Two variants of the operon existand the more common one additionally contains hblB downstream of hblCDA. Up to now, it was completely unclear whether the corresponding protein, Hbl B’, is widely expressed among B. cereus strains and if it has a distinct function. In the present study, it was shown that the hblB gene is indeed expressed and the Hbl B’ protein is secreted by nearly all analysed B. cereus strains. For the latter, a detection system was developed based on monoclonal antibody 11A5. Further, a distinct reduction of cytotoxic and haemolytic activity was observed when recombinant (r)Hbl B’ was applied simultaneously with L2, L1 and B. This effect was due to direct interaction of rHbl B’ with L1. D−6B. cereusAltogether, we present the first simple tool for the detection of Hbl B’ in B. cereus culture supernatants. Moreover, an important regulatory function of Hbl B’ in the mechanism of Hbl was determined, which is best described as an additional control of complex formation, balancing the amounts of Hbl B-L1 complexes and the corresponding free subunits.The genes  Bacillus cereus group. It is involved in endophthalmitis [B. cereus culture supernatants showed that hemolysis depends on concentration and diffusion velocity of Hbl B. While rHbl L2 does not seem to have an impact on the outer hemolysis ring, it is enhanced by additional rHbl B and hindered or disrupted by additional rHbl L1 [Hemolysin BL is a primary virulence factor produced by members of the halmitis ,2 and cahalmitis . Furtherhalmitis , hemolythalmitis , active halmitis  and it shalmitis ,7–9. Hblhalmitis , then thhalmitis . In theshalmitis ,10,11. N rHbl L1 .E. coli [Hbl is a pore forming toxin, which was first shown by osmotic protection assays . The HblE. coli , which fE. coli . NonetheE. coli  and thusE. coli ,12. A deE. coli ,21. The E. coli . The speE. coli .hbl operon encoding the toxin components is found in approximately 40–70% of all known enteropathogenic B. cereus strains [hbl operon. The wider spread one consists of the genes hblC (encoding Hbl L2), hblD (encoding Hbl L1), hblA (encoding Hbl B) and hblB (encoding Hbl B’). The rarer variant misses the hblB gene. Several B. cereus strains harbor both variants and thus, express homologous sets of Hbl proteins [B. cereus strain F837/76 only the hblCDAB operon exists [hblC is the transcription start [hbl operon is further regulated, probably in a concerted action, by CcpA, CodY, SinR, ResD and Fnr [hblC, D, A, and B have a size of 1320, 1221, 1128, and 1398 bp. Between hblC and D is a space of 61 nucleotides, between hblD and A 36, while the genes hblA and B are separated by 402 nucleotides. Upstream and downstream of hblB, a stem loop has been identified, which could function as transcriptional terminator [hblB has been suggested [hblB was first found by sequencing the hblA gene of B. cereus strain F837/76 [hblB and hblA differs significantly, it was stated that hblB emerged from duplication of hblA and a C-terminal fusion with another ORF [The  strains . There aproteins ,29–31. In exists ,32, whicon start . The Plcon start . Next to and Fnr . The genuggested . hblB wather ORF .Figure hblCDA was found to end within [hblB, until Clair et al. demonstrated that the gene is transcribed, translated, and the protein exported [B. cereus supernatants in lower amounts than the other Hbl components, which might be a reason that it has not been detected earlier. Furthermore, a 2-fold upregulation of hblB in hyperflagellated swarm cells of B. cereus strain ATCC 14579 was found [B. cereus, it is still unclear if it has a certain function. Due to the sequence homology of Hbl B, it has been speculated that Hbl B’ could complement or reinforce the function of Hbl B under specific growth conditions [Transcription of d within ,37,38 ord within  of hblB,exported . The stuas found . Althougnditions .E. coli and to study the mode of action of Hbl in more detail [B. cereus culture supernatants and (ii) to investigate its putative involvement in Hbl activity and thus, in virulence of B. cereus.Just recently, we were able to overexpress and purify fully functional recombinant Hbl components (rHbl) from e detail ,20,21. TB. cereus strains were used in this study: F837/76, F837/76 ∆nheABC, F4430/73, 14294–3 (M6), SDA KA 96, INRA A3, INRA C3, 6/27/S, F3175/03 (D7), RIVM BC 934, F528/94, RIVM BC 126, NVH 0075–95, and MHI 226 [E. coli strain BL21 (DE3) transformed with the corresponding pASK-IBA plasmids was grown in LB medium with 100 µg/ml ampicillin.The following, previously well-characterized  MHI 226 ,23,40–432 culture flasks in a humidified incubator at 37°C and 7% CO2 as recommended by the supplier.Vero cells were purchased from ECACC (European Collection of Cell Cultures), CaCo-2 and A549 cells from DSMZ . They were maintained in 80 cmTotal RNA preparation and on-column DNase digestion were performed using peqGOLD Total RNA and peqGOLD DNase I Digest Kit  according to the instructions of the manufacturer. Electrophoresis on agarose gels and testing in a spectrophotometer revealed the quality of the RNA. RNA purity was confirmed via PCR for a 241 bp 16S rRNA fragment using the primers 16S_fw (GGAGGAAGGTGGGGATGACG) and 16S_rev (ATGGTGTGACGGGCGGTGTG).hblB using the primers hblB-3'-fw (CATAACG CATACACTTTTGAAATAAAG) and hblB-3'-rev (CCGCAAATTCATCATTTGGATTG). The fragment represents the 3' part of the gene not identical to hblA.Using 100 ng RNA as template, double-stranded cDNA was synthesized with the ProtoScript first-strand cDNA synthesis kit  and random primer mix according to the instructions of the manufacturer. The cDNA was subsequently applied to a PCR for a 166 bp fragment of hblB gene, encoding the putative fourth component of the B. cereus Hbl toxin, was amplified from chromosomal DNA of strain F837/76 using the primers HblB’-fw-KpnI (ATATGGTACCCGCAATTGAAATTCAACAAACG) and HblB’-rev-NcoI (ATATCCATGGTCAGTTCAT TATATTTTGTACTTTG). The amplified gene lacked the sequence for the N-terminal signal peptide for secretion [CCATGGTCAATTCAAAAAATCCTGTTCTTTTTTCTC) as reverse primer was used. Similarly to the full-length hblB of F837/76, the hblB genes of the other 10 B. cereus strains (see above) were amplified and cloned. Overexpression and purification of the rHbl B’ proteins were performed analogously to Hbl L2, L1, and B as described before [Recombinant Hbl L2, L1, and B were produced as described before . The hblecretion . It was d before .2O, fluorescence signals were detected on a UVP ChemStudio imager .SDS-PAGE was run on a PhastGel gradient (10–15%) minigel system . For Sypro staining, proteins were fixed on the gel for 2 × 30 min in 50% MeOH and 7% acetic acid. The gel was then incubated with 2 ml Sypro Ruby protein stain  overnight at room temperature. After 30 min washing in 10% MeOH and 7% acetic acid and additional washing for 10 min in HB. cereus culture supernatant were applied to Criterion XT precast gels . After electrophoresis, proteins were blotted to a PVDF-P membrane . The membrane was saturated with 3% casein-PBS and incubated for 1 h at room temperature with 2 µg/ml Strep-MAB-Classic  or monoclonal antibody (mAb) 11A5 [For Western blotting, 30 µl protein solution or Ab) 11A5  for dete1.5 pmol/µl stock solutions of the rHbl components L2, L1, B, B’, and B’(tr) were used in toxicity assays. If not stated otherwise, they were added in 1:40 dilutions or as dilution series to the cells after pre-mixing in appropriate ratios. WST-1 bioassays and propidium iodide (PI) influx tests were performed as previously described ,42,46,47B. cereus supernatants or rHbl components (1.5 pmol/µl each) were applied as mixtures or individually into 3.5 -mm diameter stamp holes on sheep blood agar plates. If not stated otherwise, they were photographed after 3–6 h of incubation at 32°C. For quantification, samples were applied to hemolysis assays with defibrinated sheep blood . The blood was applied to centrifugation (5 min at RT and 590 g) and washed three times in 150 mM NaCl, 5 mM Tris-HCl, pH 7.2. After that, it was diluted to 2% in the buffer. Two µl B. cereus supernatant or 10 µl rHbl samples were added to 800 µl total volume. As negative and positive controls, erythrocytes in 800 µl buffer and in 800 µl H2O were carried along, respectively. Samples (triplicates) were incubated for 20 min (supernatant) or 30 min (rHbl) at 37°C and subsequently centrifuged for 1 min at RT and 8000 g. Subsequently, an Infinite F50 photometer  was used to determine the optical density at 562 nm. Results were compared to the positive control, which was set to 100%.Ten µl of 6 Vero cells in 500 µl buffer  per sample were used. rHbl B, rHbl B’, and rHbl B’(tr) were applied at a concentration of 9.38 pmol/ml in the cell culture medium to a total volume of 1 ml and incubated for 1 h at 37°C under gentle agitation. Similarly, 1:1 mixtures of rHbl B and L1, L2, or B’ were used. After two washing steps in 2 ml 1% BSA-PBS and centrifugation for 5 min at 800 rpm, 5 µg/ml mAb 11A5 was added for 1 h at RT. Samples were again washed twice and subsequently incubated with 2 µg/ml Alexa Fluor® 488 goat antimouse IgG  for 45 min at 4°C. Alternatively, directly labeled mAbs 1B8- or 1G8-Alexa Fluor® 488 were used. Two additional washing steps were carried out. After that, cell pellets were resuspended in 500 µl 1% BSA-PBS and transferred to flow cytometry tubes. FACS Calibur and CellQuestPro software  were used for fluorescence measurements. Results were analyzed using Flowing Software . Cell populations were visualized in the FSC SSC (forward scatter – side scatter) dot plot. Fluorescence was recorded in FL1 (fluorescence channel 1) and is shown as histogram.1 × 102 solution has been used for rHbl L1 and 0.5 M NaCl solution for rHbl L2 and B. In order to subtract any background noise from each experiment, all samples were also run over an unmodified sensor chip surface. Data analysis was performed with Biacore S200 evaluation software v1.1. For each measurement, the equilibrium dissociation constant (KD) was calculated using a 1:1 binding model. The KDs from three experiments were used to calculate the mean values of these variables and the SEM.Measurements were performed on a Biacore S200 instrument . rHbl B’ was diluted to a final concentration of 150 nM in 10 mM sodium acetate, pH 4.8, and chemically immobilized  onto series S sensor chip CM5 . rHbl L1, L2, and B protein samples were diluted in a running buffer  to the final concentration of 7.8125 nM, 15.625 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, 1 µM, and 2 µM and injected over the sensor chip surface at 30 µl/min at 20°C from the lowest to the highest concentration. Injection of 250 nM protein concentration was always performed in duplicate within each experiment. For the sensor chip regeneration after each injection, 1 M MgClB. cereus strain F837/76 were gained using SWISS-MODEL [DNA and protein sequence alignments were generated using clustal omega . StructuSS-MODEL .hblB gene is generally transcribed and the corresponding protein expressed in B. cereus. To answer that, total RNA was prepared from different B. cereus strains with known enterotoxin gene profiles [hblB. The hblB fragment could be detected for all tested B. cereus strains  [hblCDA operon [Earlier, contradictory studies ,35 raiseprofiles ,40,42, w strains , indicatosphere) . As a poosphere)  as well A operon  revealedhblB gene from B. cereus strain F837/76 was cloned into the vector pASK-IBA5plus. Thereby, the N-terminal signal peptide for secretion was dismissed, and an N-terminal strep-tag was added. E. coli BL21 (DE3) was used for protein overexpression. Purification was conducted via affinity chromatography. The 460 amino acid protein with a predicted molecular mass of 51.5 kDa was detected by Western blotting using an anti-strep mAb . Protein concentrations were calculated according to rHbl B and rHbl B’ concentration standards  and MHI 226 (truncated hblCDA operon) were again applied as negative controls.For the detection and estimation of the amounts of Hbl B’ in tandards . Hbl B wbased detection system for Hbl B,’ the recombinant protein was also used in different (cell culture) assays to determine its influence on Hbl activity. Pore formation and cytotoxic activity of the rHbl components L2, L1, and B have already been shown in PI (propidium iodide) influx tests and WST-1 (water-soluble tetrazolium) bioassays on target cells, as well as on planar lipid bilayers ,20,21. BB. cereus strain F837/76 ∆nheABC  was initially used for the experiments and especially for cloning of hblB, as it is well sequenced and annotated. Nevertheless, in the course of this study, it was shown that in this particular strain, the hblB gene is indeed transcribed, but the corresponding Hbl B’ protein is not expressed in detectable levels in the supernatant (see above).It is noteworthy that strain F837/76  of rHbl B’ was produced lacking the C-terminal 91 amino acids. Cloning, protein overexpression and purification were performed analogously to the full-length rHbl B’ . SimilarB. cereus strains, we were interested to see if these proteins exhibit comparable or varying inhibitory activities toward Hbl toxicity. As the hblB genes and thus, the Hbl B’ proteins of strains 6/27/S, RIVM BC 934, and RIVM BC 126 are identical , the Hbl B’ proteins of eight further strains were recombinantly expressed and purified analogously to F837/76 (see above). nheABC supernatant until a dilution of 1:4, as well as hemolysis of rHbl L2+L1+B (data not shown). Strain-specific differences were first observed when 10 µl rHbl B’ were pre-incubated in the stamp holes for 1 h before application of 10 µl rHbl L2+L1+B. rHbl B’ of strains INRA A3 and RIVM BC 934 strongly inhibited hemolysis, while only a weak effect was observed for rHbl B’ from strain 14294–3 (M6). Inhibition of hemolysis with rHbl B’ of all other strains was interjacent . Differences between the rHbl B’ proteins of the nine strains were most obvious at rHbl B:’F837/76 ∆nheABC = 1:1 as well as 5x surplus of rHbl B’ compared to rHbl L2+L1+B , F4430/73, and F3175/03 (D7) showed the least inhibition, while pore formation started most tardily under addition of rHbl B’ from strains INRA A3, F837/76, and RIVM BC 934  and Hbl were the only known pore forming toxins consisting of three individual protein components [Serratia marcescens, Erwinia mallotivora, and Aeromonas hydrophila after extensive in silico search, and the mechanism of the tripartite α-pore forming toxin AhlABC of A. hydrophila was characterized [Many years of research have led to a better understanding of the mode of action, especially the mechanism of pore formation, of the enterotoxin complex Hbl from ed order ,12. A deed order ,21. But ed order ,50. Untimponents ,51. Thencterized .hbl operons among different strains or also within a single strain, the more common form hblCDAB and the rarer variant hblCDA missing Hbl B’ [B. cereus strains. One innovation is the development of an easy and functional tool for the detection of Hbl B’ in B. cereus culture supernatants. In a previous work, mAbs with specific affinity to Hbl L2, L1, and B were generated [hbla operon in strains 14294–3 (M6), 6/27/S, and RIVM BC 126, as well as a very rare, second nhea operon in 14294–3 (M6) and MHI 226. Though initially suspected, the hbl duplication could not be confirmed in the strain RIVM BC 934 [The fourth protein component, Hbl B,’ seems not to be necessary for pore formation and cytotoxic activity of Hbl. This is justified by the occurrence of two distinct g Hbl B’ ,29–31. Mg Hbl B’ ,20,21. NM BC 934 ,40,42.B. cereus strains, but with varying concentrations. Interestingly, strain F837/76 seemed not to express Hbl B’ in a detectable amount, although hblB mRNA existed. In general, concentrations of Hbl B and presumably Hbl B’ in the B. cereus supernatants obtained in Western blots in this study are higher than concentrations determined earlier using EIAs and purified proteins as concentration standards. In the latter one, average Hbl B concentrations of approx. .5 ng/µl were obtained (unpublished data). Just recently, we gained evidence that the concentration of Hbl B in B. cereus culture supernatants might be drastically underestimated when tested with EIAs, as the protein is highly complex, with itself as well as with Hbl L1 and L2 [With this tool, Hbl B’ could also be detected in the supernatant of 10 1 and L2 ,21.B. cereus culture supernatants was also minimized. To our knowledge, this is the first time that a distinct activity of the hblB gene product was observed. Furthermore, we showed that rHbl B’ cannot bind to Vero cells and thus, does not compete with Hbl B for binding sites at the target cell surface. Moreover, it does not seem to influence the binding of B to the cell surface. On the contrary, it rather inhibits the interaction between Hbl B and L1. SPR measurements showed binding of rHbl L1 to rHbl B’ with a KD value of 131 nM  was completely abolished when either rHbl B or L2 or L1 was replaced with rHbl B'. Thus, hypotheses were disproved that Hbl B’ might exhibit a similar function than Hbl B  due to t1 nM see , which if 473 nM ), but st 0.48 nM ). Thus, hblB has originated by duplication of hblA and a C-terminal fusion with another ORF [hblB has originated last in the evolution of the enterotoxins [It has been suggested that ther ORF . Interacrotoxins ,34 and irotoxins . On the rotoxins ,20, simirotoxins . Althougrotoxins . The rolB. cereus strains decreased the hemolytic and pore-forming activity of Hbl. Only a truncated version of rHbl B’ of strain F3175/03 (D7) could be recombinantly expressed and purified. Since it was detected by StrepMAB-Classic and mAb 11A5 in Western blots . Initially, it has been suggested that hblB is transcribed together with hblCDA as a polycistronic mRNA [hblB from B. cereus ATCC 14579 [hblB might be transcribed as a single unit [hblB of our 11 strains. The predicted −10 and −35 regions as well as the predicted transcription start site were identified for nine strains. In general, the length of the hblB upstream regions varies and is less conserved than the hblB genes . At this point, it can only be concluded that strain-specific variations in Hbl B’ production and in its inhibitory activity exist, as well as in the sequences of hblB and especially the hblA-hblB intergenic region. Further in-depth analyses are necessary in order to connect those variations.In the present study, it was further shown that rHbl B’ proteins from nine lots see a, it canlots see b-d. Thislots see , is respnic mRNA . Recent CC 14579  and ATCCgle unit . Both ofhbl operon, Hbl B,’ in B. cereus culture supernatants. We found the protein to be secreted by 10 different strains. More importantly, we could verify an inhibitory function of Hbl B’ in the mechanism of Hbl, which is most likely the balanced orchestration of free Hbl B and Hbl B-L1 complexes in solution.Nevertheless, this study is the first one to present a simple tool for the detection of the fourth component of the Click here for additional data file.


Trichomonas tenax is a flagellated protozoan that inhabits the human and canine oral cavity in patients with poor oral hygiene and periodontal disease. The loop-mediated isothermal amplification (LAMP) assay could provide clinicians with a quick, cheap and reliable diagnostic test used for the detection of T. tenax in various settings. In this study, we aimed to develop a LAMP assay that can detect T. tenax with high sensitivity and specificity. A set of LAMP primers were specifically designed to detect the ITS and 5.8S rRNA gene of T. tenax. The newly developed LAMP assay was 1000 times more sensitive than conventional PCR. The limit of detection of the LAMP assay was 10 fg of genomic DNA, or 0.2–1 cell. Moreover, the LAMP assay was specific, resulting in no cross-reaction even with a closely related protozoan T. vaginalis or other microorganisms  used. The present LAMP assay can be performed directly without prior DNA extraction, making the assay an easy, fast, cheap, specific and sensitive diagnostic tool for the detection of T. tenax at the point-of-care of both medical and veterinary clinics in developed and developing countries. Trichomonastenax is a flagellated protozoan that inhabits the human and canine oral cavity in patients with poor oral hygiene and periodontal disease , 6 U of Bst polymerase large fragment , 2 µL of template DNA and nuclease free water. Reactions were performed at 60 °C for 60 min in the Mastercycler® Nexus Thermal Cyclers .The optimized LAMP reactions for the assay were performed in a 25 µL reaction mixture containing the following reagents: 0.8 µm FIP, 0.8 µm BIP, 0.4 µm LF, 0.4 µm LB, 0.2 µm F3, 0.2 µm B3, 4 mm MgSOThe amplified products were visualized using gel electrophoresis and SYBR Green I . For gel electrophoresis, 10 µL of the product was separated on 1% agarose gel containing 0.5 mg/mL ethidium bromide with a 100 bp ladder of DNA marker . For detection using SYBR Green I, 1 µL of SYBR Green I was added to 10 µL of the product and examined under UV light. Fluorescent green color is an indication of a positive reaction and orange color is an indication of a negative reaction.Taq buffer with KCl, 0.2 mm dNTPs, 3 mm MgCl2, 1 µm F3, 1 µm B3, 2.5 U of Taq DNA polymerase , and nuclease-free water. PCR was performed in Mastercycler® Nexus Gradient Cyclers. The thermal cycle was optimized for annealing temperature as follows: initial denaturation for 5 min at 95 °C, 35 cycles of denaturation for 1 min at 95 °C, annealing at 53 °C for 30 s, elongation at 72 °C for 1 min and a final step at 72 °C for 5 min.Primers F3 and B3 shown in T. tenax genomic DNA in Tris-EDTA (TE) buffer without a carrier, ranging from 50 ng/μL to 5 pg/μL was used. The results were compared to conventional PCR using the LAMP F3 and B3 primers. Analytical specificity of the newly developed LAMP assay was evaluated using a close relative , along with other microorganisms . Negative and positive controls using water and T. tenax genomic DNA, respectively, were included. Each experiment was run three times for repeatability and reproducibility.To determine the limit of detection (LD) of the LAMP assay, a 10-fold serial dilution of T. tenax cells were used to investigate the limit of detection of the spiked samples. They were diluted in canine saliva, TE buffer or phosphate buffered saline (PBS) in serial dilutions from 2 × 105 to 2 × 10−1 cells. These cells were subjected to boiling at 100 °C for 30 min followed by cooling down to 4 °C on ice. Alternatively, similar prepared spiked samples were subjected to no pre-treatment at all and were directly used to perform LAMP. Positive and negative controls using T. tenax genomic DNA and water, respectively, were included. The experiment was run three times for repeatability and reproducibility.Cultured T. tenax. The cells were centrifuged, collected and stored at −80 °C until use. To perform LAMP on these samples, the frozen cells were taken by a sterile pipette-tip without thawing and immediately suspended in PBS for quantification. The sample was applied directly to perform LAMP without DNA extraction or boiling after resuspension in TE buffer followed centrifugation. In total, 2 cells per reaction were used to perform LAMP. Positive and negative controls using T. tenax genomic DNA and water, respectively, were included. The experiment was run three times for repeatability and reproducibility.LAMP was performed on eight oral swabs that were collected from client owned pet dogs that presented to the RUSVM Veterinary Clinic. These swabs were subjected to culture up to 5 days as mentioned above for the presence of 4 concentration and time were optimized during the development of the LAMP assay. To optimize temperature, the reaction was incubated at 50, 53, 55, 58, 60, 63, and 65 °C, respectively, for 60 min. Amplification took place at 58, 60, 63 and 65 °C, and 60 °C was chosen as the optimal temperature based on the criteria of maximum LAMP amplification at the lowest temperature in order to extend the enzyme half-life as long as possible . To optimize the reaction time the reaction was incubated at 15, 30, 45, 60 and 75 min, respectively. Amplification was seen at 30 min and beyond, and the optimal time chosen for the assay was 60 min , or even with T. vaginalis, a close relative of T. tenax. This high specificity of LAMP is the result of 4–6 primers targeting 6–8 regions of the target DNA [T. tenax, T. vaginalis and T. brixi . Alternatively, a clinical sample of human or canine origin, or of other mammals as sources, can be put into the physiological saline upon collection. The sample is then centrifuged, and the pellet is resuspended in TE butter, of which 2 µL can be directly applied to LAMP mixture for testing. In short, this newly developed LAMP assay has shown how applicable and convenient it is for use at point-of-care of both medical and veterinary clinics. The newly developed assay does not require DNA extraction, cells can be directly applied to perform LAMP and the results can be obtained within one hour. At point-of-care, this LAMP assay could be a powerful diagnostic tool to effectively and rapidly detect T. tenax.Although LAMP has many advantages, there are some disadvantages as well. Due to the high sensitivity of LAMP in comparison to PCR, reports have shown that LAMP reactions have a higher possibility of getting contaminated or producing false-positive results ,30,31. DT. tenax is rapid, specific, sensitive and easy to perform. The assay designed is an affordable and powerful diagnostic test for the detection of T. tenax at the point-of-care in medical and veterinary clinics in both developed and developing countries. Further studies on clinical samples are needed to validate its diagnostic sensitivity and specificity.The LAMP assay developed in this study to detect


Psychrobacter arcticus strain 273-4. The mannan fromthe cold-adapted bacterium was compared with its dephosphorylatedderivative and the commercial product from Saccharomycescerevisiae. Starting from the chemical structure,we explored a new approach to deepen the study of the structure/activityrelationship. A pool of physicochemical techniques, ranging from small-angleneutron scattering (SANS) and dynamic and static light scattering to circular dichroism (CD) and cryo-transmissionelectron microscopy (cryo-TEM), have been used. Finally, the ice recrystallizationinhibition activity of the polysaccharides was explored. The experimentalevidence suggests that the mannan exopolysaccharide from P. arcticus bacterium has an efficient interactionwith the water molecules, and it is structurally characterized byrigid-rod regions assuming a 14-helix-type conformation.Extracellularpolysaccharides are widely produced by bacteria, yeasts, and algae.These polymers are involved in several biological functions, suchas bacteria adhesion to surface and biofilm formation, ion sequestering,protection from desiccation, and cryoprotection. The chemical characterizationof these polymers is the starting point for obtaining relationshipsbetween their structures and their various functions. While this fundamentalcorrelation is well reported and studied for the proteins, for thepolysaccharides, this relationship is less intuitive. In this paper,we elucidate the chemical structure and conformational studies ofa mannan exopolysaccharide from the permafrost isolated bacterium  These polymers are the most abundantcomponents of biofilm, assuming a crucial role in the pathogenic bacteria.3 The EPSs participate in the adhesion of bacteriato many surfaces, play a role in the protection of the microorganisms,and boost the biochemical interactions between the bacteria and thesurrounding environment.5Microbialextracellular polysaccharides (EPSs) are high-molecular-weightpolymers7 Therefore, besides theinvestigation of the primary structure of the polysaccharide, thestudy of the physical/chemical properties and the shape the polysaccharidesadopt in solution could represent the key to understand their bioactivity.Although the variety of exopolysaccharides that can be foundinnature is substantial, the biotechnological application of these polymersis often limited due to the lack of information regarding their structureand biological activity. To our knowledge, only a few papers havedescribed how the structural features of the exopolysaccharides areresponsible for such activities.9 as well as constituents of glycoproteinsin yeast cell walls.10 Mannans differ significantlyin their structure: the main backbone can consist of α-1,6-linked d-mannose residues or β-1,4-linked d-mannose,and could be linear or branched polysaccharides. The degree of branchingand the type of substituents represent another source of variability.Mannan polysaccharides have been found to be mainly delivered in thegrowth medium of pathogenic fungi11 andin some cold-adapted bacteria.14 In the case of the polymers producedby fungi, such as, for example, Candida albicans, it has been demonstrated that they are constituents of biofilmand might have a role in the biofilm adhesion and in the drug resistancemechanisms.15 The role assumed by the mannansfor the cold-adapted bacteria has not been well defined yet, sinceonly in one case, they have been suggested to be cryoprotectants.13The mannose-type polysaccharides, usually referred to as mannans,are widely distributed in nature as part of hemicelluloses in planttissue16 and atomic force microscopy.17 Some of them, such as UV circular dichroism (CD), are adoptedfrom protein analyses, but this technique is much less developed foranalysis of polysaccharides than for proteins and nucleic acids.18 The CD technique is often hampered by the absenceof suitable chromophores in most natural polysaccharides.19 To the best of our knowledge, all of these techniqueshave never been applied to study mannan polysaccharide.Few physicochemical methods applied tothe conformational studyof polysaccharides are available, such as static light scatteringPsychrobacterarcticus 273-4.20 The investigationof mannan polysaccharides is often restricted to a shallow analysis,and the absence of a well-defined repeating unit hampers the conformationalstudy through a molecular dynamic calculation approach. In the presentwork, a physicochemical approach has been undertaken by exploitationof small-angle neutron scattering (SANS), dynamic and static lightscattering , CD, and cryo-transmissionelectron microscopy (cryo-TEM), a combination of techniques that isnot usually employed for analysis of polysaccharides. We comparedall of the results with the commercial mannan from Saccharomyces cerevisiae and with the dephosphorylated P. arcticus mannan. Finally, following on our previouspapers reporting the weak ice recrystallization inhibition (IRI) activityof extracellular polysaccharides from cold-adapted bacteria,7 the IRI response of these mannan polysaccharides obtained from differentsources was evaluated.Here,we report the complete structural characterization and aphysicochemical conformational study of a mannan extracellular polysaccharideproduced by the cold-adapted permafrost isolate 22.1P. arcticus strain 273-4 was isolated from permafrostsoil located in Siberia. Shake flask cultivation was performed at4 °C, in Luria-Bertani broth added of 5% of NaCl, and under aerobiccondition. When the liquid cultures reached late exponential phase, cells were collected by centrifugationfor 15 min at 7000 rpm at 4 °C. The exopolysaccharide contentwas separated from the supernatant through the addition of three volumesof cold ethanol followed by 72 h of precipitation at −20 °C.The precipitated was then collected after centrifugation at 8000 rpmat 4 °C, dissolved in water, and lyophilized (0.5 g L–1). The mixture was purified through a Sephacryl S400 gel filtrationcolumn   eluted with 50 mM ammonium hydrogencarbonate. The chromatographic system was equipped with a Knauer RIdetector 2300 and a Gilson FC203B fraction collector. The obtainedhigh-molecular-weight fraction was freeze-dried (3.3%). The proteinconcentration was estimated using the Bradford method (Bio-Rad).2.221 Briefly, the sample (1 mg) wasdissolved in 1 mL of 1.25 M MeOH/HCl solution and kept at 80 °C for 16 h. After the methanolysis reaction,the sample was evaporated to dryness and dissolved in 200 μLof pyridine and 100 μL of acetic anhydride (100 °C for30 min). The obtained sample was evaporated, dissolved in chloroform,and extracted three times with water. The final organic phase wasevaporated, dissolved in acetone, and analyzed by GC-MS. The linkageposition was obtained after derivatization of the sample in partiallymethylated acetylated alditols.22 The samplewas methylated with 100 μL of CH3I and then hydrolyzedat 120 °C with trifluoroacetyl (TFA) 2 M for 2 h. After neutralization,it was reduced with NaBD4 and finally acetylated and injectedinto the GC-MS. All of the derivative samples were analyzed usingan Agilent Technologies gas chromatograph 7820A equipped with a massselective detector 5977B and an HP-5 capillary column . Acetylated methyl glycosides and partially methylatedalditol acetates were analyzed using the following temperature program:140 °C for 3 min, 140 → 240 °C at 3 °C min–1 and 90 °C for 1 min, 90 → 140 °Cat 25 °C min–1, 140 → 200 °C at5 °C min–1, 200 → 280 °C at 10°C min–1, at 280 °C for 10 min.The sugar compositionwas determined by gas chromatography-mass spectrometry (GC-MS) analysisafter derivatization of the sample as acetylated methyl glycosides(AMG).2.3P. arcticus (MannanP.arc) was hydrolyzed with 4 mL of 48% HF at 4 °C for 48 h.23 The mixture was neutralized and purified ona Sephacryl S400 gel filtration column   eluted with50 mM ammonium hydrogen carbonate. The obtained fraction was analyzedby 1H NMR spectroscopy, to confirm that the reaction occurred.Purified EPS (40 mg)from 2.42O at a 600 MHz Bruker  instrument equippedwith a cryogenic probe. The spectra were recorded at 298 K using acetoneas external standard . Spectra were processed and analyzed using BrukerTop Spin 3.1 software. Double-quantum-filtered phase-sensitive correlationspectroscopy (1H–1H DQF-COSY), totalcorrelation spectroscopy (1H–1H TOCSY),and nuclear Overhauser enhancement spectroscopy (1H–1H NOESY) experiments were executed using 256 FIDs of a 2028complex point. TOCSY and NOESY experiments were recorded with a mixingtime of 100 ms. Heteronuclear single quantum coherence (1H–13C DEPT-HSQC) and heteronuclear multiple bondcorrelation (1H–13C HMBC) experimentswere acquired with 512 FIDs of a 2048 complex point. 1P-and 1H–31P HMBC spectra were recordedat 298 K using a Bruker Ascend 400 MHz spectrometer. The 1H–31P HMBC experiment was acquired with 512 FIDsof a 2048 complex point.One-dimensional(1D) and two-dimensional (2D) NMR experiments were acquired in D2.524 DLS measurements wereperformed on both diluted  and concentrated (1.0 and 6.0 mg mL–1) polysaccharide samples. For SLS measurements, stocksolutions of pure Mannanyeast, MannanP.arc_HF, and MannanP.arc, at 2.0, 2.0, and 1.0 mg mL–1, respectively, were used. Deionized water filtered through a 0.22μm membrane was used in all of the cases. The mass-averagedmolecular weight Mw and the second virialcoefficient B of each polysaccharide were determinedby means of Zimm plot analysisc is the sample massconcentration; n0 = 1.33,the refractive index of water, dn/dc = 0.185, the refractive index increment with concentration;26NA is Avogadroʼs number, λis the laser wavelength in vacuum, and Rθ is the excess Rayleigh ratio at 90°. The value of Rθ was obtained from Is isthe scattered intensity of the solution, Is,0 is the scattered intensity of water, Is,R is the scattering intensity of toluene (the standard), and nR = 1.496 and Rθ,R = 2.85 10–5 cm–1 are the refractiveindex and the Rayleigh ratio of toluene, respectively.27Static and dynamic light scattering measurements were performed at scatteringangle θ = 90°, using a homemade instrument composed ofa photocor compact goniometer, an SMD 6000 Laser Quantum 50 mW lightsource operating at 532.5 nm, a photomultiplier (PMT120-OP/B), anda correlator (Flex02-01D) from Correlator.com. Measurements were performedat 4 and 25 °C with the temperature controlled by means of athermostat bath.28 The proper diffusion coefficients were determinedthrough a final assessment by the “regularization” procedure.29 Diffusion coefficients were then employed tocalculate hydrodynamic radii by means of the Stokes–Einsteinrelationk is the Boltzmannconstant, T is the absolute temperature, and ηis the medium viscosity, whose mean value was assumed to be 0.89 cPfor each aqueous mixture.In the case of DLS, the data were treatedwith CONTIN: namely, the measurements, at least five independent measurementsfor each sample, were analyzed with “Precision Deconvolve”,a program based on the approach of Benedek and Lomakin.2.6P.arc, MannanYeast, and MannanP.arc_HF was measured at 25 °Cwith a Sigma 70 tensiometer  using the DuNoüy ring method as described elsewhere.30 γ was correlated with the force required to raisethe ring from the surface of the air/liquid interface. Successivealiquots of a stock polysaccharide solution were added to the vesselwith a known volume of water. After each aliquot addition, the samplewas mixed using a magnetic stirrer and allowed to equilibrate 3 minprior to measuring the surface tension.The surfacetension, γ, of aqueous mixtures of Mannan2.7P.arc and MannanYeast were performedwith the KWS2 instrument located at theHeinz Meier Leibtnitz Source, Garching Forschungszentrum (Germany).31 Neutrons with a wavelength spread Δλ/λ≤ 0.2 were used. A two-dimensional array detector at differentwavelength, collimation, sample-to-detector distance combinationsmeasured neutrons scattered from the samples. We chose configurationsthat allowed collecting data in a q range of 0.0018–0.45Å–1. The samples were contained in a closed-quartzcell, to prevent the solvent evaporation, and all measurements wereperformed at 25 °C. D2O samples at 1.0 and 2.0 mgmL–1 concentrations for MannanP.arc andMannanYeast, respectively, were analyzed. Each measurementlasted a period sufficient to obtain ∼2 million counts.SANS measurements of the samplesof Mannan33 The absolutescattering cross-sectional data d∑/dΩ were plotted asa function of q.Raw SANS data were corrected for background and empty cell scattering.Detector efficiency correction, radial average, and transformationto absolute scattering cross sections d∑/dΩ were madewith a secondary plexiglass standard.2.8–1 solution of MannanP.arc or a6 mg mL–1 solution of MannanYeast ona Quantifoil Multi A carbon-coated copper grid. After a few seconds,excess solution was removed by blotting with filter paper. The samplewas cryo-fixed by rapidly immersing into liquid ethane at −180°C in a cryo-plunge (EMGP Leica GmbH). The specimen was insertedinto a cryo-transfer holder  andtransferred to a JEM 2200 FS EFTEM instrument .Examinations were carried out at temperatures around −180 °C.The transmission electron microscope was operated at an accelerationvoltage of 200 kV. Zero-loss-filtered images were taken under reduced-doseconditions (<10 000 e– nm–2). All images were recorded digitally by a bottom-mounted 16 bitCMOS camera system . To avoidany saturation of the gray values, all of the measurements were takenwith intensity below 15 000, considering that the maximum valuefor a 16 bit camera is 216. Images have been taken withEMenu 4.0 image acquisition program  and processedwith a free digital imaging processing system ImageJ.35Cryogenic transmission electron microscopy (cryo-TEM)images were carried out at the Heinz Maier-Leibnitz Zentrum, Garching,Germany, on a JEOL 200 kV JEM-FS2200 with a field emission gun (FEG).Samples for TEM were prepared by placing a 5 μL drop of a 6.3mg mL2.9–1 concentration in water,with 0.5 nm data pitch, 2 nm bandwidth, and 20 nm min–1 scanning speed. Each spectrum was obtained as the average of threescans.Circular dichroism(CD) spectra were recorded at 4, 20, and 37 °C using a JascoJ-715 spectropolarimeter equipped with a Peltier thermostatic cellholder (Model PTC-348WI). CD measurements were carried out in the250–190 nm range, using a 0.1 cm path length cell and polysaccharidesolutions at 0.5 mg mL2.10A 10 μL droplet of sample in phosphate-buffered saline (PBS)solution was dropped from 1.4 m onto a glass microscope coverslip,which was placed on top of an aluminum plate cooled to −78°C using dry ice. The droplet froze instantly upon impact withthe plate, spreading out and forming a thin wafer of ice. This waferwas then placed on a liquid nitrogen-cooled cryostage held at −8°C. The wafer was then left to anneal for 30 min at −8°C. The number of crystals in the image was counted using ImageJ,and the area of the field of view divided by this number of crystalsgives the average crystal size per wafer and is reported as a percentage(%) of area compared to PBS control.33.1P. arcticus 273-4 was grown at 4°C as already reported.22 After centrifugation,the cells were removed and the supernatant was incubated at −20°C for 72 h with three volumes of cold ethanol. The precipitatewas then separated from the supernatant by centrifugation at 4 °C,redissolved in water and freeze-dried.Figure S1). The GC-MS glycosyl analysis as AMGof fraction A indicated the occurrence of mannose (Man) and glucose(Glc). The analysis of partially methylated acetylated alditols (PMAA)revealed the occurrence of terminal nonreducing Man (t-Man), terminalnonreducing Glc (t-Glc), 2-substituted Man (2-Man), 3-substitutedMan (3-Man), 6-substituted Man (6-Man), and 2,6-disubstituted Manunits   see 1.S. cerevisiae (MannanYeast), revealing the presence of mannose and glucose. Furthermore,the linkage analysis revealed the same points of attachment as in P. arcticus polysaccharide.36The AMG analysis was also performed on the commercialmannan fromthe yeast P.arc and MannanYeast weretestedfor the presence of proteins by the Bradford assay. No proteins weredetected in both samples.Both Mannan3.23.2.1P.arc polysaccharide, the complete setof 2D NMR experiments was performed . The 1H–13C DEPT-HSQC experiment , 5.04/103.5 (B), 5.01/99.4 (C), 4.99/99.4 (D),4.94/103.4 (E), 4.93/103.5 (F), 4.79/100.6(G), 5.34/97.5 (H) ppm, all belonging tomannose units, and the signal at δ 4.36/102.9 ppm (I) attributable to the glucose residues.To entirelycharacterize Mannan1JC1,H1 values obtained from thecoupled F2-coupled DEPT-HSQC experiment allowed us to assign the configurationsα and β for mannose and glucose units, respectively . The results obtained from methylation analysisand the correspondence of chemical shifts with those reported in theliterature37 support the hypothesis of a sugar backbone consisting of α-(1→6)-linkedmannopyranose units branched at C-2. The branches are constitutedby 2- and/or 3-linked mannose units ending with mannose or glucose,as deduced from NMR data . NOE contacts between both H1-C and H1-D with H6-C confirmed thishypothesis . The finding of both signalsof C-2 of C and D at δ 80.0 ppm indicatedtheir substitution. Residue A substituted both residues C and D, as revealed by the long-range scalarcorrelation of H1-A and C2-C/C2-D .The latter showed an additional correlation with a proton signal atδ 4.01 ppm 6-α-Man-(1→ units), highly branchedat position O-2  of commercialMannanYeast showed the lack of signal at δ 5.34 ppm,attributable to the phosphorylated mannose residues.38Indeed, the 1H NMR1 and 1H–3.2.2P.arc was subjected to acid hydrolysis by hydrofluoric acid(HF) to remove the phosphate groups, obtaining MannanP.arc_HF. This reaction allowed us to compare the shapes of mannans in solutionby considering the presence or absence of phosphorylation on the polymers.The occurrence of the reaction was checked by 1H  and two-dimensional NMR experiments.The anomeric signals of 1-P mannose units at δ 5.34/97.5 ppmwere absent in the 1H–13C DEPT-HSQC spectrum and a new anomeric signalappeared at δ 4.86/99.0 ppm. The last was assigned to 6-substitutedmannose units since C-6 of these residues were downfield-shifted toa value of δ 66.7 ppm. In addition, the methylation analysisof MannanP.arc_HF revealed a decreased amount of 2-substitutedmannose, confirming that the phosphodiester linkage involved theseunits in the arms.Mannan3.3Besides the basic structure of the polysaccharides,theinter- and intramolecular forces, such as hydrogen bonds, can remarkablyaffect their conformation, and consequently, their different activity.P.arc, MannanYeast, andMannanP.arc_HF, was carried out.Hence, an in-depth physicochemical characterization of the threepolysaccharides, Mannan3.3.1P.arc, MannanYeast, and MannanP.arc_HF at concentrations of 0.2, 0.2, and 0.1 mg mL–1, respectively, and at 4 and 25 °C reveals that all of the systemsare characterized by a monomodal distribution independent of the temperature tobe adopted to establish the molecular weight and the second virialcoefficient for each of the three polysaccharides. These parameterswere determined through a Zimm plot analysis at 4 and 25 °C,ntration 3.40P.arc, MannanYeast, and MannanP.arc_HF are 13.0± 0.9 × 104, 4.1 ± 0.4 × 104, and 10.2 ± 0.8 × 104 Da, respectively  of MannanP.arc, MannanYeast, andMannanP.arc_HF were determined at both 4 and 25 °C. Interestingly, the presence of MannanYeast does notaffect the surface tension of water. On the contrary, both MannanP.arc and MannanP.arc_HF cause a sensible increaseof the surface tension, with a ratio Rγ = γmannan/γ0 of about 1.02, anincrease of surface tension such as that observed for a NaCl 1 molL–1 water solution.30 This result suggests that MannanP.arc and MannanP.arc_HF have a marked hydrophilic character.To further investigatethe hydrophilic character of the three polysaccharides, surface tensiontitration was performed at 25 °C up to a polysaccharide concentrationof 0.1 mg mL3.3.3P.arc, MannanYeast, and MannanP.arc_HF in solutionwere obtained by means of UV circular dichroism. Samples at 0.5 mgmL–1 in water were analyzed in the far-UV regionat three different temperatures, 4 °C (growth temperature of P. arcticus), 20 °C (room temperature), and37 °C (growth temperature of S. cerevisiae).Insights intothe local conformation of the three polysaccharides MannanFigure S17).CD spectra are reported in P.arc is characterized by medium-intensityspectrawith a deep minimum at 205 nm, a shoulder around 215–220 nm,and a maximum below 190 nm. Such signals may be indicative of a helicalconformation, even if they cannot be associated straightforwardlywith a specific kind of helix. Indeed, they are not typical of α-helicesin proteins, for which two minima with comparable intensities at 222and 208 nm are expected. On the other hand, the presence of two minimawith different intensities, and in particular, one at 208 nm deeperthan that at 222 nm was reported in the case of 310 helices,in particular, a ratio [θ]222/[θ]208 ≈ 0.4 was taken as an indication of the presence of 310 helices.45 In our case, thisratio is about 0.5, but the shifted position of the minima, i.e.,205 nm and 215–220 nm, seems in contrast to this possibility.In the case of proteins, a minimum ranging between 195 and 205 nmis often reported as a spectral feature of polyprII helix, but inour case, the lack of the maximum at 220 nm, the other signature featureof this secondary structure,46 seems topoint against such a conformation. A deep minimum ranging between205 and 210 nm has been reported as a signature spectral feature ofcollagen fibrils, a peculiar supramolecular architecture formed bythe triple helix of collagen.47Mannan48 The presence of an extra carbon atom, as wellas the possible introduction of constraints like cyclic ring systemscould make β-amino acids look more like sugars than αamino acids. CD spectra of MannanP.arc can indicate thepresence of helical structures usually formed by β-peptides,such as the 14-helix. In this respect, it is interesting to note thata 14-helix-bundle formed by a β-peptide has a CD spectrum almostidentical to that of our polysaccharide. Therefore, we can infer thatMannanP.arc adopts a local helical conformation, but itsstructural features cannot be univocally defined. Moreover, inter-and intramolecular interactions between different helices are likelyformed determining modification of the spectra with respect to thoseof known secondary structures.Notably,CD spectra with a marked minimum ranging between 205 and215 nm have been recorded for β-peptides with different helicalconformations. β-Peptides are composed of β amino acids,having an additional carbon atom in the backbone of each residue.They have higher conformational flexibility than α amino acidsand may have access to additional secondary structures.Yeast, we observe much less intenseminima than those of MannanP.arc that are positioned at222 and 208 nm and a maximum below 190 nm. Signals at these wavelengthsare usually associated with α-helical conformations, as saidbefore. However, the very low intensity of the minima points towarda very low degree of structuration. Finally, spectra of MannanP.arc_HF have a single minimum centered at 200 nm that is muchmore intense than minima in the spectra of the other polysaccharides,typical features of disordered random coil conformations. It clearlyemerges that dephosphorylation treatment completely changes spectralfeatures of mannan from P. arcticus, likely breaking hydrogen-bonding interactions giving rise to thehelical conformation.In the case of MannanP.arc and MannanP.arc_HF, whereas it increaseswith increasing temperature for MannanYeast, in agreementwith the different origin of the polysaccharides.Finally, for what concerns the effectof temperature, it is worthnoting that the intensity of spectra, that is associated with thedegree of structuration, decreases with increasing temperature forMannan3.3.4–1 for MannanP.arc, MannanYeast, and MannanP.arc_HF, respectively,were analyzed by means of DLS. At these high concentrations, correlationfunctions particularly of MannanP.arc do not reach zerovalues at long times , indicating the presence of suspended very large particles andthe beginning of a precipitation process that do not allow determinationof DLS profiles. So, samples at about 1 mg mL–1 wereused for DLS analysis . DLS profiles of these samples recordedat 25 °C  at 6.3 mg mL–1 confirmthe self-aggregation process evidenced by DLS and indicate the formationof large ribbon structures with length in microns and a diameter ofabout 40 nm, which were likely responsible for the behavior of thecorrelation function at long times. Such a structure is evocativeof a fibril, similar to that of the collagen, which is indeed characterizedby a diameter of the order of tens of nanometers.50 This finding is quite interesting, also considering indicationsfrom CD spectroscopy that could suggest supramolecular aggregationof helical segments into fibril-like arrangements. In the case ofMannanYeast  7 at abouHigh statistical averaging and short wavelength typical of SANSmake direct structural investigations possible on characteristic lengthscales of a polymer chain, from 1 to 100 nm.P.arc and MannanYeast, thescattering profile is characterized by three distinctregions: one at small q, which corresponds to largescale in direct space and describes the objects or their aggregatesas a whole; one at an intermediate q range, correspondingto a characteristic length scale in direct space, where one probesthe Flory exponent ν describing global chainconformation; and finally one at large q values,where the local conformation of the chains is probed. The main differencefeature between the two systems is in the region around q = 0.03 Å–1, where for MannanYeast, a flat region is observed, and for MannanP.arc, a profilewith a slope of −1.8 is visible. At a small q, the scattering profile decays with −2.4 and −2.9slopes for MannanYeast and MannanP.arc, respectively.In both cases, this is an indication of the clustering phenomena ofthe chains. At the higher q range, both profilespresent a power law with a slope of about −1. In this region,the q–1 scattering intensity decayindicates that on a local scale, the polysaccharide chain has a rigid-rodbehavior, which can be also confirmed by the analysis of the standardKratky plot, reporting q2*I(q) vs q . It is to note that small deviations from the −1scaling in this region are a likely result of noise in the data and/orbackground subtraction; indeed, the order of magnitude of the I(q) values in this range of q makes them very sensitive to data processing.Inspection of q one, where the two polysaccharideshave very different behaviors:while the MannanYeast profile presents a shoulder thatis probably due to a lack of an efficient hydration of chain, as SANSinvestigation on similar system suggests,54 in the case of MannanP.arc, a slope of −1.8 indicatesthat the chain is well hydrated, and that on a large scale, it hasa flexible behavior. In this case, we can determine the Flory exponent ν from qν–1/ obtaining ν = 1/1.8 = 0.56, which isvery close to the 3/5 value typical of flexible polymer chains ina good solvent.55The most interestingregion is the intermediate Yeast, the following equation wasfitted to the scattering profile using a modified correlation function.A and B, the incoherent background bgk, and the exponent n are used as fitting parameters. The exponent n canbe related-to the interaction between the polysaccharide chain andthe solvent. The fitting procedure produced values for the correlationlength ξ = 25 ± 1 Å and for n = 2.4± 0.1. The latter value is related inversely to the excludedvolume parameter, in particular to the Flory coefficient ν = 1/n = 0.42. A v value between0.42 and 0.33 suggests a self-attractive interaction within the chainand nonefficient interactions with the solvent.54In the case ofMannanP.arc, a characterizationwas obtainedthrough a q1.8*I(q) vs q representation , which has a typical correlation distance ξ, and to the q-limit of the regime, for which the scattering exclusivelyarises from the stiffness (q1). From qξ ≈ 0.011 Å–1, we can calculate a correlation distance that resulted to be notlarger than ∼600 Å by means of ξ = 2π/qξ. The upturn below qξ depends on the presence of aggregates and, inthis respect, it should be noted that the qξ value could be somewhat affected by the scattering intensity bylarge aggregates. On the other hand, as said, the q–1 decay proves that the MannanP.arc chains have a local stiffness and a rodlike behavior; therefore,they are semiflexible chains. From the q1 value, it is possible to evaluate the local rigidity, representedby the persistence length lp, throughthe relation for polymer chains in good solvent (ν = 0.56) q1 ≈ 3.5/lp. The calculated lp ∼40 Å corresponds to 9/10 sugar units, that is, about two repeatingunits of MannanP.arc.In the case of Mannanentation 8,55 whic3.456 IRI was determined by the splat assay, wherebya polynucleated ice wafer was allowed to grow at −8 °Cfor 30 min, and the relative size of the crystals compared to a PBScontrol was evaluated. Smaller ice crystals indicate greater ice recrystallizationactivity. The results highlighted some weak ice recrystallizationinhibition activity of the MannanP.arc polymer, which wasslightly higher than the IRI activity of MannanYeast .It is important to note that sufficient concentrations of any macromoleculecan slow down ice growth and that conducting the assays in the presenceof saline  is essential to remove false positives.59To determineif these polysaccharides had the capacity to modulate ice growth andice recrystallization inhibition (IRI), assays were undertaken.nanYeast 9. It shoP.arc_HF was then tested for the IRI activity before being lost completely at 1.25 mg mL–1, but this is not a significant difference. The bioassays indicatedthat the lack of phosphate groups did not substantially affect theactivity and do not determine the lack of the activity comparableto that of MannanYeast.Mannanactivity9. Intere58 The exact relationship between IRI and cryoprotectantoutcomes is also not fully understood, and hence these (or other)extracellular polysaccharides from cold-acclimatized bacteria whichdo not have significant IRI, may provide protection by another mechanism.While the activity for allsamples seen in this work is weak, severalpolysaccharides, such as those based on fucose, are emerging as potentcryoprotectants and hence the study of polysaccharides with any activitymay form the basis for discovering new cryoprotectants.4Pseudomonas mutabilis,60 the isolated mannan showed high-viscositysolution, whereas in Rhodopseudomonas palustris,61 the polysaccharide solutions havebeen reported to influence the growth of beneficial gut microbiota.Neither these studies nor those reporting the structure and the activitiesof mannans isolated from fungi have considered the structure/activityrelationships. In addition, up to now, only a few papers describingconformational analysis of microbial extracellular polysaccharidesare known; therefore, there is a need for bridging this gap.Mannan polysaccharides can befound as extracellular microbialcomponents. They are usually highly branched polymers, characterizedby the absence of a defined repeating unit. Therefore, they are mainlyinvestigated for their activity, whereas the deep structural investigationis often overlooked. For example, in P. arcticus 273-4 (MannanP.arc) displayingweak to moderate IRI activity higher than that of S.cerevisiae (MannanYeast). The MannanP.arc structure is constituted of a backbone of 6-substitutedmannose residues, which is highly ramified at C-2, with di- and trisaccharideside chains containing 2- and 3-substituted mannose units, respective.Glucose residues can be found as a terminal unit of some arms. Thephosphodiester linkage connects some terminal mannose units to the2-substituted ones. Our results indicate that this structural featureagreed with previously published data.62In this study, we present the structure and conformational analysisof a mannan extracellular polysaccharide purified from the cold-adapted P. arcticus 273-4produces both CPS63 and mediumreleased polysaccharides, and only the last have found to displaymoderate IRI activity (this study). To date, the production of mannanpolysaccharides is documented for other psychrophiles,64 even if to our knowledge, no reports about the crucial role of theconformation that directly affects the activity have been found inthe literature.It isworth noting that P.arc, MannanYeast, and MannanP.arc_HF, and compared.The latter was obtained from MannanP.arc using hydrofluoricacid, to evaluate the role of the phosphorylated mannose in definingthe observed shape.Therefore, we aimed to evaluate several physicochemicalpropertiesof the mannans in solution to figure out their possible differentshapes. The three polymers were considered, i.e., MannanP.arc is more marked and increases with decreasingtemperature, as shown by the second virial coefficient values. Moreover,both MannanP.arc and MannanP.arc_HF were characterizedby a distinct hydrophilicity typically observed in moderate concentratedsalt-water solution, as highlighted by surface tension measurements.All of the three molecules showed an efficienthydration, whichfor MannanP.arc and MannanYeast isalso proved by the SANS profiles at low q values.SANS analysis also indicates that both polysaccharides adopt a localrigid structure along the chain. MannanP.arc is a semiflexiblechain characterized by a rigid part of the chain that, according tothe calculated persistence length value, is not especially large,as, in line with those of most polysaccharides, extends up to about9–10 residues of the molecules. According to CD measurements,these rigid-rod regions are likely to adopt a helical conformationand interact by forming hierarchical organization. These helical regionsencompass a relatively few residues, presuming a conformation similarto that of helices formed by β-peptides, and it is worth sayingthat, in contrast to α-helices, these may be stable even whenformed by only a few residues.65At high concentrations, all of the three polysaccharides showedthe tendency to form a larger structure, even if a significant concentrationof free chain is still present, as clearly indicated by intensity-weightedand number-weighted DLS profiles, respectively. The presence of aggregatesin the case of Mannan19 and contributeto the overall conformation of the polysaccharide or to the formationof aggregates. Cryo-TEM images seem to confirm the latter hypothesis,showing the formation of very large ribbon-like structures evocativeof fibrils in the case of MannanP.arc. On the other hand,MannanYeast seems to be characterized by a very low degreeof structuration in α-helical conformation. Finally, CD showsthat dephosphorylation destroys the local organization of the polysaccharidechain, with the CD spectra of MannanP.arc_HF being typicalof disordered random coil conformations, by altering intramolecularinteractions stabilizing the helical segments or the supramolecularassembly. Indeed, charge modifications can destroy superhelical organizationand isolated helices may become unstable and unfold.19On the other hand, the organization in hierarchical architecturesmay stabilize helices that are intrinsically unstable, such as thecase of other polysaccharides,P.arc_HF didnot show significant differences with respect to the intact polymer,we concluded that the phosphate groups did not display a relevantrole in the bioactivity of the P. arcticus polymer. However, the presence of helical domains in MannanP.arc can be crucial for other activities since these domainsare often responsible for interchain associations giving rise to athree-dimensional network with viscoelastic behavior, a gel, by providingnoncovalent cross-linking in the junction zones.Finally, since the IRI assays of Mannan67 other macromolecules, such as EPS, may accomplish their protectiverole through increased rigidity and structuring.Our resultsmay suggest a significant difference between “functional”and “structural” cold adaptation mechanisms that needfurther investigation: while enzymes and membranes of psychrofilicorganisms preserve their functional role at cold temperatures throughan either overall or local increase of flexibility and disorder,5P. arcticus bacterium, its dephosphorylated derivative, and the mannan from S. cerevisiae.This study presents a methodology that is not commonly used forestablishing a correlation between the structure and the shape ofpolysaccharides in solution in the absence of a defined oligosaccharidicrepeating unit. The approach exploits different physicochemical methods,some of which, like cryo-TEM, have never been used for obtaining suchinformation. We employed this approach to understand the differencesamong the mannan from the Siberian permafrost P.arcticus bacterium has an efficient interaction withthe water molecules, and it is structurally characterized by a rigidpart of the chain, about 9/10 sugar units, i.e., two repeating unitsof MannanP.arc, that assumes a helical conformation. Dephosphorylationseems to destroy this local organization of the polysaccharide chain.Finally, the mannan from S. cerevisiae seems to be characterized by a very low degree of structurationin α-helical conformation. The psychrophilic mannans showeda weak IRI activity if compared with the nonactive mannan’syeast. Future studies will clarify which structural feature is responsiblefor the different behavior.The collected data reveal thatthe mannan from


Through high-throughput calculation of Fe/BTS ATMTD, a maximum of calculated transverse thermoelectric figure of merit of 0.15 was obtained at a thickness ratio of 0.49 and a tilted angle of 14°. For fabricated ATMTD, the whole Fe/BTS interface is closely connected with a slight interfacial reaction. The optimizing Fe/BTS ATMTD with 12 mm in length, 6 mm in width and 4 mm in height has a maximum output power of 3.87 mW under a temperature difference of 39.6 K. Moreover the related power density per heat-transfer area reaches 53.75 W·m−2. This work demonstrates the performance of Fe/BTS ATMTD, allowing a better understanding of the potential in micro-scaled devices.Transverse thermoelectric performance of the artificially tilted multilayer thermoelectric device (ATMTD) is very difficult to be optimized, due to the large degree freedom in device design. Herein, an ATMTD with Fe and Bi Thermoelectric (TE) technology is well-known for its capability to directly convert heat into electricity, and it has a great value in power generation, cooling, thermal detection, etc. ,2,3. Thex on the length to height ratio is greatly beneficial in developing the micro/nano-scaled devices. It works best for the extremely long and thin device [Transverse TE counterparts have been proposed as an alternative approach, whereby it can generate off-diagonal element I and Q ,17. The n device ,22,23,24zx) in ATMTD is defined as ZTzx = σxxSzx2T/κzz, where the ZTzx, σxx, Szx, κzz and T are the transverse figure of merit, transverse electrical conductivity, transverse Seebeck coefficient, transverse thermal conductivity and absolute temperature, respectively [zx of ATMTD, such as Al/Si [2Te3 [0.5Sb1.5Te3 [3/Bi0.5Sb1.5Te3 [0.5Sb1.5Te3 [zx, since it takes advantage of the transverse TE effect in an artificially tilted multilayer structure. A rational design for the given combinations that guides the ZTzx optimization is highly desired.One of the transverse TE counterparts, the artificially tilted multilayer thermoelectric device (ATMTD), has gained increasing attention, because, in addition to the three distinct advantages above, the artificial combination can be almost any material ,26. Thisectively . Recentlas Al/Si , Bi/Cu [as Al/Si , Pb/Bi2TSi [2Te3 , Ni/Bi0.Sb1.5Te3 ,32,33, YSb1.5Te3  and Bi/BSb1.5Te3 . However2Te2.7Se0.3 (BTS) and p-type pure irons (Fe) were selected as the combinatorial materials for a new ATMTD. Because BTS has a large Seebeck coefficient, small electrical and thermal conductivities near room temperature, Fe has a small Seebeck coefficient, large electrical and thermal conductivities [zx [zx value is in good agreement with theoretical ones. The maximum output power and related power density per heat-transfer area are about 3.87 mW and 53.75 Wm−2 under a temperature difference of 39.6 K, respectively.In this study, n-type Bitivities . The larties [zx . To explFe and dBTS. After the stacking layer plane is tilted at an angle, θ, against the ATMTD surface (θ ≠ 0° or 90°), the anisotropy of transport parameters comes from two alternately stacked layers, parallel and perpendicular stacking-layer planes, as well as tilt. This means that the Seebeck coefficient of Fe/BTS ATMTD is anisotropic and the off-diagonal Seebeck coefficient of Fe/BTS ATMTD is non-zero, which provides the theoretical background to see transverse TE effect. As shown in z) in Fe/BTS ATMTD is established along the z-axis under the hot-side temperature (TH) and cold-side temperature (TC), it will yield a transverse TE electromotive force (ΔVx) along the x-axis. The ΔVx can be expressed as follows:zx is the difference of Seebeck coefficient in the z-axis and x-axis direction of Fe/BTS ATMTD.The structure of ATMTD contains two alternately stacked layers, Fe and BTS, with thickness dx and other TE performance of Fe/BTS ATMTD are relevant to ZTzx = σxxSzx2T/κzz. To maximum ZTzx values, we begin with a description of the off-diagonal elements  along the different directions. It can be described by the tensors (M):The ΔVxx, κzz and Szx can be given as a function with tilted angle, θ, of the stacking layer plane in Fe/BTS ATMTD, which includes σ//, σ⊥, S//, S, κ//, κ⊥ and θ.Then, the σ//, σ⊥), Seebeck coefficient  and thermal conductivity  parallel and perpendicular to the stacking layer plane become as follows:⊥” denote the directions parallel and perpendicular to the stacking layer plane of the Fe/BTS ATMTD. Moreover, σFe, σBTS, SFe, SBTS, κFe and κBTS are the electrical conductivity of Fe, electrical conductivity of BTS, Seebeck coefficient of Fe, Seebeck coefficient of BTS, thermal conductivity of Fe and thermal conductivity of BTS, respectively; and δ = dFe/(dFe + dBTS) represents the thickness ratio of Fe layer in Fe/BTS ATMTD.To illustrate how transport behaviors arise in alternately stacked layers, we considered the simplified model below. Assume that the electrical and thermal contact resistances in the Fe/BTS ATMTD could be ignored. Combined with Kirchhoff’s theory , the eleFe, σBTS, SFe, SBTS, κFe and κBTS) of constituent materials and the geometrical configuration (δ and θ) of Fe/BTS ATMTD are the key factor to maximum ZTzx values. The solution for ZTzx is complex. A high-throughput calculation to investigate the relevance between the transverse TE properties is very important.Note that both the TE parameter  and two-step wire-cut electrical discharge machining (WEDM) method. The specific processes are presented in The microstructure and element distribution at the interface were characterized by electron probe microanalysis  equipped with an X-ray spectroscopy detector. The power generation performance of Fe/BTS ATMTD was evaluated at a temperature difference from 10 to 40 K, using a self-made measuring equipment that was presented in our previous report ,34.zx from a large number of combinations from δ and θ, a high-throughput calculation for σxx, Szx, κzz and ZTzx was implemented under different δ and θ values. The room-temperature electrical conductivity (σ), Seebeck coefficient (S), and thermal conductivity (κ) of Fe and BTS in the calculation model refer to To quickly screen the maximum ZTxx gradually increases with increasing the δ value from 0 to 1, due to the increased thickness of high conductivity Fe layer in Fe/BTS ATMTD , which results from the interfacial reaction between Fe and BTS.The BEI and line distributions of Fe, Bi, Te and Se elements were also conducted on the Fe/BTS interface by a wave-dispersive spectrometer (WDS), as shown in Prior to recording the power-generation performance, a V–I measurement was performed for the packaged Fe/BTS ATMTD at a fixed ΔT of 20 K in the self-made measuring equipment . The insz values. The theoretical ΔVx values were estimated according to the equation ΔVx = SzxΔTzl/h, where Szx is the transverse Seebeck coefficient of the as-prepared Fe/BTS ATMTD, l is the length of the as-prepared Fe/BTS ATMTD, h is the height of the as-prepared Fe/BTS ATMTD and ΔTz is the temperature difference along the z direction. It can be seen that a ΔVx along the x direction has generated when a ΔTz applies along the z direction, clearly demonstrating the transverse Seebeck effect  and output power (P) as a function of work current (I) for Fe/BTS ATMTD under different ΔT are depicted in −2.To characterize the power generation performance of the as-prepared Fe/BTS ATMTD, the electrical output characteristics were evaluated with the self-made measurement system under various ΔTk effect a. The ex 30 μV/K . The ΔVxx of 0.15 when δ = 0.49 and θ =14°. According to the optimized δ and θ, a Fe/BTS ATMTD of 12 mm in length, 6 mm in width and 4 mm in height was fabricated via the SPS and two-step WEDM method. Our microstructure analysis showed that the whole Fe/BTS interface is closely connected with a slight interfacial reaction. The thickness of interface reaction layer is only 8 μm. The maximum ΔVx value is up to 0.7 mV and 3.4 mV when ΔTz = 9.4 K and 42.2 K, respectively. The Fe/BTS ATMTD achieved a maximum P of up to 3.87 mW under a ΔT of 39.6 K, and the related power density per heat-transfer area reached 53.75 W·m−2. In the future, the performance of Fe/BTS ATMTD will be further improved if the height of the device is in the order of micron. This work demonstrates the versatile application of Fe/BTS ATMTD in power generation.A high-throughput calculation was developed to rationally design the geometrical configuration of high-performance Fe/BTS ATMTD. It was revealed that the Fe/BTS ATMTD exhibits a maximum ZTz


Brachytherapy (BT) has long been used for successful treatment of various tumour entities, including prostate, breast and gynaecological cancer. However, particularly due to advances in modern external beam techniques such as intensity-modulated radiotherapy (IMRT), volume modulated arc therapy (VMAT) and stereotactic body radiotherapy (SBRT), there are concerns about its future. Based on a comprehensive literature review, this article aims to summarize the role of BT in cancer treatment and highlight its particular dosimetric advantages. The authors conclude that image-guided BT supported by inverse dose planning will successfully compete with high-tech EBRT in the future and continue to serve as a valuable modality for cancer treatment. Brachytherapy (BT) has a long history of successful use in the treatment of various cancers with excellent clinical outcomes, but its use rate within radiotherapy has varied over time. During the first decade of this century there was a steady increase in prostate, breast and gynaecological BT in Europe , but sinHowever, other opinions have also been expressed in support of BT. Evidence suggests that BT is considered mandatory in the treatment of locally advanced cervical cancer, and concerns have been raised that some physicians are trying to replace it with an EBRT boost . In cervThe aim of the present study is to provide a dosimetric overview of the role of BT in the treatment of different anatomical sites of cancer patients in the age of advanced external beam radiotherapy. Following a thorough literature search, the dosimetric assessment was based on a review of relevant publications and our own experience.Studies comparing the dosimetry of BT and EBRT were collected from the literature and the advantages and disadvantages of BT compared to EBRT were evaluated. Data collection was performed by an online search of the PubMed database and a systematic review of the tables of contents of standard radiation oncology journals. For the online search the following keywords and their various combinations were used: “brachytherapy”, “conformal external beam radiotherapy”, “3D-CRT”, “intensity-modulated radiation therapy”, “IMRT”, “volumetric modulated arc therapy”, “VMAT”, “stereotactic body radiotherapy”, “SBRT”, “CyberKnife”, “dosimetry” and “dosimetric comparison”. Studies from 2000 onwards were accepted for evaluation and the last search was carried out in June 2021.Only studies with at least five patients were selected for detailed analysis. Articles were grouped and screened by treatment site.1 × c2, where c1 is the ratio of the volume of the PTV receiving at least the PD to the volume of the PTV, and c2 is the ratio of the volume of the PTV receiving at least the PD to the volume receiving at least the PD.Volume (Vx) and dose (Dx) parameters were recorded and compared. Vx is the percentage of the organ volume receiving at least x% of the prescribed dose (PD), and Dx is the minimum relative or absolute dose of the PD delivered to x% of the organ. Some studies have also compared the conformal index (COIN). The COIN is cBT has been used for more than a century to successfully treat various cancers, and numerous studies have investigated its dosimetric characteristics compared to new emerging EBRT techniques. Table Six articles with very few patients were found that examined technical considerations and/or the feasibility of using EBRT as an alternative to BT. Three studies, using only one patient, investigated whether 3D-CRT and IMRT can achieve a brachy-like dose distribution for cervical cancer –70. BT iAs there are no generally accepted and commonly used dose–volume parameters for evaluation of the plans, different quantifiers have been used in the studies, making comparisons difficult. The most commonly used parameters in the reviewed studies are shown in Table In a study by Milickovic et al. , treatmep < 0.05). The same was true for the heart, with values of 0.4 Gy and 0.7 Gy, respectively (p < 0.05). The median V2.8 Gy was also less with BT , and its maximum value was only 0.2 Gy in BT compared to 5.9 Gy in VMAT. There was no difference in doses to skin represented by D1cm3, (15.9 Gy vs. 15.2 Gy), but the contralateral breast received less dose with BT . The authors concluded that both techniques are feasible for this two-dose level, single-fraction ablative radiotherapy for early-stage breast cancer, but the PTV is overdosed with BT. We note that this is a natural consequence of an inhomogeneous dose distribution with hotspots around the sources. A detailed dosimetric comparison of multicatheter interstitial BT (MIBT) and IMRT plans for partial breast irradiation was performed by Major et al. [p < 0.05, V50: 13.7% vs. 25.5%, p < 0.05). The ipsilateral lung was better protected with BT, all parameters were significantly smaller . The heart dosimetry was generally better for IMRT. The mean heart dose for left-sided lesions (n = 21) was 2.0% for IMRT and 4.5% for BT (p < 0.05), but the D2cm3 was lower with BT . For skin the D1cm3 was 60.2% for BT and 87.8% for IMRT (p < 0.05), and regarding the ribs, all parameters were significantly lower with BT. Dose to contralateral breast and lung was also smaller with BT, with a significant difference for D0.1 cm3 and D1cm3.BT has been used for partial breast irradiation for decades, initially only in clinical trials, but it is now a standard treatment method in selected patient groups due to its good clinical results . Boost dr et al. . For 34 p < 0.05). In another study, treatment plans of 30 patients treated with interstitial BT and another 30 with 3D-CRT were compared [p = 0.05) and the V50 was 25% vs. 50% (p < 0.05). For ipsilateral lung the V30 was 1% vs. 8% (p < 0.05), and for heart the Dmax was 25% vs. 49% (p < 0.05), respectively.Interstitial BT was also compared with 3D-CRT for breast cancer , 21, 76.1cm3 was 86.1% vs. 46.1% (p = 0.0018), ipsilateral lung D1cm3 45.0% vs. 38.4% (p = 0.0272) and ribs D1cm3 73.6% vs. 49.0% (p = 0.0013), but the heart was slightly better protected with CK . Then, in another study, MIBT and CK were dosimetrically compared using two separate patient cohorts [1cm3 for skin was 58.4% vs. 79.0% (p = 0.0007), and for ribs 43.1% vs. 69.0% (p = 0.0004) in the MIBT and CK plans, respectively. The mean doses to the ipsilateral lung were the same (4.9%), but doses to most exposed small volumes were significantly smaller for MIBT. The heart parameters were smaller with CK, but the differences were not significant. Doses to contralateral breast and lung were small with both techniques, but CK resulted in smaller values. We note that in both studies [Following the previous study with IMRT, the same group investigated the dosimetric characteristics of stereotactic irradiation with CyberKnife (CK)  in treatment of the partial breast. First, virtual MIBT plans were created on CT images used for CK treatments . In the  cohorts : 32 APBI studies , 23, CK 1cm3 values for the ipsilateral lung are 37.8% vs. 48.2%, D1cm3 values for the skin are 54.9% vs. 84.3%, and D1cm3 values for the ribs are 45.9% vs. 70.6% in favour of BT.Analysis of the pooled data from the three studies , 22, 23 1cm3 and D2cm3 for rectum, bladder and sigmoid were very similar for all plans, but at the cost of lower D90 of the PTV in EBRT plans compared to BT. The total volumes irradiated with doses higher than 3 Gy, 5 Gy and 7 Gy were about twice as large for EBRT as for BT. From their study, the authors conclude that both IMRT and IMPT appear to be inferior to BT. In another study, for 10 cervical cancer patients, Shwetha et al. [1cm3 and D1cm3 for the rectum were significantly lower with BT , and the bladder also received less dose with BT . There was also a significant difference in the volume of normal tissue receiving 10% of the PD . It was concluded that IMRT may be the treatment of choice for cervical cancer when HDR-BT is not available or when a noninvasive treatment technique is preferred. IMRT was compared to interstitial BT for cervical cancer by Sharma et al. [p = 0.034), the coverage was better, and the doses to bladder and maximal dose to rectum were significantly lower in BT plans. The authors conclude that interstitial BT provides better dosimetry compared to IMRT and recommend it as a standard treatment for cervical cancer patients who are not suitable for intracavitary BT. SBRT with CK was also compared to BT. In a feasibility study, Cengiz et al. [Gynaecological cancers are the most common treatment sites for BT. For the first time, Schefter et al.  simulatea et al.  generatea et al. . Twelve z et al.  generatez et al.  investig2cm3 for rectum, bowel and bladder were 47.9%, 39.1% and 30.9% for BT and 89.5%, 91.1% and 78.6% for IMRT, respectively. The NTCP  values were higher for 3D-CRT plus BT than for 3D-CRT plus IMRT, but it should be noted that BT included three different fractionation schemes with both HDR and PDR treatments.In a recent paper, a dosimetric and radiobiological comparison of IMRT and intracavitary (IC) BT boost following 3D-CRT whole-pelvis irradiation for gynaecological cancer was performed . IMRT bop = 0.05). The dose to the bladder was slightly lower for SBRT but higher to rectum and sigmoid. For bowels and femoral heads, BT resulted in significantly lower doses compared to SBRT. Based on these results, the authors conclude that SBRT with HT is a feasible treatment option and is regarded as a reasonable potential back-up alternative to IC BT for endometrial cancer. In another comparison of HDR-BT and SBRT for inoperable endometrial cancer, doses to normal tissues were higher for SBRT plans [Jones et al.  evaluateRT plans .mean: 19.6% vs. 33.5%) and bladder (Dmean: 25.9% vs. 32.5%), with similar target coverage and less inhomogeneity. However, the integral dose was 7.2% higher for IMRT. For most parameters, Yildrim et al. [2cm3, which was significantly lower for BT. Recently, a study on irradiation of the vaginal cuff with VMAT and BT in postoperative endometrial cancer was published [0.1 cm3: 131% vs. 112% for rectum and 129% vs. 108% for bladder). However, BT was superior in terms of dose falloff outside the CTV, dose to femoral heads and integral body dose. These results are in very good agreement with the findings of Pedicini et al. [Dosimetric comparisons for vaginal irradiation have also been published , 71, 72.m et al.  found veublished . Using Bi et al. . In cont0.1 cm3: 77.0% vs. 85.4%, p < 0.05; D2cm3: 48.4% vs. 68.4%, p < 0.05 for mandible; D0.1 cm3: 9.7% vs. 12.3%, p < 0.05 and D2cm3: 9% vs. 10.0%, p < 0.05 for spinal cord for BT and VMAT, respectively). Doses to the parotid salivary glands were generally lower for BT. Six out of ten parameters were significantly lower with BT for both ipsilateral and contralateral glands, for example, D0.1 cm3  and D2cm3  for ipsilateral glands. All parameters for the ipsilateral submandibular glands were lower with BT, but the differences were not significant, whereas for contralateral submandibular glands, all but one parameter was significantly lower with BT. Note that the volumes of the PTVs in this study were exactly the same for both treatment modalities, as there was no additional margin around the CTVs for VMAT planning. However, with EBRT, the target volume is usually larger due to setup errors and patient movement. It follows that if larger PTVs had been used in VMAT plans, the dose to OARs would have been higher, which would have further highlighted the superiority of BT.A comparative dosimetric analysis of target volume and OARs between interstitial HDR-BT and VMAT for localised head and neck cancer was performed at our institute . Thirty-3 (BT) to 73.2 cm3 (SBRT). The PD was the same (15 Gy or 20 Gy depending on histology), and the same OAR dose constraints were used for both techniques. With BT, the target coverage was better, especially in the 20-Gy PD group. The mean D99.9, which is effectively the minimum dose in the PTV, was 19.9 Gy in BT and 17.5 Gy in SBRT plans (p < 0.001). D90 was significantly higher in the BT plans (24.3 Gy vs. 16.5 Gy for 15-Gy PD and 29.2 Gy vs. 20.6 Gy for 20-Gy PD) due to a more heterogeneous dose distribution of BT. For BT, the dose to the liver was lower. In the 20-Gy PD group, the mean V5Gy was 611 cm3 in the BT and 694 cm3 in the SBRT plans (p = 0.001). These volumes correspond to 41.8% and 45.9% of the liver volume (p = 0.007), respectively. In the 15-Gy PD group, the same volume parameter was lower in the BT plans, with no statistical difference, but for the whole patient cohort, BT was significantly more favourable. In a recent study, Walter et al. [In one study, virtual BT plans were created on CT images taken for stereotactic ablative radiotherapy (SABR) of liver metastasis, and the BT and SABR plans were compared dosimetrically . PTV wasr et al.  comparedp = 0.03), but there was no difference between the two modalities for V95 and V90. Conformity was better  and the dose gradient was significantly higher  for BT. The maximum spinal cord dose was 12.8% in BT and 21.1% in SBRT plans (p = 0.022). Differences in the other dosimetric parameters were not significant for OARs, but BT resulted in lower values for three out of five parameters. Chan et al. [3. Since the most advanced technology of real-time tumour tracking was used for SBRT, there was no difference in PTV volumes (no CTV–PTV margin). Two VMAT planning approaches were used, a margin-based and a robust optimization method. Dose distributions were calculated using the same category of dose calculation algorithms (collapsed cone convolution), with tissue heterogeneity corrections also in BT planning [p < 0.05, respectively) and the R50 dose gradient (volume ratio of 17 Gy to 34 Gy) was better . The results from the margin-based method for SBRT plans are given below. In the low dose range, the whole lung received a higher dose with BT (V5Gy: 239 cm3 vs. 237.7 cm3), but the trend was reversed at higher doses (V7Gy: 153 cm3 vs. 156 cm3 and V20Gy: 34.7 cm3 vs. 36 cm3). Dose constraints per the RTOG 0915 protocol were achieved for all critical organs except in some cases for the chest wall in BT. However, the overall mean Dmax and D1cm3 were lower in the BT plans . All other DVH metrics showed no statistical difference between BT and SBRT. Based on their results, the authors conclude that SBRT using an advanced MLC tracking technique and non-coplanar VMAT can achieve dosimetric quality comparable to HDR-BT, and that the high GTV dose in BT may be beneficial for enhanced killing of radioresistant tumour cells.In one study, Milickovic et al.  selectedn et al.  performeplanning . The PD planning . In the mean: 3.9 Gy vs. 6.6 Gy in IMRT and 5.6 Gy in HT). The bladder was also better spared by BT, and V10% for normal tissue was more than eight times lower with BT than with IMRT or HT. In another early study, treatment plans of HDR-BT and IMRT for localised prostate cancer were compared using two separate patient cohorts [mean for rectal wall was 10.5 Gy for HDR-BT and 28.2 Gy for VMAT, and in between for the other techniques. The V50 in cm3 was 1.5, 4.1, 7.2, 7.0 and 4.7 for HDR-BT, LDR-BT, VMAT, IMPT and IMIT, respectively. In the same order, the V50 for bladder wall was 0.6, 2.8, 5.5, 5.3 and 3.2 cm3, respectively. For the five techniques, the volumes of normal tissues that received 30% of the PD were 42, 120, 487, 198 and 253 cm3. The Dmean for urethra was lowest for HDR-BT (62.9 Gy) and highest with LDR-BT (106.6 Gy). The BT techniques resulted in negligible doses to femoral heads (Dmean < 2 Gy), while the EBRT techniques resulted in a Dmean of nearly 20 Gy on both sides. In another study by Fukuda et al. [For localized prostate cancer, BT has been the treatment of choice for decades, including LDR seed implantation and HDR monotherapy, as well as for a boost following or during teletherapy. But various EBRT techniques are also available and have excellent results in clinical applications. In an early study, HDR-BT dose distributions for prostate cancer were approximated by CK with very similar dosimetry but using identical PTVs for both techniques . HDR-BT  cohorts . In the  cohorts  investig cohorts  performe cohorts . For eaca et al. , when HD1cm3 was lower with BT. Sannamed et al. [p < 0.05). Due to its dosimetric characteristics, the authors consider HDR boost as the best technique to deliver boost dose to the GTV located near critical structures. In a recent study, Chatzikonstantinou et al. [p < 0.0001 and 32.7 Gy vs. 37.4 Gy, p = 0.0004, respectively). However, in terms of urethral dose, HDR-BT was worse, with a maximum dose of 45.6 Gy compared to 40.8 Gy (p < 0.0001) for SBRT. From these results, the authors conclude the superiority of HDR-BT for rectal and bladder dosimetry. Fröhlich et al. [2cm3 for the rectum and bladder being lowest in HDR-BT (36 Gy and 51.4 Gy), while D0.1 cm3 for the urethra was lowest (79.9 Gy) in VMAT. The hips and sigmoid were best protected by HDR-BT. In a feasibility study, Willigenburg et al. [p < 0.001) for the urethra, and D2cm3 was 8.6 Gy vs. 10.7 Gy (p < 0.001) for the rectum in the BT and SBRT plans, respectively.Dosimetry of boosting of the dominant intraprostatic lesions (DIL) with VMAT, intensity-modulated proton therapy (IMPT) and HDR-BT was investigated by Andrzejewski et al. , and HDRd et al.  used sinu et al.  comparedh et al.  performeg et al.  created 0.1 cm3 was very similar. Similar observations were found for the leg cases, except for the D0.1 cm3 of normal tissue, which was lower for BT. In a recent study, Wills et al. [2cm3 values were comparable, but doses to smaller volumes  were higher with BT. For complex targets, BT provided excellent target coverage, as the dose could be adjusted to irregular surfaces using flexible flap applicators. There were complex cases where only BT provided clinically acceptable plans. For smaller, flat targets, electron beams provided plans similar to BT, with better dose homogeneity.Six studies have been published on the dosimetric comparison of surface mould HDR-BT and VMAT for the treatment of skin lesions on the scalp and the extremities –67. Bomas et al.  comparedThe dose calculation algorithm is not always reported in publications. In most cases, only the name of the planning system is mentioned, especially for EBRT. In ten studies, neither the calculation algorithm nor the planning system was given. In nine cases the algorithm  and in the other cases only the name of the planning system was given. As for BT, the TG-43 formalism was used in all but six studies. In four papers, no information on the method of dose calculation was provided and more advanced model-based dose calculation algorithms (MBDCA) were used only in two studies , 63. ChaIn HDR-BT, different irradiation times can be used in different source positions using the stepping-source technique. Because of this feature, this technique can also be called intensity-modulated brachytherapy (IMBT) . In modeCurrently, the TG-43 protocol is the standard calculation formalism in clinical BT planning systems and was the most common calculation method in the cited studies. The vast clinical experience gained in recent decades is based on this method. It is important to note, however, that the MBDCAs using radiation transport in heterogeneous medium are already available for BT, but their application is still mainly limited to the field of research . The resComparative dosimetric studies of BT and EBRT fall into two categories: either they use two separate groups of patients, or the planning is performed using the same CT image set and the same contours Table . In the One of the advantages of BT is that due to the absence of a CTV–PTV margin , the target volume to be irradiated is smaller. This fact probably contributes to the favourable dosimetric characteristics of BT, in particular to the improved OAR protection. In EBRT plans, depending on the treatment site and the irradiation technique, with or without motion management, PTV is generally larger than CTV. Table In cervical cancer, intracavitary BT is mandatory, as confirmed not only by dosimetry but also by clinical results , 13. In Interestingly, for irradiation of the vaginal cuff, EBRT techniques performed better than BT in terms of dose to OARs –40. ThisSBRT is the standard technique for ablative treatment of liver cancer. However, with interstitial BT, a more favourable dose distribution can be achieved, with better target coverage and a lower liver dose . The betA systematic literature review has shown that from a dosimetric point of view, BT can compete with even the most advanced EBRT techniques, in particular with a higher dose centrally within the target volume and sparing of adjacent OARs. The advantage is particularly pronounced for interstitial BT, where adequate geometrical target coverage can be achieved with a large number of needle/source dwell positions, allowing for a conformal and relatively homogeneous dose distribution. If the source positions are outside the PTV , due to the larger distance, the inverse square law is not as effective as in interstitial cases.https://www.brachyacademy.com) as well as different multimedia and social media platforms for patients can be used [The recent decline in BT can be attributed to a number of factors , includi be used , 83, 86.A limitation of this work is that only a dosimetric comparison between BT and modern EBRT techniques was performed, without examining clinical outcomes. Our intention, however, was to evaluate only the physical characteristics of BT against emerging external beam radiation techniques, including IMRT, VMAT and CK-based robotic radiotherapy. The strength is a comprehensive literature review of the different treatment sites where a planning comparison between BT and EBRT had been published, and our final conclusions on the role of BT were based on these.The history of BT has been constantly accompanied by technological developments, and further advances are expected in the future in imaging, dose calculation algorithms, physical and biological dose optimization, new applicators, electromagnetic tracking, 3D printing and new isotopes –89. We f