d0 bits.It should be noted that both the distribution of the values of equences  and the ferences  have meaU test [p-values are effectively zero). This result shows that, even though each individual position in the artificial random sequences has nearly maximum possible entropy, and thus has a large potential to generate considerable values of mutual information with positions in genuine protein sequences due to statistical noise, the observed values of equences  and appeequences . However .It is worth noting that the values of While most studies of coevolution to date . In addition, a pair of domains could, in principle, be coevolving with some other conserved non-domain region, such as a linker, and these effects could not be filtered out even if an appropriate method existed. It is worth noting that most  architectures studied in this work consist of only two protein domains, which rules out the possibility of domain-related transitivity effects.We explain the correlation between the values of al noise . As the relation .In addition to the random noise factor described above, there is another contribution to the correlation observed in The value of  greater . This revia inter-domain interactions. These interactions may enable direct or allosteric modulation of the function of the complete protein or its individual domains. If a vital function of a multi-domain protein depends on the cooperative action of its domains, evolution may opt to distribute mutations needed to preserve this function across the domains in a coordinated fashion. Residues preferentially mutated in this way may constitute nodes of energetic connectivity in the protein structure analogous to those observed at the single domain level. [Coordinated evolution can be intuitively understood by the need to preserve essential protein function in cases in which multiple domains form a ligand-binding or catalytic site . 12, 2121 We pron level.  It shoulIn either case, additional domains act as buffers or reservoirs of evolutionary capacity that can be utilized to either mitigate the impact of mutations required to maintain proper protein function or, alternatively, to optimize the respective functions of individual domains. The precise mechanism through which this functional modulation is realized and its full impact on protein evolution remain to be established.We showed that, in the context of multi-domain proteins, evolution of domain sequences proceeds in a coordinated fashion. We proved this by comparing a mutual information-based measure between native multi-domain sequences and artificial sequence constructs which share no common evolutionary history and further showed that the observed evolutionary coupling is distinct from statistical noise.S1 FileThis archive contains a file presenting the list of UniProt identifiers of the URPs sequences included in the multi-domain MSAs for each of the 2,063 studied multi-domain architectures, and files containing the values of (GZ)Click here for additional data file.S1 FigN = 2, 063.Total number of architectures (MSAs) (TIF)Click here for additional data file.


Vip3 proteins are increasingly used in insect control in transgenic crops. To shed light on the structure of these proteins, we used the approach of the trypsin fragmentation of mutants altering the conformation of the Vip3Af protein. From an alanine scanning of Vip3Af, we selected mutants with an altered proteolytic pattern. Based on protease digestion patterns, their effect on oligomer formation, and theoretical cleavage sites, we generated a map of the Vip3Af protein with five domains which match some of the domains proposed independently by two in silico models. Domain I ranges amino acids (aa) 12–198, domain II aa199–313, domain III aa314–526, domain IV aa527–668, and domain V aa669–788. The effect of some mutations on the ability to form a tetrameric molecule revealed that domains I–II are required for tetramerization, while domain V is not. The involvement of domain IV in the tetramer formation is not clear. Some mutations distributed from near the end of domain I up to the end of domain II affect the stability of the first three domains of the protein and destroy the tetrameric form upon trypsin treatment. Because of the high sequence similarity among Vip3 proteins, we propose that our domain map can be extended to the Vip3 family of proteins. Bacillus thuringiensis and are of practical interest because of their insecticidal activity against Lepidoptera [B. thuringiensis Cry proteins, they are considered an excellent complement to Cry proteins in crop protection and resistance management. Some commercial Bt-crops  combine Cry and Vip3 proteins, and this strategy of pyramiding proteins with different modes of action is expected to continue in the future [Vip3A proteins are produced during the vegetative phase of growth of idoptera . Becausee future .Despite the increasing interest in Vip3 proteins, their mode of action is not completely understood, and their 3D structure still remains unknown. Recently, a number of studies have provided valuable information toward the structure of these proteins. Multiple-sequence alignments of Vip3 proteins have shown that they contain between 786 and 803 amino acids (corresponding to a molecular weight of around 89 kDa), with a highly conserved N-terminal part (up to residue 334) and a highly variable C-terminal region . ProteolTo date, a high resolution 3D structure of a Vip3 tetrameric protein is lacking, though low resolution images have been obtained ,10. In aWith the aim of shedding light on the putative functional and structural domains of Vip3 proteins, we have made use of selected Vip3Af alanine mutants (Ala-mutants) from a previous work  which drChanges in protein conformation may expose potential cleavage sites otherwise buried inside the protein which, when exposed to proteases, give rise to altered patterns of fragments compared with that of the wild type protein (WT). Proteolytic patterns may thus unravel structural domains in the Vip3Af protein.We confirmed the altered proteolytic patterns obtained before with Ala-mutants . To bettThe mutants selected in this study had been shown to have decreased insecticidal activity when tested as protoxins . Here weResidue substitutions may affect the capacity of the Vip3Af protein to form the tetramer , the forWith mutant P171A (which gives rise to pattern “d”), the protoxin eluted at 24 min, revealing a tetrameric form; however, just small fragments (eluting at 34.8 min) were observed after trypsinization B. SDS-PANeither the protoxin nor the trypsin-treated mutant E483A (which gives pattern “f”) showed any tetramer in solution D; the maMutant W552A (which gives rise to pattern “b”) formed a tetramer both as a protoxin and after trypsin treatment E; howeveMutant I699A (which renders pattern “c”) also tetramerized as a protoxin and after trypsin treatment F; howeveFrom the chromatographic analysis, we can conclude that the 27 and 17 kDa fragments, once cleaved by trypsin, are released from the structure and no longer form part of the oligomer. However, the tetrameric structure of Vip3Af can still be maintained in the presence of fragments 38, 35, and 19 kDa  or fragments 53, 35, and 19 kDa (such as mutants rendering pattern “c”). Residues F229 and E483 must have a key role in the oligomerization of the Vip3Af protoxin, since their exchange for alanine prevents tetramer formation even before trypsin treatment.The identification of the tryptic fragments was performed after the separation of the fragments by 2D gel electrophoresis and/or size filtration chromatography followed by SDS-PAGE. The results of the peptide fingerprint were matched with those of the tryptic sites in the primary sequence of Vip3Af, and the estimated size of the fragment was taken into account to set the fragment limits. Through 2D gel electrophoresis, we could separate and analyze the 17 and 27 kDa spots from the trypsin-treated F229A mutant. The results of the peptide fingerprint, along with the tryptic sites in the sequence of Vip3Af, indicated that the 27 kDa fragment corresponded to residues from 523/526 to probably the end of the protein. The same type of analysis with the spot of 17 kDa indicated that it corresponded to residues from 523/526 to 661/663. The 17 kDa fragment from the trypsin-treated I699A mutant was analyzed from the chromatographic fraction B15 (peak 34.8 min) of this mutant. The results indicated the same match as the 17 kDa fragment from mutant F229A. The identity of the 38 kDa fragment was determined from the chromatography fraction A11 (24.2 min) from trypsin-treated W552A; the peptide fingerprint indicated that the fragment corresponded to residues from 313/315 to 661/668.Banyuls et al.  defined In a previous study, Banyuls et al.  identifiThe results from gel filtration chromatography of the Ala-mutants shed light on the structural role of the proposed domains. Mutants rendering patterns “b”, “c”, and “e” are found forming a tetramer both as protoxins and also after trypsin treatment. Since trypsin digests the 27 kDa fragment (the only one containing domain V), we can conclude that domain V is not necessary to maintain the oligomeric structure. All these mutants, after trypsin treatment, have in common fragments of 19 kDa (domain I) and 35 kDa (domains II and III), plus another larger fragment (either of 38 or 53 kDa) which includes domain IV. Despite the fact that the 17 kDa fragment (which corresponds to domain IV) elutes separately from the tetramer in the chromatography of mutants with patterns “c” and “e”, the tetramer contains domain IV in the structure as part of the 53 kDa fragment. Therefore, according to the results, domains I–III are required to form the tetrameric structure, the need for domain IV is not clear, and domain V is not necessary.An interesting observation from patterns “b” and “c” is that, in addition to the 19 kDa band, the sum of the remaining main bands gives an apparent molecular weight exceeding that of 65 kDa. In the case of pattern “b”, these bands correspond to fragments of 35 and 38 kDa. In the case of pattern “c”, the strongest bands are those corresponding to fragments of 15, 17, 19, 35, and 53 kDa. Therefore, there must be an alternative splicing of the 65 kDa fragment in mutants rendering these two patterns.The requirement of domain I to form the tetramer, along with domain exchange studies between the 19 kDa fragment and the rest of the protein with Vip3Ab and Vip3Bb . HoweverFrom the distribution of Ala-mutants with decreased insecticidal activity in the primary structure of Vip3Af , we can Using the approach of the trypsin fragmentation of mutants altering the conformation of the Vip3Af protein, we have defined five domains in the structure of Vip3Af which match some of the domains proposed independently by two in silico models. The effect of some the mutations on the ability to form a tetrameric molecule reveals that domains I–III are required for tetramerization, while domain V is not. The involvement of domain IV in the tetramer formation is not clear. The overlapping fragments in the proteolytic patterns suggest a tetramer with a distinct disposition of the monomers, in such a way that the tryptic sites exposed in two molecules are different to those exposed in the other two. Residues around the primary cleavage site are important for maintaining the structure of the protein, since trypsin processing in mutants of pattern “d” digests most part of the protein and destroys the tetrameric form. Mutants in domain V belonging to pattern “c” destabilize this domain, though they not affect the tetrameric structure after trypsin processing. Because of the high sequence similarity among Vip3 proteins, we think that our domain map proposal may be valid for the Vip3 family of proteins. The information provided here may help to further clarify the 3D structure and its implications in the mode of action of Vip3 proteins.The source of the 788 amino acid protein Vip3Af1(WT) (NCBI accession No. CAI43275) and that of its mutant proteins has been described in Banyuls et al. [w/w), in a TNE buffer was incubated at 30 °C for 24 h. Aliquots (10 µL ) of the trypsinized proteins were subjected to 12% SDS-PAGE. Prior to electrophoresis, the samples were made 1 mM with an 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) protease inhibitor , left standing for 10 min at room temperature, and then heated at 100 °C for 5 min with a loading buffer  . The trypsin-treated samples to be used for chromatography and bioassays were stored at −20 °C for less than one week.The purified Vip3Af protoxins were subjected to proteolytic activation with commercial trypsin . A mixture of protein:trypsin  kills 100% of the larvae. A Tris buffer  was used as a blank control. Once the surface was dry, a neonate S. frugiperda larvae was gently placed into the well and then sealed. The number of dead and 1-instar larvae were recorded after 7 days. A larva was considered dead if it did not respond to mechanical stimulation. The mean mortality and functional mortality (dead larvae plus larvae that had not developed beyond the first instar) were determined from two replicates of 32 insects each.Insect rearing and bioassays were carried out in a rearing chamber maintained at 25 ± 2 °C, 70 ± 5% relative humidity, and 16:8 h light:dark on a semi-synthetic diet based on corn flour and wheat germ that contained yeast, ascorbic acid, and nipagin. Surface contamination assays were performed with 50 μL of protein sample on 2 cmGel filtration chromatography was performed with an ÄKTA explorer 100 chromatography system in a Superdex 200 10/300 GL column  at a flow rate of 0.5 mL/min of a Tris buffer , unless otherwise indicated. To estimate the molecular weight of the peaks, the column was calibrated with the following mix of standards: 4 mg/mL ovalbumin (44 kDa), 3 mg/mL conalbumin (75 kDa), 4 mg/mL aldolase (158 kDa), 0.3 mg/mL ferritin (440 kDa), 5 mg/mL thyroglobulin (6690 kDa), and Blue Dextran 200 (exclusion limit), dissolved in water.Major bands (27 and 17 kDa) from the trypsin-treated F229A mutant were identified after separation in a 2D-gel. The 17 kDa band from the trypsin-treated I699A mutant was first separated by chromatography in the Superdex 200 column and then by SDS-PAGE. The 38 kDa band from the trypsin-treated W552A mutant was first isolated by Superdex 200 chromatography and then by SDS-PAGE. https://web.expasy.org/compute_pi).For the peptide identification, protein bands were directly cut out from the gel and digested with trypsin. The peptide mass and sequence were determined by liquid chromatography and tandem mass spectrometry (LC-MS/MS) in a nanoESI qQTOF . The mass transitions were scanned first from 350–1250 m/z and then followed by a second scan from 100–1500 m/z. The peptides sequence identified were compared to the Vip3Af1(WT) protein sequence to match the region corresponding to each SDS-PAGE proteolytic band. Expected molecular weights were calculated using the online SIB Compute pI/Mw tool (


It interacts with the ubiquitously conserved bacterial master replication initiation protein DnaA at the oriC but structural and functional details of this interaction are lacking, thus contributing to our incomplete understanding of the molecular details that underpin replication initiation in bacteria. DnaD comprises N-terminal (DDBH1) and C-terminal (DDBH2) domains, with contradicting bacterial two-hybrid and yeast two-hybrid studies suggesting that either the former or the latter interact with DnaA, respectively. Using Nuclear Magnetic Resonance (NMR) we showed that both DDBH1 and DDBH2 interact with the N-terminal domain I of DnaA and studied the DDBH2 interaction in structural detail. We revealed two families of conformations for the DDBH2-DnaA domain I complex and showed that the DnaA-interaction patch of DnaD is distinct from the DNA-interaction patch, suggesting that DnaD can bind simultaneously DNA and DnaA. Using sensitive single-molecule FRET techniques we revealed that DnaD remodels DnaA–DNA filaments consistent with stretching and/or untwisting. Furthermore, the DNA binding activity of DnaD is redundant for this filament remodelling. This in turn suggests that DnaA and DnaD are working collaboratively in the oriC to locally melt the DNA duplex during replication initiation.The homotetrameric DnaD protein is essential in low G+C content gram positive bacteria and is involved in replication initiation at  DnaA filaments also invade and bind ssDNA generated during melting of the DUE, through a series of α-helices forming a ssDNA-binding staircase inside the central channel of the filament (Escherichia coli (Origin recognition complex (ORC) and Cdc6 proteins play crucial roles in eukaryotic replication initiation whereas the ubiquitous, strictly conserved AAA+ (ATPases Associated with various cellular Activities) DnaA protein is the replication initiator in bacteria . DnaA coaA-boxes , and thefilament ,13. The filament . Domain filament , HobA, Hfilament  and the hia coli ,21.Firmicutes requires two essential primosomal proteins DnaD and DnaB, which are not found in E. coli and related bacteria. DnaD interacts with the oriC-DnaA complex and sequentially recruits DnaB and the DnaI–DnaC complex to load the replicative helicase DnaC at the oriC  domains  we also show that binding of DnaD to DnaA–DNA filaments induces conformational filament changes consistent with filament stretching/untwisting. The significance of this in terms of DnaD interacting with the DnaA–DnaD filament and orisome assembly is discussed.The formation of nucleoprotein structures by DnaA and DnaD and their interaction provide the foundation for orisome assembly and initiation of DNA replication in B. subtilis DnaA protein was cloned into the pET28b+ vector for expression as an N-terminal histidine-tagged protein. The pET28b+ construct to express and purify the DDBH2 domain (residues 129–232) of the B. subtilis DnaD protein was supplied by Dr Jeremy Craven (2 competent BL21(DE3) cells were used for expression of these constructs. BL21(DE3) cells were grown in M9 minimal media supplemented with 15N ammonium chloride and/or 13C glucose, for use in NMR spectroscopy, where isotopic labelling was required. Soluble protein was obtained by induction with IPTG (1 mM) at mid-log growth phase (OD595 0.6–0.8) followed by expression for 16 h at 30°C. Soluble protein was obtained by induction with IPTG (1 mM) at mid-log growth phase (OD595 0.6–0.8) followed by expression for 4 h at 30°C or 16 h at 20°C for DnaA and DnaD constructs, respectively. Sonication  followed by centrifugation  was used to obtain the soluble fraction of the cell lysate. Ni-affinity chromatography  was used as a first stage of purification followed by thrombin cleavage of the His-tag  leaving behind a residual N-terminal Gly-Ser-His-tag. Ni-affinity chromatography was repeated for further separation of the His-tagged from the un-tagged protein. This was followed by size exclusion chromatography using a Superdex 75 26/60 column  as the final purification stage. DnaA constructs were desalted into pure MilliQ-water and lyophilized for storage whereas DnaD constructs were frozen in solution.Domain I (residues 1–81) of the y Craven  and DnaD13C15N labelled DnaA domain I at 600 μM . Backbone resonances were assigned using HNCO, (HCα)CONH, CβCαNH and CβCα(CO)NH experiments with watergate suppression.NMR experiments were recorded at 25°C on a Bruker 800 MHz Avance III spectrometer with a QCI cryoprobe. Data acquisition and processing were carried out using Topspin 3.1 software and were further compared within the CCPNMR software. 3D NMR experiments used 15N HSQC spectra. This approach was successful for unlabelling of lysine, arginine, asparagine, and histidine residues. Similarly, to aid assignment, single point mutations of a number of surface exposed residues  were used to locally perturb signals in the 15N HSQC experiment. The combination of sequentially linking amino acids from the 3D spectra and these additional approaches allowed 100% assignment of non-propyl residues 1–81 of DnaA domain I 15N HSQC spectrum. Three residues remained unassigned corresponding to a short, flexible leader sequence Gly-Ser-His resulting from the thrombin cleavage of the N-terminal His-tag.Backbone assignment was facilitated through selective unlabelling experiments to resolve some spectral ambiguities, signal overlap and exclude particular reside types from the 1H and 15N shifts according to the equation CSP = √ H2 + (0.14 x δN2)]. Significant CSP effects were determined using the standard deviation (σ) from the mean (μ) and residues with CSP values above ‘μ + σ’ and ‘μ + 2σ’ . 15N HSQC spectra were collected at 0.5 molar ratio intervals from 0:1 to 8:1 excess of DDBH2 or domain I.Chemical shift perturbations (CSPs) where calculated based upon a weighting of ‘μ + 2σ’  were use15N isotopically labelled DnaA domain I (100 μM) in 50 mM potassium phosphate pH 7.4, 100 mM NaCl, 10% (v/v) D2O, 0.02% (w/v) NaN3. A DnaD DDBH1 stock solution (400 μM) was also prepared in the same buffer. Aliquots of the DnaD DDBH1 solution were then titrated into the DnaA domain I solution to yield solutions at various stochiometric ratios.For the HSQC titration of DnaD DDBH1 into DnaA domain I, a stock solution of HADDOCK 2.2 (High Ambiguity Driven protein-protein Docking) accessible via the WeNMR  server wN191C and DnaAN198C in 50 mM Tris pH 7.5, 100 mM NaCl. The reaction mixture was flushed with nitrogen and the reaction was left to proceed overnight at 4°C with rotation mixing, prior to being quenched with 1 mM DTT. The reactive dyes were shielded from direct light throughout the labelling procedure. Atto647N-labelled and Cy3B-labelled DnaAN191C and DnaAA19C were separated from excess fluorescent dyes by extensive dialysis overnight at 4°C (into an appropriate buffer) was repeated until no further excess dye molecules were present in the exchanged buffer.Single cysteine DnaA mutants N191C and A198C were labelled with each of the Atto647N-maleimide and Cy3B-maleimide dyes. Atto647N-maleimide or Cy3B-maleimide (10 mM in DMSO) was added dropwise at 10 x molar excess to proteins DnaAdnaA and dnaN genes (2 and 2 mM ATP. BMOE (2 mM) was then added and the mixture was incubated at 37°C for an additional 5 min before the addition of cysteine (50 mM) to quench the cross-linking reaction for 5 min. Proteins were then resolved through SDS-PAGE and visualized via Coomassie staining.DnaA–DNA filaments were formed using the DnaA(N191C/A198C) mutant protein and 819 bp DNA fragment containing the half origin with the DUE and four DnaA-boxes between the aN genes . BrieflydnaA and dnaN genes -1-piperazineethanesulfonic acid (HEPES)-NaOH, pH 7.3, 10 mM MgCl2, 1 mM DTT, 100 μg ml−1 bovine serum albumin, 5% (v/v) glycerol, 1 mM mercaptoethylamine, containing ∼100 pM DnaA.DnaA–DNA filaments for FRET experiments were formed using the DnaA(N191C) and DnaA(A198C) mutant proteins and 819 bp DNA fragment containing the half origin with the DUE and four DnaA-boxes between the aN genes . A totalE* for each burst as E* = DA/(DD + DA), where DA is the number of photons in the red detection channel after green excitation and DD the number of photons in green detection channel after green excitation.Single-molecule FRET measurements were performed at room temperature using a home-built confocal microscope  allowed 97% of the non-prolyl residues of the DnaA domain I amide backbone to be assigned (The DnaA domain I (residues 1–81) was assigned . The det15N-labelled sample of DnaA domain I (100 μM) up to an 8:1 excess and the binding interaction monitored incrementally in 2D 1H/15N HSQC spectra. Statistically significant perturbations for nine residues within the DnaA domain I were observed  between DnaA domain I and the DnaD DDBH2 domain when averaged over five well resolved residues   .180IDRI184LFEW188) along with a region of the unstructured C-terminus (residues 206–215) appear to be essential for DNA binding . Selective CSP effects mapped to a similar binding patch on DnaA with the cluster of residues Glu48, Phe49, Asp52, Trp53, Leu54, and Ser56, located on helix α3 helix, along with Ser23 on helix α2 , helix α3  and helix α4 (His60) with an additional perturbation to Leu69 at the C-terminus of helix α4 also evident  was created to abolish DNA binding activity and investigate whether the C-terminally truncated DnaD maintained its DnaA binding activity without the region associated with binding nucleic acids. The truncated and unlabelled DnaD DDBH2 domain was titrated into t Figure . These r15N-labelled DnaD DDBH2 domain (100 μM) up to an 8:1 excess now enabled us to map the complementary binding surface on DnaD DDBH2, using the same CSP methodology. A binding patch of nine residues on the DnaD DDBH2 domain was identified within the structured N-terminal region and involved two clusters of residues  is fully consistent with the earlier estimate.We have previously described the assignment of the backbone of the DnaD DDBH2 domain using a similar methodology to that already described . The revs Figure . The firThe ternary complex of DnaA–DnaD interaction was further investigated using ssDNA (5’-GTTATTGCTC) in 8-fold excess. The addition of ssDNA to the experiment produced poorer quality HSQC spectra, independently of the addition or not of DnaA domain I, with weaker and broadened peaks displaying increased overlap between backbone amide residues . In partB. subtilis DnaA domain I was solved by X-ray crystallography  mutant protein and repeated the BMOE cross-linking assays in the presence of an 819 DNA fragment containing the half origin with the DUE and four DnaA-boxes between the aN genes . We wereaN genes .dnaA and dnaN genes  and DnaA(A198C) single mutant proteins, fluorescently labelled them with the FRET pair Cy3B and Atto647N, and carried out single-molecule FRET experiments in the presence of an 819 bp DNA fragment containing the half origin with the DUE and four DnaA-boxes between the aN genes . We hypoWe used a model of a mini DnaA–DNA filament with four DnaA molecules reported before  to assesE* ∼0.7  added to the DNA either before or after the addition of DnaA in order to assess the effect of DnaD on the formation of DnaA–DNA filaments and also the effect of DnaD on pre-formed DnaA–DNA filaments Figure –F. In th7 Figure  whereas 5 Figure , F. ThesdnaA and dnaN (see Further evidence that DnaD does not induce the abolition of DnaA–DNA complexes was provided by electrophoretic mobility shift assays (EMSA) using a 120mer synthetic double stranded oligonucleotide containing the four DnaA boxes within the half origin between dnaN see .E* ∼0.7 in their absence to E* ∼0.5 in their presence. This suggests that both DnaD and DnaD196 untwist the filament to a similar extend  of DnaD ,29, alsoA recent bacterial two hybrid study suggested that the DDBH1 domain of DnaD interacts with the DnaA domain I while the DDBH2 does not , contradHowever, the interaction of DDBH2 with the DnaA domain I was studied in detail by NMR and HADDOCK modelling using the experimental restraints from the CSP analysis of the NMR data and revealed two possible overlapping, but distinct, families of conformations Figure . In addiThe DnaA-interaction patch of DDBH2 contained residues  within the amino acid region 133–140 that was previously identified as essential by yeast two hybrid analysis . This reCritically, the interaction of full-length DnaD with DnaA appears to be cryptic and could not be detected by bacterial two hybrid  suggestioriC.Interestingly, the DDBH2 domain was also able to untwist the filament but to a lesser extent than full-length DnaD Figure –D, suggeIn vivo, the functional interaction of the DnaA and DnaD proteins within the oriC likely involves the DnaA–DNA filament and the DnaD scaffold. DnaD tetramers on the surface of the scaffold could potentially interact with the DnaA–DNA filament using both the DDBH2 and DDBH1 domains (Figure DnaD is a homotetramer and forms scaffolds mediated by inter-tetramer interactions through the DDBH1 domains ,31,32. Ts Figure . Althougs Figure . DnaD its Figure ,31 and ts Figure .The DnaA domain I interacts with the replicative helicase and a number of client proteins involved in the regulation of replication initiation  and as sB. subtilis cells committed to sporulation. It has been suggested before that SirA may inhibit the DnaA–DnaD interaction arresting assembly of the initiation complex (Our data show the DnaA–DnaD binding interface of the DnaA domain I overlaps with the DnaA–SirA interface determined by X-ray crystallography . This di complex ,43. This complex . TherefoSupplementary DataClick here for additional data file.


Ipomoea batatas Lam) is a nutritious crop abundant in calories and bioactive compounds such as beta-carotene, ascorbic acid, polyphenols, and dietary fibre. This study investigated the effect of pretreatments on the physicochemical and functional properties of Ndou sweet potato (NSP) flour. Flour samples were prepared by randomly assigning NSP slices to two treatments (citric acid and sodium metabisulphite) at 5, 10, and 15 g/L concentration for 10 min. Distilled water was used as control. The moisture content (7.70%) of NSP flour treated with citric acid was significantly (p < 0.05) higher than that of the flour treated with sodium metabisulphite (5.54%) as the concentration level increased. The treatments did not significantly (p < 0.05) affect the protein and fat contents of the NSP flour and protein increased from 2.54 to 2.82%, while fat decreased from 0.69 to 0.61%. Sodium metabisulphite treated samples had a higher L∗ value, ash, and pH level than citric acid treated samples. However, pH was slightly decreased by both treatments from 6.05 to 5.09. Citric acid treated samples had higher a∗ and b∗ values than sodium metabisulphite treated samples. In terms of the functional properties of NSP flour, the treatments significantly (p < 0.05) affected the water absorption capacity, viscosity, swelling power, solubility index, and thermal properties although the bulk density and least gelation concentration were not significantly (p > 0.05) affected. Sodium metabisulphite was very effective in improving physicochemical and functional properties of NSP flour as compared to citric acid. The findings of this study show the possibilities of using NSP flour in food systems as gelling agent, fat replacer, and thickeners.Sweet potato ( Ipomoea batatas Lam) has been identified as a food security crop because it contains reasonable amounts of bioactive compounds such as b-carotene, ascorbic acid, polyphenols, dietary fibre as well as vitamins, minerals, and proteins [Ndou is one of the sweet potato cultivars grown in Limpopo province, South Africa. Ndou sweet potato (NSP) cultivar is high-yielding and is mostly bred by the Agricultural Research Council in South Africa. It has a dark cream skin colour and flesh colour as well as a round and long elliptic storage root shape.Sweet potato  and sodium metabisulphite (Na2S2O5) have been used to prevent or eliminate discolouration of sweet potato flour [There are several drawbacks that have been identified during the preparation of sweet potato products and one of these is discoloration which affects the quality of products . Variousto flour , 10. Proto flour .Ipomoea batatas Lam) tubers were purchased from ARC in Pretoria, Gauteng province, South Africa. Chemicals used in this study were commercially purchased from Sigma-Aldrich, Johannesburg, South Africa.Fresh NSP  and 5, 10, 15 g/L of citric acid for 10 min (experiment 2). Each experiment was done in triplicate.Ndou sweet potato tubers were weighed, washed, peeled, and cut into 4 mm size. Sliced sweet potatoes were divided into two batches, and randomly assigned to two treatments  as follows: sodium metabisulphite and citric acid at 5, 10, and 15 g/L concentration for 10 min. The untreated (control) slices were immersed in distilled water for 10 min at room temperature. The sweet potato slices immersed in the two chemical solutions were allowed to drain for 2 min after which the pretreated and control NSP slices were put on a tray and oven dried for 10 h at 70°C. The pretreated and control NSP slices were milled into flour  . The moiL∗ (Lightness/darkness), a∗ (redness/green) and b∗ (yellowness/blue) were recorded [The colour of NSP flour samples was measured using a Hunterlab LabScan XE Spectrophotometer CIELAB. The black and white plates were used to calibrate the spectrophotometer and zero calibration followed. The NSP flour was put in a glass cell and placed above the light source of the instrument and colour coordinate values recorded .About 10 g of each sample of NSP flour samples was mixed with 100 mL of distilled water in order to measure the pH values of flour. The mixture was left at room temperature for 30 min. The pH meter that had already been standardised with buffer solutions of pH 4.0 and 7.0 was then used to measure the pH values of the supernatant.The method of Onwuka  was foll3).The method of Konak et al.  was follThe method of Sathe et al.  was adopApproximately 10 gram of the NSP flour sample was mixed with 90 ml water at 30°C  and allowed to hydrate for 30 min with occasional stirring. Brookfield viscometer  was used to measure the viscosity of the samples.The method of Anyasi et al.  was adopT0), peak time (Tp), conclusion temperature (Tc) and enthalpy (ΔH). Pyris software  was used for calculation of obtained data.The thermal properties of NSP flour samples were investigated as described by Sun et al.  with somp < 0.05 level of significance.All measurements were done in triplicate. The data collected were subjected to statistical analysis to test for variance (ANOVA) using SPSS version 23.0 with Duncan test at p < 0.05 in the moisture content of citric acid treated samples at different levels. A higher level of 7.70% of moisture content was obtained from citric acid pretreatment of 10 g/L concentration, while the lowest moisture content of 5.54% was obtained for sodium metabisulphite pretreatment of 15 g/L concentration. This might be due to the ability of sodium metabisulphite to cause dehydration in sweet potato slices. The lower moisture content of the NSP flour samples will give a better shelf life and denser nutrient [The moisture content of untreated (control) NSP flour was 6.50% and it ranged from 5.54% to 7.70% for variously treated samples as shown in nutrient .p < 0.05, ash content was significantly affected by sodium metabisulphite treatment and it increased as the concentration of sodium metabisulphite increased from 1.77 to 2.54%. The increase in ash content might be due to diffusion of solutes from sodium metabisulphite solution to sweet potato slices during soaking. Ahmed et al. [p > 0.05) affect the ash content of untreated (control) and treated NSP flour samples. The low values of ash content on 15% sodium metabisulphite treated sample might be due to the leaching out of minerals during the pretreatment of NSP slices. Jangchud et al. [p < 0.05) affect the protein content of NSP flour. Untreated sample (control) had the value of 2.64% while treated samples ranged from 2.54% to 2.82%. This was lower than 5% which has been reported as the crude protein content of sweet potato flour [p < 0.05) difference in sodium metabisulphite treated samples. Ahmed et al. [p < 0.05) affected the fat content of NSP flour and it ranged from highest (0.68%) to lowest (0.61%). This is similar to Haile et al. [All the sweet potato flour samples had the moisture content within a favourable range for effective storage of the flour with minimal risk of microbial contamination. The moisture content of flours below 14% is capable of resisting microbial growth resulting in shelf stable flour . The phyd et al.  observedd et al.  and Osund et al.  revealedto flour , 27. Howto flour . NSP floto flour . There we et al.  observatp < 0.05) in pH values of NSP flour. The values ranged from 5.09 to 6.42. The untreated sample had a pH level of 6.05. A decrease in pH level was observed across the pretreatment concentration of sodium metabisulphite and citric acid which decreased from 6.42 to 5.82 and 5.64 to 5.09 respectively as concentration increased. Low pH values have been reported to be caused by high amylase activity which increases the level of acidity and this was similar to observations of Nabubuya et al. [The pH of a NSP flour suspension  is impora et al.  who founa et al. . Citric p < 0.05) different in lightness (L∗), redness (a∗) and yellowness (b∗) values for all pretreated flour samples. Sodium metabisulphite increased the lightness of the flour with an increase in concentration. The increase in the L∗ value might be due to the potential of the pretreatments to retard both enzymatic and nonenzymatic reactions in the sweet potato flour. Pretreatment such as sulphite and its salts are used to preserve the colour of fruits and vegetables because of their ability to retard the reactions of both enzymatic and nonenzymatic [L∗ values) might also be due to oven drying temperature of the NSP since higher temperature inactivates phenolase enzyme. Phenolase activities have been reported to be deactivated at high temperatures [L∗ values of sweet potato flour. Compared to sodium metabisulphite, the L∗ values of citric acid treated samples were least ranging between 69.27 and 78.82 across all pretreatment concentration. Changes in carotenoids, oxidation, caramelisation, or phenolase [L∗ values of the flour.The results of colour measurement, as presented in nzymatic . The inceratures . Moreoveeratures . Ahmed eeratures  also rephenolase  might hab∗ values for pretreated Ndou sweet potato flour was higher than the control sample in all pretreated concentration. However citric acid had a greater effect on this chroma than sodium metabisulphite. In citric acid and sodium metabisulphite, b∗ values increased as concentration level increased, ranging from 20.88 to 22.41 and 20.04 to 21.73, respectively. High yellowness values in pretreated samples can be due to the higher amount of beta carotene in NSP flour [a∗ values indicated that redness (a∗) was significantly (p < 0.05) affected by treatments. Sodium metabisulphite showed a decrease in redness values which ranged from 1.59 to −0.16 as concentration increased. A higher redness value of 3.74 was obtained at 10 g/L of citric acid, showing a greater intensity of redness of the flour. The higher redness value could be due to the presence of the anthocyanins pigments in the NSP flour since β-carotene is 6708 μg/100 g in fresh sweet potatoes while anthocyanin is 525 mg/100 g fresh weight (unpublished data) and it might also have been influenced by milling conditions and other contaminants [Conversely, the SP flour . Beta-caaminants .p < 0.05) in BD for all citric acid treated samples across all pretreatment concentrations while sodium metabisulphite did not have any significant difference (p < 0.05) with values ranging from 0.86 to 0.87 g/ml. This was similar to the value of 0.92 g/ml obtained by Eleazu and Ironua in 2012 [The bulk density (BD) of NSP flour is shown in  in 2012  for a cr in 2012 , 35.The results of BD revealed that it depends on the particle size and initial moisture content of flours . The incp < 0.05) higher than that of control samples (1.44 ml/g) which ranged from 1.63 to 2.03 ml/g. The higher values of WAC recorded for the flour samples might have been caused by high polar amino acid residue of protein having affinity for water molecule [The water absorption capacity (WAC) of NSP flour is shown in molecule . Since tmolecule .The WAC might also be influenced by nonidentical structure of flours. The high WAC of sweet potato flours shows that they can be useful in the formulation of various foods such as meat sausage, bakery products, dough, and processed cheese .p > 0.05) affect the LGC of NSP flour as shown in Both pretreatments did not significantly (Akubor  reportedp < 0.05) higher than that of control samples (7.18 g/ml) which ranged from 8.47 to 7.57 g/ml except for citric acid pretreated sample at 15% which had a lower value of 6.30 g/ml. Structural changes within the starch granules might be responsible for the increase in SC of pretreated samples [Viscosity, swelling power, and starch solubility index of NSP flour are shown in  samples . When st samples . The pre samples .p < 0.05) higher than that of control samples (7.20 g/ml), ranging from 7.3 to 9.6 g/ml. The difference in the SI is related to the difference in the extent of starch degradation in NSP flour samples. High SI in pretreated samples could be due to high amount of amylose which leaches out easily during the swelling process [The decrease in SP could also be due to the interactions that might have occurred between amylose–amylose and amylo-pectin–amylopectin chains . A low S process . At a hi process .T0), peak temperature (TP), conclusion temperature (Tc), and gelatinization ΔH values of NSP flour samples are given in To) ranges from 51.26 to 63.65°C, peak temperature (Tp) from 67.58 to 75.72°C, conclusion temperature (Tc) from 74.73 to 85.28°C and enthalpy of gelatinization (ΔH) from 8.48 to 12.78 J/g. A significant difference (p < 0.05) was observed in To, Tp, and Tc and of pretreated and control NSP flour samples.Thermal properties of NSP flour are shown in H values recorded in NSP flour samples might be attributed to large quantities of short amylopectin chains present in the starch of the flours [Low transition temperatures and Δe flours , 50 while flours . Shinoj e flours  reportedThe correlation between functional and physicochemical properties of NSP flour is represented in Correlation among proximate composition of NSP flour is shown in Correlation between proximate composition, physicochemical, and functional properties of NSP flour is shown in NSP flour was prepared by pretreating the tubers with citric acid and sodium metabisulphite at three different concentrations of 5, 10, and 15 g/L for 10 min. In the conversion of dried pretreated NSP slices to flour, citric acid had a significant effect on moisture, bulk density, pH, and colour while sodium metabisulphite had a significant effect on moisture, ash, pH, and colour. Both citric acid and sodium metabisulphite did not have any significant effects on protein, fat, water absorption capacity, and least gelation concentration. Pretreatments did improve physicochemical and functional properties of NSP flour. However, sodium metabisulphite was more effective than citric acid at 15 g/L. It is, therefore, recommended that a further study should be conducted on the effect of pretreatments on some bioactive compounds and other functional properties such as oil absorption capacity, emulsion activity, and foam capacity and stability of NSP flour.


Volcanic phenomena are currently monitored by the detection of physical and chemical observations. Generally, the ground deformation field is the most relevant shallow expression of the geometric and physical parameters variations in the magmatic reservoir. In this study, we propose a novel method for the direct estimation of the geometric parameters of sources responsible for volcanic ground deformation detected via the DInSAR technique. Starting with the biharmonic properties of the deformation field, we define an approach based on the Multiridge and ScalFun methods to achieve relevant information about both the positions and shapes of active sources, such as the Mogi source. Our methodology is definitely different from the methods currently used for modeling ground-deformation sources, mainly based on forward or inverse techniques. In fact, (i) it does not require any assumptions about the source type, and (ii) it is not influenced by the distribution of medium elastic parameters or (iii) the presence of high-frequency noise in the dataset. For synthetic cases, we accurately estimate the depth to the source within a 3% error. Finally, we study the real case of the Okmok volcano ground-deformation field and achieve results compatible with those in previous works. Specifically, the Differential SAR Interferometry (DInSAR) technique has significantly contributed to the characterization and imaging of the deformation of Earth’s surface, allowing for the analysis of a large variety of geological phenomena, such as landslidesP)7. The estimation of these parameters is performed using two different approaches: (i) forward modeling9, which is based on a trial-error procedure and strongly depends on the experience of the scientist, and (ii) inverse modeling12, which is faster than the previous approach. Current methods of inversion are essentially based on optimization techniques, where the final solution is that obtaining the best-fit between the measured and modeled data; this optimization/inversion procedure is performed by using algorithms such as Monte-Carlo, Gibbs, Simulated Annealing, and Levenberg-Marquardt14. In the case of the optimization/inversion of geodetic data, several authors have evaluated model parameters via posterior probability density distributions16. However, we remark that these methods are often unable to provide unique solution17.In a volcanic environment, the accumulation processes of shallow magma in the crust often cause slight movements at the surface, which can be measured using standard land-surveying techniques (leveling) or satellite geodesy methodologies . In this context, these aforementioned processes often result from variations in several geometric and physical parameters of magmatic sources .Starting with these considerations, we propose a novel method based on the theory of Laplace’s equation:We study the conditions under which the theory of the deformation field reduces to the PFT and then show how to obtain the main geometric parameters of the active source.19. We further assume that modeling the magmatic reservoir is performed through the approximation of the elasticity theory7 and under the assumption that an active volcanic source is not affected by shape changes20. Among the sources of deformation, we first consider those that also satisfy the homogeneity equation18, such as a Mogi source21. In this framework, we use methods that have been applied to analyze potential fields22, namely, the Multiridge23 and ScalFun24 methods. By using these, the geometric features of the active source, such as the position and source type, can be easily detected; the main advantages of the proposed methods are that they are determined independently from (i) other physical features of the source  projection of the deformation field. Finally, we consider the deformation field of the Okmok volcano (Aleutian Islands), which has already been interpreted with standard methods in other works29. In particular, we consider the Multiridge23 and ScalFun24 methods, which are two well-assessed methods, to analyze homogeneous potential fields. For the first approximation, we assume that the ground deformation field results only from volume changes in subvolcanic bodies  in a linear elastic medium.The method is applied to interpret the large number of deformation points achieved by the SBAS-DInSAR technique18. In particular, a potential field , we specify that we are clearly not considering that the deformation field could propagate into the air. The upward-continued deformation field instead corresponds to that which would have been produced by the same source in a region that was upper extended by the same amount of upward continuation. For example, if the analyzed field is observed by a source with a depth of n Figure .22. In our analysis, we have selected the ridges of subsets For the concerns of this study (2), we clarify that more than one subset can be defined: a multiridge subset, 2, where R is the correlation coefficient, which represents a statistical measure of how the data (edges) are close to the fitted regression line (ridges). Moreover, we evaluate the solution uncertainties (intersection at the ridges) by considering the error on the best-fit linear regression coefficients (intercept and slope constants).For the representation and continuation of the ridges (3), we specify that each ridge is determined by a best-fit linear regression within a 95% confidence interval; in particular, we calculate the determination coefficient R31. The accuracies and details of this procedure are shown in the Supplementary Information  data measurement levels, we have to perform another procedure before applying the Multiridge method. Since the method is based on a level-to-level algorithm, and the ground deformation field is measured at the topographic surface , the dataset must be processed to numerically generate a ground deformation field that could have been measured in the case of constant distance between the source and measurement surface. This transformation relocates the analyzed field onto the constant reduction level, which is performed in this study by using the CWT-domain algorithm Figures  and S3.Consider again the ground deformation field modeled through the Mogi source, and let us evaluate its homogeneity properties.18:The function 24. By inserting equation  and achieve a homogeneity degree of 32, which is in agreement with the features of the true source.We generate the vertical component of the ground deformation field on a flat surface  Fig. . The resion Fig. . Then, wrce Fig. .Figure 2For the third case study, we consider the same source model parameters as those of the previous tests, but it is embedded in a heterogeneous elastic space. In particular, using a 3D finite element approach, we run the model by considering the heterogeneities of the elastic parameters (increasing shear modulus with depth) of the medium Figure . The modion Fig. . The appN≈3 Fig. .Figure 3) and, finally, the high-frequency noise is added  Fig. .Then, we consider the previous test configuration projected along the descending LOS by changing only the surface measurements from =−2 Fig. . This vaFinally, we consider the aforementioned test configuration, with 10% high-frequency noise , on the descending LOS deformation field Fig. . The achWe point out that for all of the performed tests summarized in Table 35 that represents the surface expression for the subduction of the Pacific Plate as it moves northwards beneath the North American Plate36.The Okmok volcano is an active caldera field located on the oceanic crust of the central Aleutian arc (Alaska – USA)26. The Okmok physiography is dominated by a central caldera, with a diameter of 10 km; the rim and caldera floor have elevations of approximately 900 and 400 m a.s.l., respectively28. This physiography is the result of two different and large (≈15 km3) caldera-forming events35 caused by catastrophic pyroclastic eruptions that occurred approximately 12.0 and 2.05 kyr ago, respectively37. These eruptions began with a small Plinian rhyodacite event, followed by the emplacement of a dominantly andesitic ash-flow unit, whereas effusive inter- and post-caldera lavas have been more basaltic35.In particular, it is a dominantly a basaltic shield volcano covering most of the northeastern end of Umnak Island in Alaska38.Subsequent eruptions produced a field of small cones, which was once entirely filled with water, and lava flows, including several historically active vents. Observed pillow lavas and other textures are consistent with the subaerial eruptions within the caldera38. The most recent eruption, in 2008, originated from several new vents and occurred near the eastern rim of the caldera28, while the three previous eruptions  originated near the southwest rim of the caldera39. In particular, the 1997 eruption began with steam and ash plumes and progressed into moderate Strombolian activity, producing explosive ash plumes and lava that flowed toward the center of the caldera. Geochemical analyses of the erupted products are consistent with the primitive magma from the depth and brief storage of the shallow reservoirs35.For the past 200 years, the Okmok volcano has had an effusive and basaltic eruption every 10–20 years, generally from the intracaldera cones41. The P-SBAS results from the ENVISAT data were spatially averaged  to obtain a pixel size of approximately 80 m by 80 m on the ground. The ENVISAT satellite look angle is 23°, and the mean values of the LOS unit vector are  relative to the satellite descending orbit.We analyze the Okmok volcano ground deformation pattern retrieved by processing the ENVISAT SAR images. The interferogram is related to the period 15 July 2003–29 June 2004, with images acquired by the ENVISAT satellite (ESA) along the descending orbit (Track 115). The ENVISAT dataset was processed by using the online P-SBAS web tool available within ESA’s Grid Processing On Demand (G-POD) environment, which is within the framework of ESA’s Geohazards Exploitation Platform (GEP)After the processing steps, the descending interferogram is unwrapped to retrieve the LOS deformation measurements Fig. . In the z-axis, which corresponded to a depth of −3.4 ± 0.06 km b.s.l. with horizontal UTM coordinates equal to 690.9 ± 0.08 km East  was a priori assumed, and the elastic parameters  were fixed before inverting the data. Only in one case28 did the authors use seismic tomography as a priori information to set the heterogeneous distribution of the elastic parameters. For these authors, the source depth ranged from 3.1 to 3.5 km, while the source position ranged from 690.3 km to 690.72 km for the East UTM coordinate and 5923.6 km to 5923.98 km for the North UTM coordinate .Our results are in good agreement with those of the aforementioned works, indicating a depth source equal to 3.4 ± 0.06 km and a horizontal position at 690.9 ± 0.08 km E and 5924 ± 0.07 km N (Table In conclusion, since our method does not depend on physical model parameters, we need to apply a subsequent optimization/inversion procedure to fully interpret the ground deformation measurements and determine the source pressure variation, Supplementary information