Scattered radiation was reduced with a different rate for each CBCT device as distance was increased. For CBCT1 the reduction was 0.047 μGy, for CBCT2 it was 0.036 μGy, and for CBCT3 it was 0.079 μGy, for every one meter from the X-ray gantry. Therefore, at certain distances from the central X-ray, the scattered radiation was below the critical level of 1 mGy, which is defined by the radiation protection guidelines as the exposure radiation limit of the general population. Consequently, an operator could stay inside the room accompanying the patient being scanned, if necessary.This study aimed to estimate scattered radiation and its spatial distribution around three cone-beam computed tomography (CBCT) devices, in order to determine potential positions for an operator to stand if they needed to be inside the CBCT room. The following devices were tested: Morita Accuitomo (CBCT1), Newtom Giano HR (CBCT2), Newtom VGi (CBCT3). Scattered radiation measurements were performed using different kVp, mA, and Field of View (FOV) options. An anthropomorphic phantom (NATHANIA) was placed inside the X-ray gantry to simulate clinical conditions. Scattered measurements were taken with the Inovision model 451P Victoreen ionization chamber once placed at fixed distances from each irradiation isocenter, away from the primary beam. A statistically significant ( Cone-beam computed tomography (CBCT) is one of the most important technological achievements in oral and maxillofacial radiology in the last forty years. In time, this has found numerous applications, from diagnostic applications to pre-implant assessment and surgical guidance using specialized software . The maiThe X-ray beam of the CBCT machine consists of primary radiation, which yields useful imaging information through the patient, and secondary radiation which is scattered radiation . The priThe health risks associated with occupational radiation exposure are either of a deterministic or stochastic nature . StochasOur hypothesis assumes that in some exceptional occasions the dentist or the staff  may need to be present in the X-ray room during the CBCT examination, to visually and verbally direct and encourage the patient to place themselves correctly. Thus, we wanted to determine if there is a safe distance from the CBCT device to stand, so to receive the lowest possible scattered radiation ,11.This original research study specifically aims to estimate the patterns of scattered radiation and its spatial distribution around three CBCT devices, in order to determine potential positions for an operator to stand if they need to be present in the X-ray room during the CBCT examination for maxillofacial diagnostics.The following devices were tested in this research study: Morita Accuitomo (CBCT1) , Newtom Giano HR (CBCT2) , and Newtom VGi (CBCT3) . Exposure measurements were performed for different kVp, mA, and Field of View (FOV) values , , ). An anthropomorphic phantom (NATHANIA)  was placed in the X-ray gantry to imitate clinical conditions .In terms of ambient dose equivalent H*(10), scattered radiation measurements were taken with a Victoreen ionization chamber (Inovision 451P), with dimensions 10 cm × 20 cm × 15 cm (451P) . Calibration of ionization chambers provides traceability to Physikalisch-Technische Bundesanstalt (PTB) through the Ionizing Radiation Calibration Laboratory of the Greek Atomic Energy Commission ). The survey meter was placed at fixed distances from each irradiation isocenter, away from the primary beam ,19. The x-axis) represented the distance (in m) of the 451P to the right and left of the CBCT device, showing positive (right) and negative (left) values, respectively. The second (y-axis) represented the distance (in m) of the 451P from the CBCT device, in front of the CBCT device, perpendicular to the x-axis  x and y axes (yellow points)  and 32 (CBCT2) measurements of scattered radiation at two different heights (1 m or 1.3 m from the floor) were carried out in the rooms of CBCT1 and CBCT2 devices at each point of intersection of the  points) . The meaMeasurements of scattered radiation from different Fields of View (FOVs) were also performed. More specifically, measurements of scattered radiation were carried out in the CBCT1 device in three different FOVs ((4 × 4), (6 × 6), (8 × 8)), in the CBCT2 device in two different FOVs ((8 × 8), (11 × 8)), and in the CBCT3 device in two different FOVs ((8 × 8), (15 × 15)).t-test were used to assess the mean difference of 451P measurements between the three rooms and between the FOVs in each room. Then, Bonferroni tests for multiple comparison corrections were applied. In order to investigate whether the position of CBCT device, in the three rooms, was related to 451P, the Euclidean distance of each measurement point was calculated as the distance in meters (m). Generalized additive models (GAMs) were applied to assess the relationship between 451P (dependent variable) and distance (m) from the CBCT device (independent variable), in each room. Mixed effect linear regression models were used to assess the relationship between 451P and the FOV, in each room. All models were adjusted for the height (m) that the measurement was carried out and for the coordinates  of each measurement point, by including a bivariate smooth function (thin plate spline) of . The spatial distribution of scattered radiation in 451P was estimated through the rigging universal interpolation method. A test for trend was applied to investigate the trend of 451P in the FOVs of each room.Data were described using mean values and standard deviation (SD) for the scattered radiation dose measurements (µGy) in the ionization chamber (451P) at three different rooms of CBCT devices . One-way analysis of variance (ANOVA) and independent samples p-values are reported. A p-value less than 0.05 was considered as statistically significant.All statistical analysis was performed using R version 4.1.3 (10 March 2022), library (mgcv) and library (lme4). All spatial analysis was conducted using ArcGIS Desktop v.10.1. . Two-tailed p < 0.001) between rooms. Specifically, after applying the Bonferroni test, differences in the mean 451P measurements were statistically significant between rooms 2 and 3 (p <0.001), rooms 1 and 3 (p <0.001), and between rooms 1 and 2 (p < 0.001). The maximum value of 451P measurements (9.03 µGy) was observed in CBCT1, in a distance of 100 cm from point “0”, while in CBCT2 and CBCT3 the maximum values were 5.70 and 8.70 µGy at a distance of 50 cm and 55 cm from point “0”, respectively (ectively .p = 0.012) and in room 3 (p = 0.001). Regarding room 1, a significant difference in 451P measurements was observed between FOVs 4 × 4 and 8 × 8 (Bonferroni multiple comparison p = 0.010). Moreover, as the FOV increased, a significant increasing trend in 451P measurements was shown in rooms 1 and 3 (p < 0.001). For example, in room 1, 451P measurements made at FOV 6 × 6, compared to FOV 4 × 4, were on average higher by 0.523 μGy : 0.139 to 0.820) μGy). Moreover, 451P measurements performed at FOV 8 × 8, compared to 4 × 4, were on average higher by 0.776 μGy (95% C.I.: 0.365 to 1.053) μGy) (53) μGy) .x, y) of measurement points, in each room. In room 1, a 1 m increase in the distance from the CBCT device, resulted in a decrease in mean 451P by 0.047 μGy (95% C.I.: −0.057 to −0.037 μGy), adjusting for the other variables. In room 2, a 1 m increase in the distance from the CBCT device, resulted in a decrease in mean 451P by 0.036 μGy (95% C.I.: −0.062 to −0.010 μGy), taking into account the other variables. Note that in both rooms, the height of the measurements did not significantly predict 451P . In room 3, a 1 m increase in the distance from the CBCT device resulted in a decrease in mean 451P by 0.079 μGy (95% C.I.: −0.115 to −0.043 μGy), adjusting for the other variables  of measurement points, in each room. A statistically significant effect between FOV and 451P was found in rooms 1 and 3. Specifically in room 1, FOV 6 × 6 and 8 × 8, compared to FOV 4 × 4, had on average a higher value of 451P by 0.480 (95% C.I.: 0.139 to 0.820 μGy) and 0.709 μGy (95% C.I.: 0.365 to 1.053 μGy), respectively. In room 3, FOV 15 × 15, compared to FOV 8 × 8, had a higher mean value of 451P by 2.005 μGy (95% C.I.: 1.453 to 2.558 μGy) (558 μGy) .The spatial distribution of scattered radiation in 451P was estimated through the rigging universal interpolation method. Color maps of dose distributions were drawn for horizontal and vertical planes. Scattered radiation dose mapping in the ionization chamber (451P) was depicted per room  on the scattered radiation dose distributions color maps . It is wCBCT: Cone-beam computed tomography device 1 (CBCT1); point “0”.x-axis defined the distance (in m) of the ionization chamber (451P) to the right and left of the CBCT1 device (point “0”), showing sometimes positive (right) and sometimes negative (left) values. In this color map, the absolute values are displayed in order to avoid confusion ((point A − point “0” = 0.7 m), (point “0” − point N = 2.10 m)).x (in m): y-axis defined the distance (in m) of the ionization chamber (451P) from the CBCT1 device in an anterior position, perpendicular to the x-axis ((point A − point D = 3 m), (point N − point Q = 3 m)).y (in m): 451P (μGy): Scattered radiation measurements (μGy) were taken with the Inovision model 451P Victoreen ionization chamber (0.04–8μGy). Red color means very high scattered radiation dose, while blue color means very low scattered radiation dose.Height (1 and 1.3 m): The measurement carried out at a height of 1 m from the floor represented the anatomical location of the gonads. The measurement carried out at a height of 1.3 m from the floor represented the anatomical location of the thyroid gland.CBCT: Cone-beam computed tomography device 2 (CBCT2); point “0”.x-axis defined the distance (in m) of the ionization chamber (451P) to the right of the CBCT2 device (point “0”), showing positive values (point “0” − point D = 1.5 m).x (in m): y-axis defined the distance (in m) of the ionization chamber (451P) from the CBCT2 device in an anterior position, perpendicular to the x-axis ((point “0” − point C = 2 m), (point D − point G = 2 m)).y (in m): 451P (μGy): Scattered radiation measurements (μGy) were taken with the Inovision model 451P Victoreen ionization chamber (0.2–2.8 μGy). Red color means very high scattered radiation dose, while blue color means very low scattered radiation dose.Height (1 and 1.3 m): The measurement carried out at a height of 1 m from the floor represented the anatomical location of the gonads. The measurement carried out at a height of 1.3 m from the floor represented the anatomical location of the thyroid gland.CBCT: Cone-beam computed tomography device 3 (CBCT3); point “0”.x-axis defined the distance (in m) of the ionization chamber (451P) to the right and left of the CBCT3 device (point “0”), showing sometimes positive (right) and sometimes negative (left) values. In this color map, the absolute values are displayed in order to avoid confusion ((point H − point “0” = 1.2 m), (point “0” − point D = 1.4 m)).x (in m): y-axis defined the distance (in m) of the ionization chamber (451P) from the CBCT3 device in an anterior position, perpendicular to the x-axis ((point H − point J = 0.55 m), (point D − point G = 2 m)).y (in m): 451P (μGy): Scattered radiation measurements (μGy) were taken with the Inovision model 451P Victoreen ionization chamber (0–6.8μGy). Red color means very high scattered radiation dose, while blue color means very low scattered radiation dose.height (1.3 m): The measurement carried out at a height of 1.3 m from the floor represented the anatomical location of the thyroid gland.In this research study, measurements of the scattered radiation dose were collected inside rooms with CBCT installations. The spatial distribution of scattered radiation measured with ionization chamber (451P) was estimated through the rigging universal interpolation method and the safest locations of the people who could be present inside the X-ray room were determined .The protection against scattered radiation is a perennial concern of the scientific community, even when the radiation dose is quite low, such as during intraoral radiography ,22,23. AAlcaraz et al. (2006) measured the scattered radiation at various distances from the patient, who was lying supine, 48 cm from the floor. The measurements were carried out at distances of 60, 90, 120, 150, and 180 cm, and at an angle of 0°, 135°, and 180°. The results of this study in relation with dose reduction of intraoral dental radiography showed that the safest position for the dentist was behind and right of the X-ray beam at an angle of 135° . These fAn increase in the scattered radiation at a height of 100 cm from the floor, at the level of the X-ray gantry, and a decrease in the absorbed dose of radiation near the gantry, were observed by other researchers . Conversp > 0.05). In addition, due to technical difficulties, a limitation of the present study was that in room 3 (CBCT3) all measurements were made at the same height (1.3 m). The present study provides future prospects for further investigation of scattered radiation distribution with more CBCT devices for maxillofacial diagnostics.Yamaji et al. (2021) measured the distribution of scattered radiation by C-arm cone-beam computed tomography (CBCT) in the angiographic suite. In this study, the measurements showed the highest radiation dose over 600 μGy by a single CBCT image acquisition at a distance of 60 cm from the beam entry site and a height of 90 cm from the floor . In the In all CBCT devices that were tested in this study, the scattered radiation that an individual may be exposed significantly decreased with distance. The Newtom VGi CBCT showed the greatest decrease with distance. In all instances the measured scattered radiation was below 1 mGy, which is defined by the radiation protection guidelines as the exposure radiation limit of the general population. Nevertheless, the scattered radiation is significantly reduced, as long as the dentist, radiology technologist, or other occupationally exposed individual stands at a safe distance and position from the patient (as determined by our results) in the X-ray room during the CBCT examination for maxillofacial diagnostics.


We establish that ρN ≥ 1 ensures that every polyplex is enveloped by a liposome,thus avoiding coexisting oppositely charged species prone to aggregation.Hybrid core–shelllipid–polycation–nucleicacid nanoparticles (LPNPs) provide unique delivery strategies fornonviral gene therapeutics. Since LPNPs consist of multiple components,involving different pairwise interactions between them, they are challengingto characterize and understand. Here, we propose a method based onfluorescence cross-correlation spectroscopy to elucidate the associationbetween the three LPNP components. Through this lens, we demonstratethat cationic lipid shells (liposomes) do not displace polycationsor DNA from the polycation–DNA cores (polyplexes). Hence, polyplexesand liposomes must be oppositely charged to associate into LPNPs.Furthermore, we identify the liposome:polyplex number ratio ( These materialsreadily associate with the oppositely charged nucleic acids (NAs),forming nanoassemblies that can be therapeutically efficient in vivo.9 Notwithstanding this success,the efficacy of nonviral gene therapeutics remains limited, particularlyfor therapeutic targets beyond the liver.11Nonviral gene therapeutics to treat geneticand acquired diseaseshave become clinical reality through the introduction of an siRNA-basedtreatment for hereditary transthyretin-mediated amyloidosis19 which consist of a core composed by polycations complexed with NAs, enveloped by a lipidmembrane ,which may dictate alternative structures depending on their relativestrengths and method of assembly.23 Namely, cationicmembranes may interact more favorably with NAs than polycations, excludingthe latter from the assembly.22 This mayeasily be missed by most common characterization approaches, likedynamic light scattering (DLS) and electrophoretic mobility, whichprovide no direct evidence for formation of LPNPs comprising all threecomponents. Hence, to this date, a suitable predictive model for LPNPformation is still missing, making the assembly of LPNPs mostly empiric,and limiting their potential.Hybrid lipid–polycation–nucleic acid nanoparticles(LPNPs),membrane 1A,are p26 to quantify and elucidate the association betweenpolyplexes and liposomes to form LPNPs. With this information, a pictureof LPNP assembly is obtained, which could allow for improved efficiencygene therapeutics. We foresee that the methodology and insights shownhere will also impact the understanding and development of other self-assembledcomposite nanosystems, including the promising cell-membrane-coated(camouflaged) nanoparticles.28Here we propose an approach basedon dual-color fluorescence cross-correlationspectroscopy (FCCS)29) with DNA at polycation–amineto DNA–phosphate molar ratios (ρP:DNA) of 1, 1.5, and 3. ρP:DNA = 1 polyplexes are negatively overchargedwith DNA, showing negative ξPotential (29ρP:DNA = 1.5 polyplexes are close to the polylysine–DNA isoelectricpoint (ρP:DNA.iso = 1.6),showing a smaller negative ξPotential and virtually no free DNA or polylysine. Conversely, ρP:DNA = 3 polyplexes are cationic, coexistingwith excess polylysine. All the starting polyplexes and liposomeshave hydrodynamic diameters (DH) below140 nm .Parts B and C of ξPotential of the starting polyplex. For cationic polyplexes(ρP:DNA = 3), neither DH nor ξPotential are significantly affected when cationic liposomes are added. Conversely,when the starting polyplexes are anionic (ρP:DNA = 1 and 1.5), the LPNP charge switchesto cationic already at the lowest ρL:DNA additions, coinciding with the observation of strongaggregation (DH > 500 nm), especiallyfor ρP:DNA = 1. Furtheraddition of liposomes leads to stabilization. Clear solutions with DH < 120 nm are obtained for ρL:DNA = 5 (for ρP:DNA = 1) and ρL:DNA = 0.75 (for ρP:DNA = 1.5) LPNPs. Since ρP:DNA = 1 polyplexes are more anionic and coexist withfree DNA , more liposomes are neededto stabilize these LPNPs than the ρP:DNA = 1.5 ones. However, at first, the factor of ca.6 times larger ρL:DNA needed to stabilize ρP:DNA = 1 polyplexes seems high.In the second step, liposomes composed of 80 mol % (molar percentage)cationic lipid and varying amounts of PEG-lipid are added to formcore–shell LPNPs at various cationic lipid to DNA phosphatemolar ratios , the lackof observable changes with ρL:DNA hints at no formation of LPNPs, but also here one shouldconsider the possibility of cationic liposomes displacing some polycationsto form LPNPs. These alternative scenarios are difficult to discernwith DLS/electrophoretic mobility and even with TEM.Negative polyplexes aggregatinginitially and then redispersingfor increasing  results 1D also s results 1A, simil32 We labeled polylysine covalently with Atto-488 (green) and likewisethe cationic liposomes with Texas Red. In the ρP:DNA = 3 system, DNA is labeled with YOYO-1(green) instead of polylysine, to avoid the complication of havinglabeled non-complexed polylysine in solution. Single channel FCS auto-correlationcurves for the different polyplex and liposome starting formulationscan be found in Figure S1.FCCS canovercome this limitation by providing a quantitative measureof co-localization between two fluorescently labeled species .FG and FR, respectively). Thisis the case for the ρP:DNA = 3 system, in which polyplexes and cationic liposomes show unsynchronizedspikes in both channels (ρP:DNA = 1.5system (ρP:DNA = 1 case(ρP:DNA = 1 and 1.5) but not when the chargeof polyplexes and liposomes has the same sign (ρP:DNA = 3).In FCCS,the confocal volumes defined by the green and red lasersof a confocal microscope are fixed in space, while labeled speciesdiffuse through the overlapping green and red region 2A. If pochannels 2B. In co5system 2C,where= 1 case2D, the s26 -dependent part, M(τ),contains information on their dynamics:w0 and wz are the lateral and axialradii of the detection volume, respectively, and D is the diffusion coefficient. By fitting DH through D, which is used to determine LPNPsize . For a more comprehensive description of FCCS,readers are referred to the Supporting Information.For a quantitative analysis,the auto- and cross-correlation curvesof the time-dependent fluorescence signals can be examined26 2E–G.In GG and GR) describethe dynamicsof species carrying the green and red labels, respectively, the cross-correlationcurve (GX) results from the correlationbetween the two signals and therefore only contains information onthe species carrying both labels. Most importantly, their amplitudescontain information on the number of each labeled species, and onthe number of species containing both labels,24 according toAG and AR are the amplitudesof the green and red auto-correlation,respectively, and AX is the amplitudeof the cross-correlation. NPPf, NLf, and NLPNP arethe average number of free polyplexes, free liposomes, and LPNPs inthe confocal volumes, respectively. Notably, the ratio between AX and AR providesa direct measure of the fraction of polyplexes converted to LPNPs(fLPNP), according to24 In sections S2 and S3 of the Supporting Information we detail why a much more complex model based on a 1:n stoichiometry with n liposomes per LPNP to estimate fLPNP is generally not required.While the green and red auto-correlation curves (ρP:DNA =3) practically do not associate with cationic liposomes to form LPNPs(Inspectionof ρP:DNA = 1.5) show much larger AX values, comparableto both AG and AR, indicating strongco-localization (fLPNP (ρL:DNA is raised from 0.5 to 2 (fLPNP ≈ 1) in detriment of the possible competing structures depictedin ρL:DNA indicatesthat some LPNPsmay have more than one liposome attached, although this deviationis only significant at the largest ρL:DNA = 2, when many liposomes are added. We will returnto this point later.In contrast, systems containingmildlyanionic polyplexes , we can affirm that NPPf = 0. This allows us to directly determine NLPNP from AG–1  is not straightforwardto obtain with most techniques but is easily accessible using FCCS.The total number of polyplexes and liposomes in solution is obtainedthrough AG–1 and AR–1, respectively have a ρN ratio greater than 1. Conversely, thesamples in the marginally stable region (ρL:DNA = 0.5) show a ρN slightly below 1. This strongly hints that ρN ≥ 1 is a criticalrequirement for robust colloidal stability, suggesting that all polyplexesare coated by one liposome and converted to LPNPs. Conversely, ρN < 1 indicates thatsome polyplexes will be transformed into cationic LPNPs while someothers will remain anionic, ultimately leading to aggregation.ρN ≥ 1 requirement is a critical insight. The reason whywe and others have failed to notice ρN earlier likely results from its hitherto intangiblenature. ρN is difficultto estimate beforehand and to determine experimentally. Furthermore,it depends on sample preparation. Hence, ρN is not salient and tends to be overlooked.FCCS then plays a crucial role in revealing and highlighting ρN by measuring both thenumber of polyplexes and liposomes simultaneously.However intuitive, this ρN = 1is reached at ρL:DNA =0.63, for a 1:1 stoichiometry and fLPNP = 1, the ratio between the total number of liposomes and polyplexesis given by ρL:DNA/0.63.Hence, the number of unused liposomes can be estimated by section S3). Both expressions are plotted in ρL:DNA = 0.5, corroboratingour interpretationthat in this regime there are not enough liposomes to coat all polyplexes.For ρL:DNA, hinting that having many free liposomes results in ahigher probability of some LPNPs being enveloped by an additionallipid bilayer. For a 1:n polyplex:liposome stoichiometry,this can be modeled by modifying the eq S11) to include the term p is theprobabilityof a free liposome enveloping an existing LPNP. The mutual best fitfor both p =0.19 , and n is slightly greater than 1. Hence, even though these polyplexesare more charged, one liposome per polyplex is, on average, stillsufficient to envelop and convert them into LPNPs. The much larger ρL:DNA ratio needed to stabilize ρP:DNA = 1 polyplexes can then be explained,on one hand, by the initial excess free DNA coexisting with polyplexes,29 which requires additional liposomes to consumeit , the number of polyplexes is greater in the ρP:DNA = 1 system by a factor of ca. 3–5times , meaning that low-PEGliposomes associate more extensively with polyplexes (probably becausePEG acts as a steric barrier against adsorption1).The findings above are largely reproduced when the PEG% in liposomesis reduced from 10 to 5 and 0 mol % 3A. Most ble ones 3B, reinf28), providing insights into their assembly, stability, and structure–functionrelations.We postulate that the generalfeatures of LPNP assembly observedhere can be extended to other LPNP systems containing reasonably strongpolycations and cationic liposomes with monovalent ionizable/cationiclipid membranes of similar rigidity. The experimental approach detailedhere can also be extended to quantify the association between differentcomponents in diverse composite materials within and beyond gene therapeutics, asopposed to the more common charge ratio, is a critical parameter topredict stable LPNP formation. We find that ρN ≥ 1 is required to ensure thatevery polyplex is enveloped by a liposome, thus avoiding the coexistenceof oppositely charged species and inhibiting aggregation. Furthermore,we have observed significant differences in polyplex behavior, dependingon the ρP:DNA ratio.When further away from the isoelectric point, polyplexes are morenumerous and coexist with free DNA, requiring more liposomes to produceLPNPs. Such particles are likely to contain smaller amounts of DNAand coexist with lipoplexes, which should be taken into considerationin formulation design. These findings were largely reproduced whenvarying the degree of liposome PEGylation, which further strengthensour observations about the assembly and stability of LPNP systems.Finally, since the chosen LPNP components are common, these observationsand FCCS methodology should be applicable to many systems, includingother self-assembly composite materials for applications beyond genedelivery.In this work we have explored the associationof polylysine–DNApolyplex cores with cationic liposome shells to form LPNPs using FCCS.With this technique, we have shown that, for polyplex cores and liposomeshells to associate and form LPNPs, they must be oppositely charged.Importantly, we have also shown that the liposome:polyplex numberratio (sn-glycero-3-phosphocholine(DOPC), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) solubilizedin chloroform were purchased from Avanti Polar Lipids and used asreceived. Poly-l-lysine solution 0.1% (w/v) in water waspurchased from Sigma-Aldrich. GFP plasmid (pCMV-GFP), obtained throughAddgene, was a gift from Connie Cepko (Addgene plasmid #11153).33 The dye Atto 488-NHS was purchased from ATTO-TECGmbH. Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine,triethylammonium salt (Texas Red-labeled lipid), and the dye YOYO-1were purchased from ThermoFisher Scientific Inc. All the aqueous solutionswere prepared with DNase-free and RNase-free Milli-Q water.Thelipids 1,2-dioleoyl-3-trimethylammonium-propane(DOTAP), 1,2-dioleoyl-ξPotential) were performed with a SZ-100 device from Horiba, measuring at ascattering angle of 173°. Transmission electron microscopy (TEM)of the samples after negative staining with UranyLess (following themanufacturer’s instructions) was performed with a JEOL 2100200 kV TEM. FCCS was performed in a confocal microscope LSM780 fromZeiss following a previously described protocol.24 Each independent sample was measured with 40 repeats of5 s each, with the final auto- and cross-correlation curves beingaveraged out. Typically, five-to-seven independent samples were preparedand measured with FCCS for each composition. The excitation laserlines used were 488 nm for Atto 488 dye and 561 nm for Texas Red.The laser power was set so that the intensity in the “red”(561 nm) channel was slightly above that in the “green”(488 nm) channel, thus minimizing overestimation of cross-correlationdue to crosstalk.24 Besides minimizingcrosstalk experimentally, the data is further corrected for crosstalkaccording to the procedure described in Bacia et al.34 The confocal volumes were calibrated using fluorophoreswith a known diffusion coefficient, and the overlap volume was determinedby employing a mixture of single-labeled green (Atto 488) and red(Texas Red) liposomes and double-labeled liposomes with the same fluorescentlabels . A related calibration approach was described recently by Werneret al.35 FCCS auto- and cross-correlationcurves were fitted to a 3D normal diffusion model using QuickFit 3.0software and home-built Matlab scripts.The different nanostructuresobtained in this work were characterized by the following techniques:DLS and electrophoretic mobility (2 groups in the polymer). After stirring inaqueous solution at room temperature overnight, the labeled polymerwas obtained. The purification was performed by size exclusion chromatographyusing a Sephadex G25 stationary phase.Polylysinewas labeled by reacting the polymer with Atto 488-NHS (ratio 1:100Atto dye:NHρP:DNA ratios (ratio between amino groups inthe polymer to phosphate groups in the plasmid) of 1, 1.5, and 3 andincubating them at room temperature for 30 min in an orbital shaker.Polyplexes were prepared by mixingaqueous solutions of plasmidic DNA and polylysine at x:x  were prepared.First, the lipids were mixed in chloroform solution, followed by evaporationof the solvent, rehydration with water, and sonication with a tipsonicator at a final total lipid concentration of 4 mM .PEGylated and non-PEGylated cationic liposomes with a lipid molarcomposition of DOTAP:DOPC:DSPE-PEG of 80:20-ρL:DNA) in the range of 0.5–5.A final polylysine concentration of 7 μg/mL was fixed for allthe prepared samples. The obtained dispersions were characterizedby ξPotential and DLS.Finally, both components (polyplex andliposomes) were mixed atdifferent proportions, with a lipid amine to DNA phosphate ratio (ρP:DNA = 3) were also prepared with YOYO 1-labeled DNA and TexasRed-labeled liposomes.ForFCCS, the LPNPs were prepared following the same protocol butemploying labeled components. Atto 488-labeled polylysine was usedto prepare the polyplexes, while Texas Red-labeled phospholipid wasincluded in the liposomal formulation . Sampleswith cationic polyplexes (


The Holliday junction (HJ) is a DNA intermediate of homologous recombination, involved in many fundamental physiological processes. RuvB, an ATPase motor protein, drives branch migration of the Holliday junction with a mechanism that had yet to be elucidated. Here we report two cryo-EM structures of RuvB, providing a comprehensive understanding of HJ branch migration. RuvB assembles into a spiral staircase, ring-like hexamer, encircling dsDNA. Four protomers of RuvB contact the DNA backbone with a translocation step size of 2 nucleotides. The variation of nucleotide-binding states in RuvB supports a sequential model for ATP hydrolysis and nucleotide recycling, which occur at separate, singular positions. RuvB’s asymmetric assembly also explains the 6:4 stoichiometry between the RuvB/RuvA complex, which coordinates HJ migration in bacteria. Taken together, we provide a mechanistic understanding of HJ branch migration facilitated by RuvB, which may be universally shared by prokaryotic and eukaryotic organisms. During homologous recombination, RuvB drives branch migration of the Holliday junction to facilitate DNA repair. With our cryo-EM structure models we have provided a comprehensive mechanism for branch migration that may be universal for all species. After DNA damage is detected, a series of steps, including strand invasion and D-loop formation, occurs before a key intermediate structure of homologous recombination, the Holliday junction (HJ), is formed2. The HJ consists of two double-stranded DNA (dsDNA) helices that are separated into four hetero-duplex strands through a crossover point. In bacteria, RuvA, RuvB, and RuvC are involved in processing the HJ. RuvA was shown to assemble into a tetramer for recognizing the HJ core and recruits RuvB. As a motor protein, RuvB can bind the dsDNA part of the HJ and promote the movement of HJ, known as branch migration. Though earlier studies showed that the RuvA-RuvB complex, but not RuvB alone, can unwind dsDNA in the presence of ATP in vitro3, later studies revealed that RuvB drives branch migration by translocating along duplex DNA without unwinding dsDNA4. Eventually, the HJ is resolved back into two complete DNA strands by RuvC, an endonuclease. In eukaryotes, there are homologous proteins of the RuvAB complex, such as Rad54 in humans, to conduct HJ branch migration8. Therefore, fundamental mechanisms of HJ migration in bacteria may be universally shared by all organisms.Homologous recombination is an omnipresent cellular process that is critical not just for the maintenance of genomic stability after DNA damage but also for generating genetic diversity and has been associated with a variety of human diseases9. Domain III of RuvA was shown to directly interact with RuvB for the loading of RuvB onto HJ10. Crystal structures of RuvB from different bacteria showed that RuvB shares a conserved architecture that is composed of three domains: N-terminal domain (NTD), Middle domain (MD), and C-terminal domain (CTD)13. A feature β-hairpin in RuvB NTD was shown to interact with domain III of RuvA through hydrophobic interactions10.Earlier structural studies have provided rich information regarding interactions of RuvA with HJ DNA and RuvB. RuvA has been shown to consist of three distinct domains . A crystal structure of RuvA in complex with HJ has revealed that RuvA forms a unique tetrameric architecture, in which domains I and II are responsible for Holliday junction binding+ (ATPase associated with various cellular activities) family, which includes many members involved in numerous physiological processes14. All AAA+ proteins share a core domain with an αβα sandwich-fold, which contains two key motifs, the Walker A motif and the Walker B motif15. Walker A motif is composed of GXXXGK(T/S), where X stands for any amino acid, and the Walker B motif consists of hhhhD(D/E), where h represents any hydrophobic amino acids. The Walker A motif is responsible for binding nucleotides, and the Walker B motif coordinates Mg2+ ions and water molecules to catalyze the hydrolysis of nucleotides. Structural studies on classical members of AAA+ ATPase, including Cdc48, p97, ClpX, N-ethylmaleimide-sensitive-factor (NSF), and FtsK, showed that AAA+ proteins tend to form ring-like structures for function24. Similarly, earlier low-resolution electron microscopy showed that RuvB assembles into a hexamer on HJ DNA, forming a tripartite complex by flanking a RuvA octamer on both ends26. However, the low-resolution structure of RuvA-RuvB-HJ limits a mechanistic understanding of Holliday junction branch migration.RuvB belongs to the AAAStreptococcus thermophilus RuvB in complex with RuvA and DNA were published in Nature13. The asymmetric hexameric assembly of RuvB, around dsDNA, provides a mechanistic understanding of Holliday junction migration, in which ATP hydrolysis drives conformational changes of RuvB, leading to the pulling and revolving of DNA inside the central pore of RuvB by two nucleotides per step. Moreover, our structural analysis also revealed that ATP hydrolysis occurs at the top position of the spiral staircase most probably in a sequential manner. Additionally, the asymmetric assembly of RuvB also indicates a potential asymmetric engagement with RuvA and an association-and-detachment mechanism for the interactions between RuvA and RuvB during HJ branch migration. Together, we propose that HJ migration includes a series of highly coordinated events, including nucleotide cycling, RuvB conformational changes that trigger DNA pulling and revolving, as well as the association and detachment of RuvA to RuvB.Here, we present cryo-EM structures of RuvB in complex with dsDNA with resolutions up to 2.9 Å, showing that RuvB forms a spiral staircase to facilitate the HJ migration. During our manuscript preparation, similar structures of Thermus thermophilus RuvA and RuvB from Escherichia coli. Then, we incubated RuvA and RuvB with DNA substrates in the presence of 2 mM ATPγS to assemble the HJ complex. We also incubated RuvB with DNA substrates and 2 mM ATPγS to assemble the RuvB-DNA complex, which was further purified by gel filtration , protomers A, B, C, and D in the RuvB hexamer display large conformational changes, in which the CTDs rotate away from the NTDs  protomer, which are critical for ATP hydrolysis, are not positioned well for catalysis in the AF dimer but primed for catalysis in the CB dimer and the BA dimer , and revolution motors generally display a large central pore (diameter >3.0 nm)28. All protein translocases with structures available for analysis, including Cdc48, p97, and Clpx, display a small pore and have five subunits engaged with their substrates20. Our structure of RuvB hexamer clearly showed that RuvB forms a ring-like structure with a large central pore (3.4 nm), which has enough space for RuvB to revolve around the DNA duplex, and that RuvB has four protomers engaging DNA, contrasting those with small pores.In earlier studies, two models have been proposed to explain the molecular mechanisms of AAA+ ATPases tend to form asymmetric spiral staircases in the presence of substrates for function. Earlier AAA+ ATPase studies revealed that most AAA+ ATPases form symmetric oligomers32. At that time, most structures of AAA+ ATPase were determined in the absence of substrates due to technical challenges in obtaining crystals of AAA+ ATPases in complex with substrates. With the advancement of cryo-EM single particle analysis, more and more cryo-EM structures of AAA+ ATPase in complex with substrates were published, revealing that a spiral assembly of AAA+ ATPase hexameric rings is universally shared to facilitate the translocation of substrates through the pore. In our case, we also observed that RuvB assembles into a spiral staircase with dsDNA in the central pore. More interestingly, four protomers of RuvB hexamer engage with dsDNA, indicating a coupled mechanism of revolution and translocation by RuvB to facilitate Holliday junction migration. In addition, the dsDNA in our structure is longer than what was observed in the recently published HJ complex structure13, which allowed us to identify two DNA binding motifs in a RuvB protomer.It may be a universal mechanism that AAA+ ATPases for triggering the “hand-over-hand” movement of the arginine finger pore loops that facilitate the unidirectional movement of substrates. At least three models have been proposed for mechanisms of ATP hydrolysis by AAA+ ATPases, including a synchronized model, stochastic model, and sequential model35. Our structural study on RuvB, together with a previous structural study on S. thermophilus RuvB, came to a census that RuvB hydrolyzes ATP most probably in a sequential manner, in which ATP hydrolysis occurs at the uppermost position of the spiral staircase, while ATP is loaded at the lowest point of the spiral staircase. In the published manuscript on S. thermophilus RuvB, it was proposed that ADP release triggers ATP hydrolysis through a signaling mechanism across protomers of RuvB hexamer13. However, from our structural analysis of RuvB hexamer, we did not see strong evidence to support this cross-protomer communication. Instead, we tend to believe that ATP hydrolysis occurs at the top position of the spiral staircase mainly because the RuvB protomer at that point and its neighboring protomer in trans is better positioned for ATP hydrolysis than the other subunits. In addition, the relatively poor density of ATPγS in the nucleotide-binding pocket of protomer D suggested that ATPγS may have relatively low occupancy. Some particles may have ADP or are empty at the position of protomer D. Considering that protomer E is occupied by ADP while protomer C has an ATP in its pocket, we posit that the exchange of ADP to ATP takes place at the position of protomer D, consistent with what was proposed in a recent publication13.Furthermore, ATP binding, ATP hydrolysis, and ADP release are key events driving the conformational changes of AAA13. Domain III of RuvA, which is linked to the CTD RuvB winged helix DNA binding domain through a flexible linker, forms interactions with the feature β-hairpin of RuvB, which is a conformationally flexible motif in RuvB. These features enable a mechanism of association and detachment between RuvA and RuvB during the revolution of RuvB, similar to the working mechanism of the carbon brush and the motor in an electric generator. Thus, the concerted events of nucleotide cycling, RuvB conformational oscillation, DNA pulling, and rotation, as well as RuvA association and detachment, ensure a precise DNA migration by a step size of 2 nucleotides per ATP hydrolyzed.Lastly, RuvB is loaded onto HJ DNA by interacting with RuvA, which assembles as a tetramer to bind to the HJ core  expression vector with an N-terminal histidine tag, using the NdeI/Sall and NdeI/XhoI restriction sites for RuvB and RuvA, respectively, using the following primers: RuvA_F (GGAATTCCATATGATCC GCTACCTCCGGGGCCTGG), RuvA_R (CCGCTCGAGCTAGCGGAGGCGCTTTAGG GCCTCC), RuvB_F (GGAATTCCATATGGAAGACCTCGCCCTTAGGCCC), and RuvB_R (ACGCGTCGACTCACGGCTCCAAGAGGGGGCCCACC). RuvB and RuvA were recombinantly expressed using E. coli BL21(DE3) RipL strain. Bacterial cells were grown at 37 °C in LB supplemented with 50 µg mL−1 kanamycin until they reached an OD600 of about 0.6. Expression of RuvB or RuvA was induced by the addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and cultures were further incubated at 18 °C for 16 h. Cells were pelleted at 2000 × g for 20 min at 4 °C, resuspended in 20 mM Tris-HCl pH 7.5, 300 mM NaCl lysis buffer, supplemented with a complete mini protease inhibitor cocktail , disrupted by sonication, incubated in a water bath at 55 °C for 30 min, and the resulting lysate was centrifuged at 40,000 × g for 60 min at 4 °C. The supernatant was loaded onto a column containing Ni-NTA beads  that were pre-equilibrated with lysis buffer. The column was washed with 10 column volumes (CV) of 20 mM Tris-HCl pH 7.5, 300 mM NaCl, 30 mM imidazole wash buffer, followed by elution of RuvB using 5 CV of 20 mM Tris-HCl pH 7.5, 300 mM NaCl, 300 mM imidazole elution buffer, in 1 mL aliquots. Eluted protein was pooled, dialyzed against 20 mM Tris-HCl pH 7.5, 150 mM NaCl gel filtration buffer, and concentrated to 2 mL using an Amicon ®Ultra 30,000 NMWL centrifugal filter , before application to a Superdex 200 10/300 GL Increased column  to purify protein to homogeneity. RuvB was eluted with gel filtration buffer on an ÄKTA go FPLC (Cytvia). RuvB and RuvA were verified by SDS-Page gel, stained with Coomassie blue, and protein concentration was determined by nanodrop A280 .RuvB and RuvA from 25: HJ1 (AGAATCTGCCGAGAGACCGAGCAGAATTCTATGTGTTT ACCAAGCGCTG), HJ2 (CAGCGCTTGGTAAACACATAGAATTCTGCTCGGTCTGAGC CGTCTAAGA), HJ3 (TCTTAGACGGCTCACTGGCTGTGGGATCCGAGCTGTCTAGAG ACATCGA), HJ4 (TCGATGTCTCTAGACAGCTCGGATCCCACAGCCAGTCTCGGCA GATTCT). The four strands were mixed at equimolar ratios and were incubated at 95 °C for 10 min before allowing to cool to room temperature. The RuvB/DNA complex was reconstituted by mixing the purified RuvB with synthesized HJ DNA at 1:6 (DNA/RuvB monomer ratio) in 20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT, and 2 mM ATP-γ-S buffer for 30 min at 37 °C. The complex was purified from monomeric RuvB via gel filtration using 20 mM Tris-HCl, 150 mM NaCl buffer. Complex purity was determined by SDS-Page gel, and DNA presence was determined by agarose gel stained with ethidium bromide. Only the peak pertaining to RuvB/DNA complex was used in further testing.The HJ DNA substrate was prepared by annealing the four following synthetic DNA oligonucleotidesThe entire complex of RuvA-RuvB-HJ DNA complex was prepared in the same conditions as the RuvB-DNA complex, using a 6:4:1 (RuvB:RuvA:HJ DNA) molar ratio.−/Å2. All data processing, including patch motion correction and contrast transfer function (CTF) calculations, were performed in cryoSPARC (ver. 3.2)36.Quantifoil 1.2/1.3, 300 mesh copper grids (Quantifoil) were glow discharged at 0.2 atm for 30 s and were loaded onto a FEI vitrobot (Thermo Fisher). 3 µL of the sample was applied to the grid face before blotting for 4 s and plunge freezing in liquid ethane. Grids were stored in liquid nitrogen prior to screening on a 200 kV Glacios (Thermo Fisher Scientific) equipped with a K3 bioquantum detector . Grids were again stored in liquid nitrogen until data could be collected using a 300 kV Titan Krios (Thermo Fisher Scientific), also equipped with a K3 detector. 6870 movies were collected, using data acquisition software EPU (v2.12.1.2782REL), and TIA (v5.0 SP4) (Thermo Fisher Scientific), at a magnification of ×81,000 and a pixel size of 1.08 Å, at a total dose of 50 e37. Heterogenous refinement of an Ab-initio volume containing 305,449 particles followed by non-uniform refinement produced a final volume at 3.2 Å, using C2 symmetry. Additionally, a mask was generated over a map volume pertaining to a single hexamer of the 3.2 Å map in UCSF Chimera (ver. 1.15), which was used in conjunction with the local refinement of a heterogeneously refined volume containing 390,143 particles to yield a final volume at 2.97 Å using C1 symmetry. The two final volume maps were used for model building, post-processing, and interpretation using Phenix (ver. 1.20.1), UCSF Chimera (ver. 1.15), chimeraX (v1.4 and v1.5), coot (ver. 0.9.8.1), ISOLDE v1.5, Molprobity (unversioned), and PyMol (ver. 2.1)40. Coordinates of the RuvB dodecamer and RuvB hexamer atomic models, 8EFY and 8EFV, respectively, have been deposited to the Protein Data Bank, and their density maps, EMD-28107 and EMD-28101 have been deposited to the Electron Microscopy Data Bank.Approximately 500 particles were manually picked from 65 exposures for initial 2D classification, creating six 2D classes that were used to train the template picker job. After manual inspection of template picker picks, 6,874,881 particles were picked from 6723 micrographs that showed clear features of target particles. Two rounds of 2D classification were performed to remove poor-quality particles. Multi-class ab-Initio reconstructions were performed using 884,750 particles and yielded four volumes for subsequent 3D classification ranging from 3.51-6.59 Å, estimated by the “gold standard” Fourier shell correlation of 0.1438GH8, have been deposited to the Protein Data Bank, and the density map, EMD-40036, has been deposited to the Electron Microscopy Data Bank.Similar procedures were employed for the reconstruction of RuvA-HJ DNA. 3,383,678 particles were initially picked, and after 2D classification 130,426 particles were used for 3D reconstruction. The volume was refined with a C4 symmetry and a final map at 4.3 Å resolution was obtained. Coordinates of the RuvA-HJ DNA atomic model, Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Reporting Summary


The long-term prognosis of heart failure (HF) in Japan, a country with a long-life expectancy and an aging population, has not been fully elucidated in recent years. Most epidemiological studies about HF have focused on HF with reduced ejection fraction (HFrEF) (left ventricular ejection fraction [LVEF] <40%) or HF with preserved ejection fraction (HFpEF) (LVEF ≥50%), and a few have focused on patients with HF with mildly reduced ejection fraction (HFmrEF) (LVEF 40%-49%).The JROADHF (Japanese Registry Of Acute Decompensated Heart Failure) is a nationwide, multicenter, retrospective observational study of hospitalized patients with HF.P = 0.007). When divided into cardiovascular and noncardiovascular deaths, the age- and sex-adjusted mortality rates for HFrEF, HFmrEF, and HFpEF were 9.2, 7.8, and 6.6 per 100 person-years for cardiovascular death and 7.6, 8.3, and 9.3 per 100 person-years for noncardiovascular death, respectively. The risk of cardiovascular death was significantly higher in HFrEF than in HFpEF . Conversely, the risk of noncardiovascular death was significantly lower in HFrEF than in HFpEF   . Of the The GWTG (Get With The Guidelines) study,


Candida albicans strains are drug-resistant, emphasizing the urgent need to discover alternative therapies. In this study, the previously characterized Octominin was modified into a shorter peptide with an 18 amino acid sequence (1GWLIRGAIHAGKAIHGLI18) and named Octominin II. The secondary structure of Octominin II is a random coil with a helical turn and a positive charge (+2.46) with a hydrophobic ratio of 0.46. Octominin II inhibited C. albicans, C. auris, and C. glabrata with minimum inhibitory and fungicidal concentrations against C. albicans of 80 and 120 µg/mL, respectively. Field emission scanning electron microscopy confirmed that Octominin II treatment caused ultra-structural changes in C. albicans cells. Furthermore, membrane permeability results for the fluorescent indicator propidium iodide revealed modifications in cell wall integrity in Octominin II-treated C. albicans. Octominin II treatment increases the production of reactive oxygen species (ROS) in C. albicans. Gene expression studies revealed that Octominin II suppresses virulence genes of C. albicans such as CDR1, TUP1, AGE3, GSC1, SAP2, and SAP9. In addition, a nucleic acid binding assay revealed that Octominin II degraded genomic DNA and total RNA in a concentration-dependent manner. Additionally, Octominin II inhibited and eradicated C. albicans biofilm formation. Octominin II showed relatively less cytotoxicity on raw 264.7 cells (0–200 µg/mL) and hemolysis activity on murine erythrocytes (6.25–100 µg/mL). In vivo studies confirmed that Octominin II reduced the pathogenicity of C. albicans. Overall, the data suggests that Octominin II inhibits C. albicans by employing different modes of action and can be a promising candidate for controlling multidrug-resistant Candida infections.Most clinically isolated  Candida spp. are opportunistic pathogens in the commensal flora, and their infections have increased over past decades, causing a hassle to the healthcare system [Candida spp. cause superficial infections known as candidiasis and systemic infections known as candidemia [Candida family, Candida albicans has been identified as the prime pathogen. The pathogenicity of Candida is influenced by different factors, such as its ability to thrive under extreme and different environmental conditions, which are supported by virulence factors, including its ability to form biofilms on various anatomical surfaces and drug resistance [Candida infections will continue to increase [Candida has shown diverse mechanisms to escape the effects of antibiotics, such as drug target alteration, drug target overexpression, and efflux pump overexpression [e system ,2. Candindidemia . Among tsistance . Candidesistance ,4,5. Curincrease ,6,7. CanAntimicrobial peptides (AMPs) have recently gained attention owing to their potential use as antibiotics to mitigate antimicrobial resistance. AMPs are small proteins of ≤50 amino acids in length that can be found naturally in all living organisms, including mammals, amphibians, insects, microorganisms, aquatic organisms, plants, and also in cancerous cells. These peptides are called ‘host defense peptides’. AMPs mostly show cationic characteristics, wherein the net charge ranges from +2 to 11 with a higher proportion of hydrophobic residues than hydrophilic residues ,9. AMPs Candida albicans [Streptococcus parauberis [Acinetobacter baumanii [Although AMPs are readily found in natural sources, their use in therapeutics is not feasible. Researchers have focused more on synthetic peptides because they can be easily designed, modified, and synthesized in large quantities with high purity, and the activity of AMPs can be predetermined using bioinformatic tools coupled with machine learning ,13. Promalbicans , Streptorauberis , and Acibaumanii , as wellbaumanii .C. albicans. The overall results of this study confirmed that Octominin II could be a potential anticandidal agent for controlling Candida spp. effectively.In this study, we modified the original Octominin sequence into a shorter fragment (18 amino acids) and named it Octominin II. The antifungal activity of Octominin II was determined by evaluating the minimum inhibitory/fungicidal concentration (MIC/MFC), ultrastructural changes, membrane permeability, transcription, and antibiofilm activities. Moreover, the internal cellular targets were studied with respect to the DNA- and RNA-binding ability of Octominin II. Additionally, the toxicity/safety level of Octominin II was determined in vitro (RAW 264.7 cells) and in vivo (zebrafish embryos and larvae) and hemolysis activity (murine red blood cells). Finally, an in vivo study was performed to investigate the therapeutic efficacy of Octominin II in adult zebrafish challenged with Octominin II was derived from a previously characterized Octominin used as a template sequence by deleting 5 AAs from its C-terminus. The amino acid sequences of Octominin and Octominin II are indicated in C. albicans, C. auris, and C. glabrata. Octominin II was found to exert species-dependent antifungal activity, based on the different MIC and MFC values obtained for each species. The following were the MIC and MFC for C. auris , C. albicans , and C. glabrata . In addition, fluconazole (positive control)-treated C. albicans showed the highest MIC (280 µg/mL). Among them, C. albicans was used for further experiments since it is the most prominent Candida spp. with a global public health priority. Time-kill kinetics assay was used to determine the growth inhibitory profile of Octominin II at different concentrations . The c20 µg/mL A,B. To ihe media C. At pH C. albicans  were observed using field-emission scanning electron microscopy (FE-SEM) to identify possible ultrastructural changes .Propidium Iodide (PI)/Fluorescein Diacetate (FDA) assay was performed to identify live and dead cells after Octominin II treatment via fluorescence-based staining. Red fluorescence indicates dead cells by staining the nuclei, and green fluorescence is emitted in FDA in viable cells via metabolism to fluorescein. The results of the PI/FDA analysis showed that cells treated with 120 µg/mL (MFC) and 80 µg/mL (MIC) Octominin II had the highest levels of red fluorescence signals when compared to the negative control . This in2−1, hydroxyl radical OH−1, and hydrogen peroxide (H2O2), which damage DNA and RNA, elevating oxidative stress in the cell. ROS levels were measured to determine whether the inhibitory activity of Octominin II was related to endogenous oxidative stress caused by ROS production. C. albicans treated with 80 µg/mL (MIC) and 120 µg/mL (MFC) of Octominin II emitted a higher level of green fluorescence than the positive control , whereas the negative control exhibited less green fluorescence  include highly reactive superoxide, such as superoxide Orescence . This suC. albicans. The DNA mobility pattern was observed by agarose gel electrophoresis, as shown in C. albicans cells, which may influence the molecular-level survival mechanisms, leading to cell death.DNA and RNA mobility shift assays were performed to investigate whether Octominin II is involved in DNA/RNA binding in C. albicans biofilms was assessed using confocal laser scanning microscopy to observe the 3D structure of the C. albicans biofilms after Octominin II treatment  and the positive control, fluconazole 280 µg/mL (3/µm3) and thickness when compared to Octominin II treated samples, as indicated in the graphs , AGE3 , and TUP1  were also downregulated. Secreted aspartic proteases (Saps) SAP2  and SAP9  genes play a key role in nutrient acquisition and virulence by promoting biofilm growth. In summary, all virulence-related genes that were tested were suppressed in 0.5 MIC (40 µg/mL) and MIC of Octominin II (80 µg/mL).qRT-PCR was performed to evaluate the transcriptional response of the genes related to virulence of albicans . CDR1 wa50 is 341.45 µg/mL (In vivo cytotoxicity was assessed using RAW 264.7 cells treated with varying concentrations (0–200 µg/mL) of Octominin II. The results revealed that Octominin II caused very low cytotoxicity in RAW 264.7 cells, wherein the calculated IC45 µg/mL A.To evaluate the hemolytic activity of Octominin II, murine red blood cells were treated with varying concentrations of Octominin II (6.25–100 µg/mL). Octominin II showed very low hemolytic activity when compared to that of the positive control  B.50 values are 52.72 and 73.56 µg/mL for zebrafish embryo and larvae, respectively.In vivo toxicity of Octominin II was measured by treating zebrafish embryos (4 hpf) and larvae (72 hpf) at different concentrations (0–20 µg/mL) of Octominin II and observed at 24 h intervals. Octominin II exhibits concentration-dependent toxicity in both zebrafish embryos and larvae. Considering the mortality and survival patterns, the highest concentration (20 µg/mL) showed high mortality during the observed period (96 h). C,D. CalcC. albicans-infected zebrafish dorsal muscle tissues were investigated using PASH staining  [Nicotiana alata [Pisum sativum [Nerita versicolor [Acanthoscurria gomesiana [patients . The majal drugs . Therefoal drugs . In our oferrin) , plant-bna alata , Psd1 fr sativum , marine rsicolor , and theomesiana , have beC. albicans has a cell wall composed of a β-glucan-chitin skeleton [C. albicans.When physiochemical parameters were considered, Octominin was found to be a cationic peptide with high hydrophobicity. Cationic peptides initiate peptide–membrane binding and lyse cells via electrostatic interactions with negatively charged bacterial membranes . C. albiskeleton ,32, whicskeleton . Octominskeleton . This suC. albicans. Pores and deformities were observed in treated C. albicans cells, but not in untreated cells. Similar cell wall damage has been reported in other AMP-treated C. albicans [C. albicans is a vital factor in host–fungus interactions leading to infection, and such defects in the cell wall can adversely affect the growth and development of Candida spp. [Candida, which can eventually lead to growth inhibition and cell death. Based on the results of PI uptake, it was evident that Octominin II altered the membrane integrity of C. albicans. PI is impermeable to live cells and only penetrates dead cells. However, it is permeable through damaged cell membranes, can bind to DNA or RNA in the cell, and emits a red fluorescence signal [C. albicans.SEM confirmed that Octominin II treatment caused ultrastructural changes on the cell surface of albicans ,36. The ida spp. . Morphole signal . Since hC. albicans, and ROS accumulation was associated with its inhibitory activity. With ROS production, mitochondria can also be negatively affected and cellular ATP production can be halted, which may indirectly trigger an inhibitory effect in C. albicans [ROS production can be induced with antimicrobial agents, which leads to cellular stress. Numerous studies have shown peptide-mediated ROS generation connected to fungicidal activity ,40,41. Oalbicans .The DNA-binding ability of Octominin II was evaluated by observing the retardation of DNA movement after treatment with Octominin II. Positively charged peptides and negatively charged DNA can be attracted, which may lead to the accumulation of peptides in cells, wherein DNA and RNA may be the intracellular targets of peptides . PreviouC. albicans. CDR1 is a prominent multidrug transporter, and its high expression levels render C. albicans resistant to drugs via ATP-binding cassette transporters. To block the activity of this gene, a theoretical approach is to reduce the intracellular ATP levels, which has not yet been clinically practiced [C. albicans by promoting ATP efflux, whereas AMP treatment lowers CDR1 and CDR2 expression. Tup1 is a repressor of the yeast-to-filamentous transition process, supporting hyphae formation [ALS3 and ECE1 [AGE3 is also associated with drug resistance; fluconazole-resistant clinical C. albicans isolates acquire fluconazole sensitivity after deleting the AGE3 gene [GSC1 is associated with echinocandin resistance and is one of the available drug groups for the treatment of C. albicans infections in humans [C. albicans and are one of the major hydrolytic enzymes secreted by C. albicans [SAP2 and SAP9 are known to degrade host proteins, promote host tissue invasion, and confer higher virulence in C. albicans [To investigate the effects of Octominin II at the molecular level, we examined the translational responses of selected genes from racticed . Tanida racticed  reportedormation . It was and ECE1 . AGE3 isGE3 gene . GSC1 isn humans ,53. Secralbicans . SAP2 analbicans . InteresC. albicans is considered as an essential factor in its pathogenicity and facilitates drug resistance. C. albicans forms complex biofilm structures that exacerbate health-associated complications, such as fatal candidemia, and escalate health-related costs due to additional medications and device renewal [Candida spp.Biofilm formation by  renewal ,56. OctoCandida spp. Patients with acquired or inherited neutrophil defects are more likely to develop invasive candidiasis [Candida infection by studying protein defects such as CAARD9 deficiency, a rare autosomal recessive primary immunodeficiency disorder [C. albicans was altered using cold atmospheric plasma, and the results revealed reduced neutrophil migration and inflammation in the vulvovaginal tissues of female mice [C. albicans when compared to that of the C. albicans-challenged group incorporated with Octominin II. Transcriptional analysis also suggests that the virulence and pathogenicity of C. albicans can be suppressed by Octominin II, which may lead to defects in the invasion and infection of C. albicans into the host tissue.Neutrophils are the first line of innate immune defense against several fungal pathogens, including didiasis . Severaldisorder . The virale mice . In our Candida was evaluated, and Octominin II was found to inhibit C. albicans via different mechanisms. Octominin II damages and alters the integrity of yeast cells, thereby increasing ROS production. In addition, it damages DNA and RNA, which may lead to defects in transcriptional regulation and affect the survival mechanisms of C. albicans. Molecular analysis of gene expression revealed that Octominin II suppresses the virulence and pathogenicity of C. albicans. Taken together, Octominin II has the potential to be used against C. albicans to mitigate the burden of Candida infections in the future.In this study, the ability of Octominin II to inhibit http://osddlinux.osdd.net/raghava/pepstrmod/index.php (accessed on 20 May 2023) and visualized using the BIOVIA Discovery Studio Visualizer . The physicochemical properties of Octominin II were determined using the Peptide R package version 2.4.5 [http://lbqp.unb.br/NetWheels/ (accessed on 20 May 2023). Octominin II was synthesized using a solid-phase peptide synthesis method  and purified by reverse-phase high-performance liquid chromatography. The peptide was dissolved in nuclease-free water (1 mg/mL) and stored at −20 °C.Octominin II (18 AAs) was designed by removing five amino acids from the C-terminus of the Octominin sequence. A three-dimensional image was generated using PEPstrMOD, choosing the cluster prediction option  were added to a 96-well plate and treated with Octominin II (0–250 µg/mL) in total volume of 200 µL. The plates were incubated at 37 °C for 24 h, and the optical density was measured at 590 nm. The MIC was determined as the Octominin II concentration at which zero visual fungal growth  was observed. The MBC was determined using the agar plating method by plating Octominin II-treated C. albicans (≥MIC). Time-kill kinetics were determined by measuring the optical density of Octominin II-treated C. albicans (1 × 106 CFU/mL) at 3 h intervals.Initially, the antimicrobial activity of Octominin II was screened against three us study . C. albiC. albicans was prepared, and 10% inoculum was added to fresh PDB. Cells were allowed to grow until they reached a density of 1 × 106 CFU/mL. After that, the cell suspension was added to a 96-well plate and treated with varying concentrations  of Octominin II and incubated at 37 °C for 24 h. The percentage inhibition was calculated and compared with the negative control.The pH-dependent activity of Octominin II was also tested. A pH gradient was prepared from pH values of 3, 4, 5, 5.5, and 7 using PDA. An overnight culture of C. albicans. Octominin II treatments were performed at MIC (80 µg/mL), MFC (120 µg/mL), and fluconazole (280 µg/mL) with C. albicans (1 × 106 CFU/mL) and incubated at 37 °C for 10 h. Fungal cells were collected by centrifugation at 1500× g for 10 min. The cell pellets were washed twice with 1× PBS, suspended in 1× PBS, and pre-fixed with 2.5% glutaraldehyde for 30 min. Then, the pre-fixed cells were obtained by centrifuging again at 1500× g for 10 min, washed with 1× PBS, and then dehydrated using serial ethanol concentrations . The fixed cells were dried and coated with platinum by ion sputtering . Finally, the cells were observed by field-emission scanning electron microscopy .FE-SEM analysis was performed to determine the morphological and structural changes in Octominin II-treated C. albicans (1 × 106 CFU/mL) was treated with Octominin II with the MIC (80 µg/mL) and MFC (120 µg/mL) as the positive control . Samples were incubated at 37 °C for 10 h, and then the cells were collected by centrifugation at 1500× g for 10 min. The cell pellet was washed with 1× PBS and resuspended in 1 mL PBS. For the PI uptake assay, 50 µg/mL of PI  and 40 µg/mL FDA  were added and incubated for 30 min at room temperature (28 °C) in the dark. Then, the excess stain was removed using 1× PBS, and cells were resuspended in 1× PBS (50–100 µL), and cell suspension (5 µL) was added onto a glass slide and observed under confocal laser scanning microscopy (CLSM). Red fluorescence was measured at excitation and emission wavelengths of 535 and 617 nm, respectively, and green fluorescence was measured at excitation and emission wavelengths of 488 and 535 nm, respectively.A PI uptake assay with fluorescein diacetate (FDA) staining was performed to identify any effect of Octominin II on membrane permeability or cell membrane integrity. Propidium iodide (PI)/FDA staining was performed according to the method described by Jayathilaka et al., 2021 [C. albicans was assessed using the 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) assay. After treatment and collection of C. albicans as described above, C. albicans cells were washed twice with 1× PBS and resuspended in 1 mL of 1× PBS. H2DCFDA staining was performed by adding 50 µg/mL H2DCFDA  to the cell suspension, followed by incubation for 30 min at room temperature in the dark. Excess H2DCFDA was washed with 1× PBS and resuspended in 50–100 µL of 1× PBS, and 5 µL of the cell suspension was added to a glass slide and observed under confocal laser scanning microscopy (CLSM). Excitation and emission wavelengths for green fluorescence were 488 and 535 nm, respectively. For H2DCFDA staining to determine ROS generation, 50 µg/mL H2DCFDA  was added and incubated for 30 min at room temperature in the dark. Excess H2DCF-DA was washed with 1× PBS and resuspended in 50–100 µL of 1× PBS, and 5 µL of the cell suspension was added to a glass slide and observed under confocal laser scanning microscopy (CLSM). Excitation and emission wavelengths for green fluorescence were 488 and 535 nm, respectively.ROS production in C. albicans. First, the genomic DNA of C. albicans was isolated by the method described by [® RNA isolation kit according to the manufacturer’s protocol . Isolated genomic DNA and RNA were treated with different weight ratios of Octominin II  in a 20 µL reaction mixture and incubated at 37 °C for 30 min. Then, the total volume of the DNA or RNA with Octominin II was loaded onto 0.8% agarose gel. The RNA Octominin II mixture was loaded onto 1% gel and electrophoresed using an electrophoresis system . The gel was visualized using a gel imaging system .DNA and RNA mobility shift assays were performed to determine whether Octominin II binds to the nucleic acids of ribed by , and RNAC. albicans were analyzed following Octominin II treatment  was treated with Octominin II with 0.5 MIC and MIC concentrations with the positive control (fluconazole 280 µg/mL) and incubated at 37 °C for 3 h. Then, the cell pellet was obtained by centrifugation at 1500× g for 10 min at 4 °C. PBS washed cell pellet was used to isolate total RNA by NucleoSpin® RNA isolation kit, according to the manufacturer’s protocol . RNA concentration and purity were determined using a NanoDrop spectrophotometer , and cDNA was synthesized using the Prime Script 1st strand cDNA Synthesis Kit , according to the manufacturer’s protocols. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the Thermal Cycler Dice Real Time System III  with the total reaction mixture (10 µL), which included cDNA template (3 µL), 1 µL of each forward and reverse primers (10 µM each), and 5 µL of TB green Premix Ex Taq II . The three-step PCR was performed with a single dissociation step. The relative mRNA expression fold values were calculated following the 2−ΔΔCT method, wherein the reference control gene was Actin 1 [The gene expression profiles of the selected genes of reatment . C. albi Actin 1 .C. albicans biofilms were prepared for CLSM, according to a previously described method with minor modifications [C. albicans was grown to 106 CFU/mL in PDB, and desired amounts of C. albicans culture and Octominin II were added to Falcon® 96-well Black/Clear Flat Bottom plates  up to a total volume of 100 µL and incubated at 37 °C for 24 h for biofilm formation. The biofilms were carefully washed twice with 1× PBS to remove planktonic cells. The biofilms were stained with 10 mM SYTO9  and kept in the dark for 30 min. Subsequently, the remaining dye was washed off and observed using a CLSM with Z-stacking . Plates were visualized at a wavelength of 488 nm. Quantification of the formed biofilms was performed using COMSTAT software [3/µm2) and the biofilm thickness were quantified.ications . For thesoftware , where tC. albicans culture with the desired amount of Octominin II was added to a 96-well plate until a final volume of 100 µL and incubated at 37 °C for 24 h. For biofilm eradication assay, 100 µL of C. albicans culture was added to the 96-well plate and incubated at 37 °C for 48 h. The biofilm formed was washed twice with 1× PBS after removing the medium. Next, formed biofilms were treated with 50, 80, 100, and 120 µg/mL of Octominin II, and fluconazole (280 µg/mL) and new media were added until a final volume of 100 µL and incubated at 37 °C for 24 h.For the biofilm inhibition assay, C. albicans biofilms after Octominin II treatment. First, the remaining culture medium was carefully removed, and the biofilm-containing wells were washed twice with 1× PBS to remove non-attached cells without disturbing the remaining biofilm. Then, crystal violet (0.1%) was added to each well and incubated for 30 min at room temperature. After that, the plate was washed with distilled water, and then 100 µL of ethanol was added to each well and kept for 10 min. Finally, the plate was read at 595 nm using a microplate reader.A crystal violet assay was performed to measure the remaining biomass of The biofilm formation inhibition percentage was calculated using the following equation:The remaining biofilm upon Octominin II treatment was calculated using the following equation:v/v) fetal bovine serum  with an antibiotic–antimycotic solution , and incubated at 37 °C in a humidified atmosphere at 5% CO2 for 24 h. The cells were then seeded into a 96-well flat bottom microtiter plate at a density of 1 × 104 cells per well (100 µL) and allowed to adhere by incubating at 37 °C for 24 h. After that, the medium was aspirated, and the cells were washed with sterile 1× PBS. Cells were treated with Octominin II (0–200 µg/mL), and untreated cells were used as negative controls. After incubation, cell viability was measured using the EZ-Cytox cell viability assay kit , according to the manufacturer’s protocol. The hemolytic activity of Octominin II was assessed using murine red blood cells (RBC), according to the method described by Jayathilaka et al. (2021), with minor modifications [The in vitro cytotoxicity of Octominin II was determined in murine macrophages (RAW 264.7 cells). Cells were cultured in Dulbecco’s modified Eagle’s medium , containing 10% (ications . BrieflyDanio rerio) embryos and larvae. Zebrafish embryos (4 hpf) and larvae (72 hpf) were placed in 96-well flat bottom microtiter plates, with one embryo/larva per well (n = 10). Embryos or larvae were treated with Octominin II (0–25 µg/mL) at 28 °C for 96 h, and mortality was determined.In vivo toxicity of Octominin II was assessed in zebrafish  negative control (PBS), (2) C. albicans challenge , and (3) C. albicans challenge with Octominin II treatment . Zebrafish (0.4 g body weight) were anesthetized using 160 µg/mL buffered tricaine , and Octominin II treatments were injected into the dorsal muscle of the zebrafish and kept in 28 °C. The fish were observed for mortality and health abnormalities. Muscle samples were collected 48 and 96 h after the challenge/treatment (n = 3 from each group). The collected tissues were subjected to periodic acid-Schiff hematoxylin (PASH) staining for histopathological observation according to the method described by Kulatunga et al. [An in vivo experiment was conducted according to the method described by Kulatunga et al. (2019) with minor modifications . C. albia et al. .t-test was used for the transcriptional analysis with significance levels of * p < 0.05, ** p < 0.01, and *** p < 0.001.All experiments were statistically analyzed using GraphPad Prism software version 8 . All in vitro and in vivo assays were performed in triplicate and analyzed using the one-way ANOVA test, while an unpaired C. albicans growth, and a positive correlation was found between the MIC and MFC of 80 and 120 µg/mL, respectively. Our results revealed that Octominin II exerts multiple modes of action to inhibit C. albicans such as physical cell membrane damage, increased plasma membrane permeability, induction of high levels of ROS, and binding with intracellular DNA and RNA. Moreover, Octominin II showed good antibiofilm activity by successfully inhibiting and eradicating C. albicans-derived biofilms. Calculated IC50 was 341.45 µg/mL (RAW 264.7 cells), and LD50 was 52.72 µg/mL (zebrafish embryos) and 73.56 µg/mL (larvae). Overall, Octominin II could be a potential therapeutic agent against the multidrug-resistant Candida species; however, further experiments are required to confirm the clinical application of Octominin II.In conclusion, Octominin II inhibited


Fructose-1,6-bisphosphatase (FBPase) deficiency, caused by an FBP1 mutation, is an autosomal recessive disorder characterized by hypoglycemic lactic acidosis. Due to the rarity of FBPase deficiency, the mechanism by which the mutations cause enzyme activity loss still remains unclear. Here we identify compound heterozygous missense mutations of FBP1, c.491G>A (p.G164D) and c.581T>C (p.F194S), in an adult patient with hypoglycemic lactic acidosis. The G164D and F194S FBP1 mutants exhibit decreased FBP1 protein expression and a loss of FBPase enzyme activity. The biochemical phenotypes of all previously reported FBP1 missense mutations in addition to G164D and F194S are classified into three functional categories. Type 1 mutations are located at pivotal residues in enzyme activity motifs and have no effects on protein expression. Type 2 mutations structurally cluster around the substrate binding pocket and are associated with decreased protein expression due to protein misfolding. Type 3 mutations are likely nonpathogenic. These findings demonstrate a key role of protein misfolding in mediating the pathogenesis of FBPase deficiency, particularly for Type 2 mutations. This study provides important insights that certain patients with Type 2 mutations may respond to chaperone molecules. Genotype-biochemical phenotype correlation suggests protein misfolding plays a key role in pathogenesis of fructose-1,6-bisphosphatase deficiency for one functional class of missense mutations reported in the associated enzyme. Baker and Winegrad first reported FBPase deficiency in 19702, and Kikawa et al. subsequently identified three FBP gene (FBP1) mutations associated with FBPase deficiency in 19973. FBPase deficiency is an autosomal recessive disorder that generally occurs concurrently with hypoglycemia and metabolic acidosis4. Although fasting and febrile infections are known to trigger life-threatening episodes of hypoglycemia and lactic acidosis in infancy, these episodes rarely occur in adults due to increased glycogen storage4. Several harmful mutations in the coding region of FBP1 have been reported5. Nevertheless, with incidence rates estimated to range between 1/350,000 and 1/900,000, FBPase deficiency remains a very rare inherited disease6. Due to its low incidence, elucidating the molecular mechanism by which the identified mutations cause the loss of enzyme activity is still challenging, particularly in terms of genotype-phenotype correlations and in adult-onset cases. Here, we present the case of a 22-year-old female patient who was diagnosed with FBPase deficiency and exhibited a compound heterozygous mutation of the FBP1 gene. Our results demonstrate that the underlying mechanism by which the identified mutations cause FBPase deficiency involves protein misfolding, and we examined and categorized the biochemical phenotypes of all previously reported FBP1 missense mutations into three functional groups.Fructose-1,6-bisphosphatase (FBPase) is a key regulatory enzyme in gluconeogenesis that catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate2 19 mmHg, HCO3− 2.5 mmol/L). The patient reported experiencing similar episodes triggered by fasting or febrile illness during her early childhood , high levels of lactate (110 mg/dL) and severe metabolic acidosis , mutant clones (G164D or F194S), or cotransfects (G164D and F194S) were transfected into FBP1-KO HepG2 cells. NADP-coupled spectrophotometric assays were used to examine the enzymatic activity of mutant FBPase. As a result, the FBPase activities of all mutants  were significantly lower than that of the WT clone (4.82 ± 1.61 mmol/min/mg protein)  Fig. . We valiin) Fig. .Fig. 2BiFBP1 gene product. We found that the mutant FBP1 with G164D or F194S aggregated in the cytoplasm, whereas the FBP1 protein of the WT clone was diffusely localized in the cytoplasm  analysis for the proteins pulled down by immunoprecipitation with an anti-Myc tag. In total, 408 proteins were detected by LC‒MS/MS analysis for the WT, G164D mutant, and F194S mutant Fig. . Next, whed Fig. . Importahed Fig. , suggest11. According to previous studies, the protein expression of FBP1 is regulated by the ubiquitin proteasome and autophagy pathways12. Thus, to address the involvement of ERAD or protein degradation pathways in FBP1 mutant protein expression, we examined the effects of various inhibitors, such as a proteasome inhibitor (MG-132), an autophagy inhibitor (3-methyladenine), and a potent inhibitor of ER mannosidase I (kifunensine), on the protein expression levels of the G164D and F194S FBP1 mutants. Immunoblot analysis revealed that MG-132 and 3-methyladenine had no impact on the protein expression of these mutants . These unfolded proteins undergo mannose trimming by ER-associated mannosidase and degradation by the proteasome , which can be suppressed via the inhibition of ER-associated mannosidase activity using kifunensine13. Moreover, missense mutations were shown to shift the folding equilibrium toward a partially folded state, thus increasing the cellular fraction of HSPs relative to the WT protein14. Based on these findings, we evaluated the binding of HSP70, HSP90, HSP60, and TCP1 to the FBP1 proteins using anti-Myc immunoprecipitation with subsequent immunoblot analysis  are a family of molecular chaperones that collectively form a network that is critical for protein foldingsis Fig. . The G16sis Fig. , indicatFBP1 mutations  have been reported5. The reported missense mutations are D119N7, P120L5, R158W6, G164S3, A177D3, F194S8, G207R5, N213K15, G260R16, E281K17, P284R8, G294E18, G294V15, and V325A3. We examined the relationship between the mutated site and the substitution of hydrophobicity in these 14 types of FBP1 missense mutations and the G164D mutation we newly identified in this patient  in addition to patient’s mutant clones (G164D or F194S). These FBP1 cDNA constructs were transfected into FBP1-KO HepG2 cells with subsequent in vitro functional analysis. Then, these clones were separated into two groups: six mutants without a change in hydrophobicity (indicated in blue) and nine mutants with a change in hydrophobicity (indicated in red)  Fig. .At the protein level, there were several exceptions, but FBP1 expression tended to be different depending on the substitution of hydrophobicity. The mutations without changes in hydrophobicity (indicated in blue), except G294V, maintained a similar level of protein expression as WT FBP1, whereas the mutations with changes in hydrophobicity (indicated in red), except G207R, exhibited decreased protein expression Fig. . ConsistFBP1 missense mutations into three functional groups  are likely to be located outside of the important amino acid residues in the functional motif Fig.  and appeRegarding G294V, the hydropathy index shows certain amount of change between glycine (−0.4) and valine (4.2), even though both glycine and valine are hydrophobic amino acids Table .3.Type 3 mutations (G207R and V325A) were structurally distant from sites associated with the enzyme activity motif and substrate binding pocket Fig. . These mAs expected, when we examined the binding ability of all mutants to HSP, the interaction with HSP70 and HSP90 was increased to a greater extent for only Type 2 mutants compared with that seen for WT FBP1 and Type 1 and Type 3 mutants Fig. . Further19. Our patient’s case and the observations in the present study illustrate how FBPase deficiency can be confused with disorders of fatty acid oxidation. While acylcarnitine profiles are useful for the diagnosis of the latter, we advocate that fructose tolerance tests and genetic analyses would be beneficial and provide informative confirmation for the diagnosis of patients with FBPase deficiency.FBPase deficiency is sometimes misdiagnosed or diagnosed in delay following initial suspicion of other energy-related deficiencies. Early diagnosis is very important in FBPase deficiencyIn general, fructose loads can lead to large, rapid expansions in the hexose- and triose-phosphate pools, potentially providing increased substrates for all central carbon metabolic pathways, including glycolysis, glycogenesis, gluconeogenesis, lipogenesis, and oxidative phosphorylation. In FBPase deficiency, a marked decrease in intracellular free phosphate due to hepatic accumulation of fructose 1,6-bisphosphate can inhibit glycogenolysis, leading to fructose-induced hypoglycemia.5. Although the FBP1 c.581T > C (which results in the missense mutation F194S) and c.490 G > A (which affects the neighboring nucleotide) mutations have been previously demonstrated8, the c.491 G > A mutation (responsible for the missense mutation G164D) identified in this study is novel to the best of our knowledge. The G164D and F194S mutations, which are located in the β-strand . HSPs are a family of molecular chaperones that collectively form a network that is critical for protein folding13. It has been suggested that HSP70 recognizes unfolded proteins, and HSP90 recognizes partially folded proteins. However, fully folded proteins do not bind with either HSP70 or HSP9014. In addition, missense mutations have been shown to shift the protein folding equilibrium toward a partially folded state, thus increasing the cellular fraction of HSP70 and HSP90 relative to WT proteins14. Some proteins can only be partially folded by HSP70 and therefore require additional assistance from HSP60 (chaperonin) to acquire a folded functional conformation13. Aberrant protein aggregation was found to be controlled by chaperonins containing TCP-1. We found that the G164D and F194S FBP1 variants increase the interactions between FBP1 and HSP70, HSP90, HSP60, and TCP1. In addition, the FBP1 proteins are partially aggregated and trapped in the ER. Thus, we conclude that the decrease in protein expression detected in FBPase deficiency is, at least in part, a result of protein misfolding.The interactome analysis based on liquid chromatography-tandem MS data for binding partners demonstrated that FBP1, particularly in its mutant forms, interacts with molecular chaperone proteins related to the unfolded protein response, including HSPs. Protein misfolding has been recognized as an important pathophysiological cause of protein deficiency in some genetic disorders, such as Fabry disease, Pompe disease, and Gaucher disease5. This variant has been previously reported in the heterozygous state with an allele frequency of 0.0001498 in 10 European (non-Finnish) individuals5, although its pathological effects have not yet been elucidated. In contrast, the V325A mutation is not recorded in the dbSNP database. Although this mutation was observed in patients with FBPase deficiency, it was found to harbor the same mutant alleles as G164S. Based on an examination of the chimeric V325A mutation, Kikawa et al. suggested that this mutation does not play a pathogenic role in FBPase deficiency3, which is consistent with the findings of our study. Thus, G207R and V325A were defined as Type 3 mutations.Finally, 15 FBP1 missense mutations Figs. , and 5 wThis finding is consistent with clinical characteristics, but further investigation is needed to uncover the role of our defined sites.7. However, G164S decreased protein expression21. In addition to missense mutations, several types of FBP1 mutations  have been reported5. Previous in vitro studies reported that c.704delC, c.838delT, and c.960dupG all decreased truncated FBP1 protein expression22. However, the mechanism that leads to a decrease in truncated FBP1 protein expression has not been sufficiently elucidated. Considering our findings, protein misfolding may be involved in the underlying disease pathophysiology in these truncated types of FBP1 mutations, similar to Type 2 mutations.In the context of genotype–biochemical phenotype associations, previous in vitro studies evaluated only two missense mutations (D119N and G164S), and consistent with our findings, D119N decreased enzymatic activity with no impact on protein expressionThe clinical applications of pharmacological chaperone therapy for Fabry disease have become increasingly popular. The chaperone molecules support the folding of mutated enzymes and increase their stability and activity, which may be broadly applicable to other protein deficiencies. Life-threatening episodes of hypoglycemia and lactic acidosis are sometimes triggered by fasting and febrile infections during childhood in cases of FBPase deficiency. However, to date, no preventive agent has been developed for these patients and their symptoms. Our findings indicate the possibility that certain patients with Type 2 mutations may respond to such chaperone molecules, although Type 1 mutations are likely to be untreatable cases of FBPase deficiency. Further studies are warranted to fully understand the pathophysiology of this rare disease as well as to clarify the usefulness and efficacy of pharmacological chaperone therapy.FBP1 gene were amplified by polymerase chain reaction (PCR) and sequenced using a 3130 Genetic Analyzer . Supplementary Table DNeasy Blood and Tissue Kits  were used to extract genomic DNA from the patient’s blood. Then, all 7 exons of the 23. The sequenced reads were aligned to a human genome reference (GRCh37) using the default parameter settings in Burrows‒Wheeler Aligner version 0.7.10, and the PCR duplicates were eliminated using Picard-tools version 1.39 (http://picard.sourceforge.net/). Candidate mutations with (i) depths ≥8, (ii) number of variant reads ≥4, and (iii) variant allele frequencies (VAFs) of 0.4–0.6 and 1 were adopted, as VAFs of pathogenic germline mutations are estimated to be approximately 0.5 in the heterozygous state and 1 in the homozygous state. Mutations were further filtered by excluding (i) variants presenting only in unidirectional reads, (ii) insertions and deletions in simple repeat regions, (iii) synonymous SNVs, and (iv) known variants listed in the 1000 Genomes Project (Nov 2010 release), Exome Sequencing Project (ESP) 6500, Human Genome Variation Database , and ExAC database with frequencies >0.001 to exclude non-pathogenic variants.Whole-exome sequencing involved the targeted capture of all exon sequences using SureSelect Human All Exon v6 , followed by massive parallel sequencing of the enriched exon fragments on the HiSeq 2500 platform  using the 125-bp paired-end mode as per the manufacturer’s protocol24.The quality control process for the whole-exome sequencing data is shown in the Supplementary Data FBP1 gene was carried out using the forward primer 5′-CACCATGGCTGACCAGGCGCCCTTCG-3′ and the reverse primer 5′-TCACTGGGCAGAGTGCTTCTCATAC-3′. The PCR procedure consisted of the following steps: (a) denaturing at 98 °C for 2 min, followed by 94 °C for 15 secs; and (b) annealing for 30 secs at 58 °C and extension at 72 °C for 1 min for 30 cycles. The PCR products were subcloned into the pGEM-T Easy Vector . Then, the FBP1 fragments (WT and 2 mutants) were cut from the pGEM-T Easy Vector using EcoRI and subcloned into a pCMV-Myc-N Vector . A corresponding pCMV-Myc-N vector was used as a negative control.Total RNA was extracted from the patient’s blood using RNeasy Kits  and reverse-transcribed using SuperScript 2 reverse transcriptase  and oligo (dT) primers. Amplification of the coding region of the D119N, P120L, R158W G164S, A177D, G207R, N213K, G260R, E281K, P284R, G294E, G294V, and V325A mutations were introduced by site-directed mutagenesis  using the primers listed in Supplementary Table 25. The backbone vectors pX459 pSpCas9(BB)-2A-Puro and pX462 pSpCas9n(BB)-2A-Puro were obtained from Addgene . The target guide RNA sequences were designed for exon 5, including c.491G, exon 6, including c.581T, and exon 8, including the Japanese common mutation site (c.960-961insG), of the FBP1 genome 3. To clone the exon 5 and exon 6 target sequences into pX462 and the exon 8 target sequence into the pX459 backbone, oligos were synthesized using Eurofins genomics  (Supplementary Table A hepatocellular carcinoma cell line (HepG2) was used in this study. HepG2 was purchased from the American Type Culture Collection. The cell line was tested for mycoplasma contamination. The protocol used for the CRISPR/Cas9 system was based on that reported by Cong et al.FBP1-KO HepG2 was cultured using Dulbecco’s modified Eagle’s medium containing antibiotics and 10% fetal bovine serum. FBP1-KO HepG2 cells were plated on 6-well plates and then transfected with plasmid DNA (2.5 µg) complexed with Lipofectamine 3000 reagent (7.5 µl) and P3000 reagent (5 µl) in 250 µl of Opti-MEM . Subsequently, the FBPase activity of HepG2 cells was measured 48 hours after transfection using a nicotinamide adenine dinucleotide phosphate (NADP)-coupled spectrophotometric assay.The human hepatocarcinoma cell line 26. The assay mixture (300 µl) was composed of 40 µg protein of cell lysate, 50 mmol/L Tris-HCl buffer (pH 7.5), 2.0 mmol/L MgCl2, 1.0 mmol/L EDTA, 0.2 mmol/L NADP, 3.5 U/mL glucose-6-phosphate dehydrogenase, 1.5 U/mL glucose-6-phosphate isomerase, and 100 µmol/L FBP, which served as the substrate. A 96-well plate reader was used to record the rate of NADPH formation.The FBPase activity was calculated from an NADP-coupled spectrophotometric assay as described by Kikawa et al.29. The FBP1 gene-specific mRNA expression values were determined and normalized to those of β-actin as an internal control. Briefly, total RNA (4 μg) was extracted using an RNeasy kit  and reverse-transcribed using a ReverTra Ace qPCR RT Kit . The cDNA products were subjected to RT‒PCR using a Step One Plus Real-Time PCR system . All primer information is provided in Supplementary Table RT‒qPCR experiments were performed as previously described30. Briefly, ECL-films were used for western blot developing. After the optimal exposure time, we open the cassette and pop the film directly into the film developer. Once our film has been developed, we overlay it back on the blot to mark the protein ladder on the film with a Sharpie marker. Western blot films with size marker labeled by a Sharpie marker as uncropped western blots are provided in Supplementary Fig. Immunoblot analyses were performed as previously describedThe extraction of the ER was performed using an Endoplasmic Reticulum Enrichment Kit  according to the manufacturer’s instructions.FBP1-KO HepG2 cells were cultured in 4-well chamber slides (2 × 104 cells/well) and then transfected with plasmids (0.5 µg). Twenty-four hours after the transfection, the cells were treated with CellLight® ER-GFP , which is a marker of ER. This was followed by fixation in 100% ethanol at −20 °C for 10 min and incubation with blocking solution and primary antibodies against FBP1 , c-Myc  and GFP  48 hours after the transfection. A confocal laser microscope  was used to obtain fluorescence images.Immunofluorescence analysis was performed to examine the cellular expression of WT and mutant FBP1. The commercially available c-Myc-tagged Protein Mild Purification Kit  was used to perform the immunoprecipitation assay. HepG2 cell extracts containing different Myc-tagged FBP1 variants were incubated with anti-Myc beads at 4 °C for 1 hour. The beads were then rinsed and eluted using a wash solution and elution peptides.FBP1-KO HepG2 cells were transfected with plasmid DNA containing Myc-tagged FBP1-WT, Myc-tagged FBP1-G164D, or Myc-tagged FBP1-F194S. These cells were treated with the mannosidase inhibitor kifunensine (200 µM) for 48 h. Myc-tagged FBP1 HepG2 cell solubilization was obtained with the following buffer: 50 mM Tris-HCl (pH 7.5), 1.0 mM MgCl2, 0.1 mM EDTA, 0.5 mM phenylmethanesulfonyl fluoride, 2.5 µg/mL leupeptin and 1.0 µg/mL antipain. Immunoprecipitation with anti-Myc beads  was performed at 4 °C for 1 h. The beads were then rinsed and eluted using a wash solution and elution peptides. Immune complexes were separated by SDS‒PAGE. Bands were removed from the gel and examined by mass spectrometry to find corresponding proteins. Respective gel pieces were rinsed two times using 100 mM bicarbonate in acetonitrile with subsequent protein digestion by trypsin. Then, 0.1% formic acid was added to the supernatant, and the peptides were subjected to liquid chromatography-tandem mass spectrometry (LC‒MS/MS) using an LTQ Mass Spectrometer . Analysis of the MS/MS dataset results was conducted using the Mascot software program . The source Mass spectrometry data is shown in the Supplementary Data 31. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation and Gene Ontology (GO) analyses were performed using the STRING interaction database.Proteomic analysis and database searches were performed as previously describedSus scrofa FBP1 complexed with fructose 6-phosphate, AMP, and magnesium32; Fig. Homo sapiens and Sus scrofa show approximately 90% similarity. In particular, the functional motifs directly associated with FBPase activity are highly conserved.Ke et al. revealed the detailed crystal structure of P < 0.05, **P < 0.01; NS: not significant P > 0.05.The results are presented as the mean ± SD. One-way ANOVA followed by Dunnett’s multiple comparison test was used to analyze continuous variables. Significance is indicated by asterisks or NS, as follows: *The experimental findings were reliably reproduced with at least two independent experiments in Figs. The genetic study and collection of patient information were approved by the ethics committee of Chiba University Graduate School of Medicine. Written informed consent for the genetic studies was obtained from the patient and her family in accordance with the protocols issued by the Chiba University Graduate School of Medicine. All relevant ethical regulations were followed for experiments with human participants.Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Reporting Summary