To compare the near and long-term outcomes of endoscopic thoracic sympathectomy (ETS) for palmar, axillary and plantar hyperhidrosis.We retrospectively analyzed the clinical data of 218 patients with hyperhidrosis who were admitted to the Department of Thoracic Surgery of Gansu Provincial People’s Hospital for surgical treatment from April 2014 to August 2021. The patients were divided into three groups according to the method of ETS and the perioperative clinical data and postoperative follow-up data were collected to compare the near and long term outcomes of the three groups.P > 0.05). There was no statistically significant difference between the three groups in terms of operative time (P = 0.148), intraoperative bleeding (P = 0.308) and postoperative hospital stay (P = 0.407). Postoperatively, all three groups showed significant relief of palmar hyperhidrosis symptoms, with the R3 + R4 group having an advantage in terms of relief of axillary hyperhidrosis symptoms, patient satisfaction and quality of life index at 6 months postoperatively and the R4 + R5 group having an advantage in terms of relief of plantar hyperhidrosis symptoms. The difference in compensatory hyperhidrosis at 12 months postoperatively was not statistically significant among the three groups (P = 0.867), but the incidence was higher in the R3 + R4 and R4 + R5 groups than that in the R4 group.There were 197 eligible patients at follow-up, 60 patients in the R4 cut-off group, 95 patients in the R3 + R4 cut-off group and 42 patients in the R4 + R5 cut-off group. There were no statistically significant differences in baseline indicators such as sex, age and positive family history among the three groups (Patients with simple palmar hyperhidrosis can first consider R4 cut-off treatment; R3 + R4 cut-off is more effective in treating palmar hyperhidrosis combined with axillary hyperhidrosis; R4 + R5 cut-off is more effective in treating palmar hyperhidrosis combined with plantar hyperhidrosis. However, patients need to be informed that R3 + R4 and R4 + R5 dissection may increase the risk of severe compensatory hyperhidrosis after surgery. Primary palmar hyperhidrosis is a condition characterized by excessive hyperhidrosis of the palms of the hands. The etiology of primary palmar hyperhidrosis remains unclear. The prevalence of hyperhidrosis in the United States was 2.8% in 2004  and incrPatients with palmar hyperhidrosis are often associated with axillary and plantar hyperhidrosis and endoscopic thoracic sympathectomy (ETS) is considered as the most effective treatment for this condition. Different surgical approaches obtain different rates of relief of hyperhidrosis symptoms, with R2-R4 ETS and R3-R4 ETS or ganglion blocks achieving higher rates of relief, but the former can lead to severe postoperative compensatory hyperhidrosis . More reClinical data of patients with palmar hyperhidrosis, axillary hyperhidrosis and plantar hyperhidrosis admitted to the Department of Thoracic Surgery of Gansu Provincial People’s Hospital who performed ETS treatment from April 2014 to August 2021 were collected.Inclusion criteria: (1) according to the clinical guidelines for minimally invasive treatment of palmar hyperhidrosis : patientDiagnostic criteria for palmar hyperhidrosis refer to the clinical guidelines for minimally invasive treatment of palmar hyperhidrosis : (1) bilAll patients underwent thoracoscopic bilateral R4, R3 + R4, or R4 + R5 ETS. Anesthesia was performed by single-lumen tracheal intubation. The patient was placed in a 45-degree semi-Fowler position with the arms abducted 90 degrees to expose the axilla. The surgical incision was chosen as a single incision . An artificial pneumothorax is created by injecting carbon dioxide into the chest cavity and maintaining the pressure at 10 mm Hg. After identifying the posterior mediastinal sympathetic chain, the mural pleura overlying the sympathetic chain at the 4th rib (R4 group) or the 3rd and 4th ribs (R3 + R4 group) or the 4th and 5th ribs (R4 + R5 group) was hooked and dissected with a conventional 5 mm electric hook and the sympathetic chain was severed by electrocoagulation. The bypassed nerve fibers, such as the Kuntz nerve fiber bundle and sympathetic nerve fibers, are also cut approximately 2–3 cm along the surface of the rib cage. The residual pleural fluid is aspirated with a suction device and the lung is observed under direct vision to reopen the lung without placing a chest drain. The procedure is then repeated on the other side.(1) Baseline information includes sex, age, age of onset, BMI, smoking history, positive family history, severity of symptoms and follow-up time; (2) the dermatology life quality index (DLQI)  was usedP < 0.05 was considered as a statistically significant difference.SPSS 26.0 software  was used for statistical analysis and measurement data conforming to a normal distribution were described by mean ± standard deviation; one-way ANOVA test for comparison between groups and LSD test or Tamhane test for two-way comparison. Categorical variables were expressed as frequencies and percentages (%) and the chi-square test was used for comparison between groups. This study has been reviewed by the Ethics Committee of Gansu Provincial People’s Hospital, approval number: 2022 − 343. All patients signed the informed consent form for surgery before surgery.P > 0.05), see Table There were no statistically significant differences in baseline indicators such as sex, age and positive family history among the three groups (P = 0.148), intraoperative bleeding (P = 0.308) and postoperative hospital stay (P = 0.407). In terms of postoperative complications, the overall incidence was 13.3% in the R4 group, 14.7% in the R3 + R4 group and 14.3% in the R4 + R5 group, with no statistically significant difference among the three groups (P = 0.964); see Table All three groups of enrolled patients completed the surgery successfully without perioperative deaths. There was no statistically significant difference between the three groups in terms of operative time (P = 0.536); no statistically significant difference in the remission of axillary hyperhidrosis in the three groups (P = 0.840), but the rate of complete remission was better in the R3 + R4 group; no statistically significant difference in the remission of plantar hyperhidrosis in the three groups (P = 0.112), but the rate of complete remission was better in the R4 + R5 group. Satisfaction was higher in all three groups without statistically significant difference (P = 0.092), with the highest satisfaction in the R3 + R4 group. In terms of DLQI at 6 months postoperatively, the difference among the three groups was statistically significant (P < 0.001) and the improvement in postoperative quality of life was better in the R3 + R4 group than in the R4 and R4 + R5 groups. All 197 patients in this study has no recurrence after surgery. The overall incidence of compensatory hyperhidrosis at 12 months postoperatively was not statistically significant among the three groups (P = 0.976), but the incidence of severe postoperative compensatory hyperhidrosis was higher in the R3 + R4 group (6.3%) and the R4 + R5 group (4.8%) than in the R4 group (3.3%); see Table There was no statistically significant difference in the remission of palmar hyperhidrosis in all three groups after surgery  this study is retrospective, the sample size of the included studies is relatively small and the data source is a single center and the results may be biased; (2) there was no objective assessment of the psychological impact of patient hyperhidrosis before and after surgery; (3) the family history of hyperhidrosis patients in this study is small, which may be related to the patients’ unfamiliarity with hyperhidrosis in family members  during the patient interview; (4) follow-up data were collected through telephone interviews, micro-surveys and outpatient visits. During the follow-up visits, patients usually need to recall their postoperative conditions, which may lead to bias.In summary, patients with simple palmar hyperhidrosis can first consider R4 cut-off treatment; R3 + R4 cut-off treatment for palmar hyperhidrosis combined with axillary hyperhidrosis is more effective; R4 + R5 cut-off treatment for palmar hyperhidrosis combined with plantar hyperhidrosis is more effective. However, patients need to be informed that R3 + R4 and R4 + R5 dissection may increase the risk of severe compensatory hyperhidrosis after surgery. Future high-quality prospective randomized controlled trials are expected to further validate the results of this study.


Host density is one of the main factors affecting the infective capacity of viruses. When host density is low, it is more difficult for the virus to find a susceptible cell, which increases its probability of being damaged by the physicochemical agents of the environment. Nevertheless, viruses can adapt to variations in host density through different strategies that depend on the particular characteristics of the life cycle of each virus. In a previous work, using the bacteriophage Qβ as an experimental model, we found that when bacterial density was lower than optimal the virus increased its capacity to penetrate into the bacteria through a mutation in the minor capsid protein (A1) that is not described to interact with the cell receptor.Here we show that the adaptive pathway followed by Qβ in the face of similar variations in host density depends on environmental temperature. When the value for this parameter is lower than optimal (30°C), the mutation selected is the same as at the optimal temperature (37°C). However, when temperature increases to 43°C, the mutation selected is located in a different protein (A2), which is involved both in the interaction with the cell receptor and in the process of viral progeny release. The new mutation increases the entry of the phage into the bacteria at the three temperatures assayed. However, it also considerably increases the latent period at 30 and 37°C, which is probably the reason why it is not selected at these temperatures.The conclusion is that the adaptive strategies followed by bacteriophage Qβ, and probably other viruses, in the face of variations in host density depend not only on their advantages at this selective pressure, but also on the fitness costs that particular mutations may present in function of the rest of environmental parameters that influence viral replication and stability. The infection cycle of any virus can be divided into two phases, an intracellular one, corresponding to the period of replication inside the cell, and an extracellular one, comprising the time between infections. In the extracellular phase, viruses behave as inert entities –the virions– that are exposed to the physicochemical conditions of the environment, which can damage the virus particles and compromise the ability to initiate new infections. The time that a virus remains in the extracellular environment is strongly conditioned by virus and host concentration. Due to mass action kinetics, host concentration is the most relevant factor influencing the number of infections that can be initiated from a viral population of a given size . VirusesSometimes, improving one of the parameters described above implies worsening others, that is, it has a fitness cost . An examEscherichia coli using as receptor the conjugative F pilus. It is a member of the Leviviridae family (genus Allolevivirus)  and the virus genomic RNA penetrates inside the central channel that traverse the plasma membrane at the basis of the pilus and is driven into the host cytosol  . It has vivirus) . It consvivirus)  that is vivirus) . Around  cytosol . Due to  cytosol , a simil cytosol .In a previous work, we studied the adaptation of bacteriophage Qβ to replicate in the presence of different bacterial densities at the optimal replication temperature for the virus (37°C) . A fractIn the experiments presented in this work, we have studied the effect of varying cell density at the same time that another essential parameter for virus replication, temperature, is also modified. Temperature largely influences the tertiary structure of proteins. Therefore, it is to be expected that all processes involving interaction between molecules and structures (for instance between phage and bacteria components) or enzymatic catalysis are affected by variations in this parameter. The impact of temperature changes on Qβ replication and evolution has been previously studied in depth in our laboratory, although always at the same bacterial density that we use as a standard. While the decrease in temperature (from 37 to 30°C) does not strongly decrease viral titers, the increase (from 37 to 43°C) produces drastic reductions in the virus yield . NeverthThe new results we have obtained show that, when bacteria availability is reduced, bacteriophage Qβ selects a common adaptive strategy both at 30 and 43°C, which is based in the improvement of its entry into bacteria. However, while at 30°C adaptation is achieved through the same mutation in the A1 protein that was selected at 37°C, at 43°C it does so through a different mutation located in the A2 protein (V256A), which has strong fitness costs at lower temperatures. It appears, therefore, that adaptation of bacteriophage Qβ to low host availability involves different mutational pathways depending on the balance between fitness advantages and costs at each replication temperature.Escherichia coli DH5-α, a strain that permits virus expression, although it cannot be infected because it lacks the virus receptor. The supernatant of an overnight culture, obtained from a transformed colony, was used to infect E. coli, strain Hfr (6 plaque forming units (pfu) were used to infect an E. coli Hfr culture under standard conditions  for 2 h. The supernatant of this culture was used as the ancestor of all the evolutionary lineages analyzed in this work. It was denoted QβAnc and its consensus sequence showed no mutations relative to the Qβ cDNA cloned in pBR322. The virus QβAnc is the same described in our previous work concerning Qβ adaptation to low host density . Infections in liquid medium were carried out using fresh log-phase E. coli cultures with an OD600 of 0.8 that were infected with the amount of pfu indicated in each experiment. Cultures were incubated at the indicated temperature for 2 h with good aeration (250 rpm). To estimate the total virus yield, cultures were treated either with chloroform  or with egg white lysozyme . Virus supernatants were harvested upon centrifugation at 13000 × g and maintained at 4°C for short-term use (less than 15 days) or at −80°C for long-term storage. Virus titers were determined by plaque assay and expressed as the number of pfu per ml of the phage suspension.Qβ was propagated in Anc was used to initiate 8 replicate evolutionary lineages differing in the bacterial density  per ml.1 The culture was serially diluted in NB medium , and 0.5 ml of either the undiluted culture or the corresponding dilution were placed in 10 ml tubes containing 400 μL of NB. Infections were initiated with 107 pfu in 100 μL of phage buffer . After 2 h of incubation under standard conditions either at 30 or 43°C, the virus supernatants were collected upon treatment with chloroform, and a fraction of the phage suspension  was used to infect fresh E. coli cultures prepared as described above. Evolutionary lineages were denoted Qβ (3 × 108)30°C, Qβ (3 × 108)43°C, Qβ (3 × 107)30°C, Qβ (3 × 107)43 ^° C, Qβ (3 × 106)30°C, and Qβ (3 × 106)43°C to indicate the bacterial density (in brackets) and the temperature used in the experiment. Lineages propagated at the bacterial density of 3 × 105 cfu/ml were extinguished before transfer 16 and were not used for further experiments. When necessary, the numbers 1 and 2 were used to distinguish the two replicas performed for each condition.The virus Qβ density . PropagaA negative control in which undiluted bacteria were incubated in culture medium in the absence of virus was set at each transfer. This control was processed and plated exactly the same as the experimental samples. When lysis plaques appeared, the corresponding transfer was discarded and repeated again.U830C), and C2011A (virus QβC2011A). The replication temperatures and the bacterial densities used are indicated in the text and in the legend of the corresponding figures.Additional evolution experiments using a similar protocol to that described above were performed with single mutant viruses containing mutation U830C  for 30 min at 37°C with shaking (850 rpm in thermoblock). Lysozyme was used instead of chloroform because of its higher efficiency to lysate cells, demonstrated in several lysis assays carried out in our lab (data not shown).The virus yield obtained in replication assays carried out in liquid medium was used as a measure of the virus replicative ability. Triplicate liquid cultures containing the indicated bacterial density were inoculated with 10U830C) or C2011A (virus QβC2011A). Mutagenesis was carried out using the QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) with the primers 5′GACAATCTGTACCCTGCCGCTGCTTACTTTAAACTGAAA3′ (for QβU830C) and 5′TGGGATTCTCGGCTTAGTTATAACACGT TCCGCGG3′ (for QβC2011A), together with their respective complementary containing the mutations to introduce in the virus genome. The procedures to build and isolate the site-directed mutants were the same as previously described for other Qβ mutants . Plaques were incubated overnight at 37°C. Images of the Petri dishes were taken with ChemiDoc MP Imaging System (Bio-Rad) using a white sample tray and “Coomasie Blue” mode. Images were analyzed with Image Lab Software (Bio-Rad) (RRID:SCR_014210), using the tool “Volume Tool-Round” to manually select 70 lysis plaques of each virus whose areas were calculated. We used the Mann-Whitney test to assess the statistical significance of the differences between different sets of data.Areas of the lysis plaques formed by the viruses Qβ8 cfu/ml) and 105 pfu of the indicated virus, were incubated at either 30, 37, or 43°C with low shaking (75 rpm) for 0, 5, 10, 15, and 20 min. At each time, 100 μL of EDTA 100 mM were added to stop virus entry. A control experiment showed that incubation of Qβ with this EDTA concentration completely inhibited its entry into E. coli, which agrees with previous results obtained for other phages , N is the number of bacteria in the sample (3 × 108 cfu), and t is the incubation time. We defined the parameter k (expressed in ml/min/cell), corresponding to c/N, as the constant of formation of infectious centers at the bacteria density assayed. Fittings were performed using the function NonLinearFit of Mathematica 9.0 (Wolfram Research). Viruses whose k-values were compared were always assayed in the same experiment, using the same cell preparation. Differences were considered statistically significant when the value obtained for a particular virus was outside the confidence interval of the one to which it was compared.where t-test.Additional experiments were performed in the same way but with a fixed incubation time (10 min) and using different bacterial concentration (indicated in the corresponding figure). The statistical significance of the differences between the number of infectious centers obtained in different sets of data was assayed by means of the Student’s 8 bacteria and 106 pfu of the viruses QβAnc, QβU830C, or QβC2011A were incubated for 5 min (curves performed at 30 and 37°C) or 10 min (curves performed at 43°C) in NB medium and 75 rpm. The reason for incubating longer at 43°C was to allow the entry of a greater amount of virus to mitigate the low replication yield at this temperature. In all cases, virus entry was stopped by a 10000-fold dilution of the cultures. Diluted cultures were incubated at the temperature assayed in a static bath, and only mixed by inversion right before taking the samples, to avoid additional encounters between cells and viruses. At different times, 250 μL aliquots were removed and centrifuged to obtain the virus supernatants that were titrated. The duration of the latent period was calculated as the intercept between the regression line of the natural logarithm of the virus titers vs. time during the exponential rise period and the regression line for the points corresponding to the pre-rise period.Duplicate 1 ml liquid cultures containing 3 × 108 bacteria and 106 pfu of the virus indicated were incubated for 10 min at either 30, 37, or 43°C in NB medium and 75 rpm to allow virus entry into bacteria. After this time, a 0.3 ml aliquot was removed to determine the number of infectious centers as described above (see section “2.6. Determination of virus entry into bacteria”). Another 0.3 ml were diluted 100000-fold (for the assays carried out at 30 and 37°C) or 10000-fold , and incubated at the same temperature used for virus entry with shaking (250 rpm). The incubation times were 75, 55, and 75 min for QβAnc and QβC2011A at 30, 37, and 43°C, respectively, and 90, 80, and 75 min for QβU830C at 30, 37, and 43°C, respectively. The times were chosen so that each virus would have reached the plateau as shown in the one step growth curves. After these times, the virus supernatants were collected and titrated to estimate the amount of extracellular virus. The burst size was calculated by dividing the virus titers  by the number of infectious centers determined in the aliquot removed after the initial 10 min allowed for virus entry.Triplicate 1 ml liquid cultures containing 3 × 10E. coli Hfr into a 50 ml flask. The culture was treated with 500 μL of chloroform at 25°C, 250 rpm for 20 min. To completely break the cells, the flask was vortexed at maximum speed for 5 extra minutes. Finally, lysates were centrifuged at 25°C, 12000 rpm for 10 min. A 300 μL fraction of the supernatant was plated on an NB plate to check that there were no live bacteria remaining that could allow virus replication. Triplicates containing 1 ml of lysate supernatant were incubated with 3 × 105 pfu for 16 h at 30, 37, or 43°C. Controls for each temperature were prepared the same way, but using NB instead of lysate supernatant. Test samples and controls were titrated by plaque assay.To study the virus interaction with cellular debris, cellular lysates were prepared by transferring 10 ml of a stationary culture of 2 Mutations relative to the sequence of the virus QβAnc were identified using the same software.Viral RNA was prepared following standard procedures to determine the consensus sequence either from biological clones or from complex virus populations. RNAs were used for cDNA synthesis with the avian myeloblastosis virus reverse transcriptase (Promega), followed by PCR amplification using Expand high-fidelity DNA polymerase (Roche). The pairs of oligonucleotide primers used for RT-PCR were the following: P1 forward (5′CTTTAGGGGGTCACCTCACAC3′) with P1 reverse (5′GGATGGGTCACAAGAACCGT3′) to amplify from nucleotide position 10 to 1595, P2 forward (5′GACGT GACATCCGGCTCAAA3′) with P2 reverse (5′CAACGGACGGAACATCTCCT3′) to amplify from nucleotide position 1109 to 2787 and P3 forward (5′GTGCCATACCGTTTGACT3′) with P3 reverse (5′GATCCCCCTCTCACTCGT3′) to amplify from nucleotide position 2254 to 4195. PCR products were column purified (Qiagen) and subjected to standard Sanger sequencing using Big Dye Chemistry with an automated sequencer . Sequences were assembled and aligned with Geneious Pro v4.8.5.t-test (Function T-Test).All statistical analyses were performed with the program Mathematica 9.0 (Wolfram Research). When calculating the statistical significance of the differences between two series of data, the Location-Test function was used to choose the most appropriate test for the comparison, which in all cases, except for the comparison of the size of the lysis plaques produced by different viruses, was the 8 cfu/ml. To analyze the effect of temperature on phage replication as a function of the host density, the phage was propagated for 16 transfers at either 30 or 43°C. For each temperature, we tested the following concentrations of bacteria: 3 × 108, 3 × 107, 3 × 106, and 3 × 105 cfu/ml, performing two replicas for each  as a function of temperature is shown in In our previous publication , we founfor each . Since aAnc), were used to determine whether the optimal bacterial density for Qβ replication depended on temperature. At 30°C, we observed that the curve flattened between 3 × 107 and 3 × 108 cfu/ml, and at 43°C the maximum virus titer was obtained at 3 × 107 cfu/ml . In that case, the virus was extinguished at transfers 9 and 10 for the replicas performed at 30°C, and at transfer 7 for those performed at 43°C. As it happened during the propagation of Qβ at 37°C at lower-than-optimal bacterial densities, the evolutionary lineages (with the only exception of the two replicas propagated at 43°C at 3 × 108 cfu/ml) became progressively enriched in viruses that gave rise to lysis plaques with smaller-than-usual size. In the following sections we will analyze this character quantitatively.The results obtained in the first transfer, which in all cases was initiated from the same virus sample . The results obtained .We then determined whether the populations obtained at transfer 16 had increased their replicative ability with respect to the ancestor. To ensure that viral production was not saturated during the assay, this was performed with 10obtained  showed tTo determine the genetic changes responsible for adaptation to the assayed conditions, we determined the consensus sequences of all evolutionary lineages obtained at transfer 16 . The mos8 cfu/ml, mutation U830C (V256A in the A2 protein) was present. In addition, other mutations, some of which had previously been identified in other experiments of Qβ adaptation to high temperature and standard bacterial density, were also detected (5 cfu/ml. It is interesting that mutation A1088G (D342G in the A2 protein), which was always selected in our lab when Qβ was adapted to 43°C using bacterial densities around 3 × 108 cfu/ml, was not selected when the amount of available bacteria was lower.The results obtained at 43°C showed interesting differences with those observed at 30 and 37°C. First, mutation C2011A was not selected in any lineage. In contrast, in all those propagated at bacterial densities below 3 × 10detected . The preU830C) or C2011A (virus QβC2011A). We determined the virus titers obtained in a replication assay carried out with the single mutants at both selective and non-selective temperatures at all the bacteria concentrations compatible with the preservation of the virus population for the 16 transfers of the evolution experiment  showed that mutation U830C only increased significantly the virus titers at 43°C at 3 × 107 cfu/ml (8 cfu/ml) or the difference was non-significant (3 × 106 cfu/ml), suggesting that the presence of this single mutation is not sufficient for the virus to adapt to both selective pressures, high temperature and low host density, simultaneously. Mutation C2011A increased the replicative ability with respect to the virus lacking the mutation at all bacteria densities assayed at 30°C.The virus yields obtained at selective temperatures , both mutations always rendered lower virus yields than the ancestor  . It is remarkable that in all assays carried out to determine the replicative ability of the mutant QβU830C  for the virus QβAnc at 30, 37, and 43°C. We observed that this virus generated infectious centers more rapidly at 43°C than at 37°C, and the lowest value was obtained at 30°C  was probably due to the poorer fit of the experimental points to the function used, resulting in wider confidence intervals.Then, we compared the value of the constants obtained for the virus Qβand 43°C . Each coAnc, QβU830C, and QβC2011A were incubated for a fixed time (10 min) with different bacterial densities, after which the number of infectious centers formed was determined. The results obtained  followed by a sharp decline until almost the total elimination of viral infectivity at 24 h. In contrast to this, different amounts of virus incubated for 24 h in NB medium at 43°C did not show any significant decrease in their titers. The result suggests that Qβ loses infectivity faster when it is replicating in a medium with bacteria than in cell-free NB medium.Another feature influencing virus fitness is the time that infectivity can be preserved in the extracellular environment in the period between infections. Previous assays carried out with the virus Qβ5pfu of the ancestral virus or the mutants QβU830C and QβC2011A for 16 h at 30, 37, and 43°C, after which the virus infectivity in each sample was assayed. The results , and incubated it with about 3 × 10 results  showed tU830C at 37°C (8 cfu/ml) or low (3 × 105 cfu/ml). In both conditions, the virus experienced an initial increase in its titers, which was greater at high bacterial density, and remained fairly constant during the rest of transfers. The consensus sequences showed the loss of mutation U830C in the two replicas carried out at high bacterial density and the permanence of the mutation when the bacterial density was low , using a high (3 × 108 cfu/ml) or a low (3 × 106 cfu/ml) bacterial density  that remained constant with some fluctuations throughout the experiment. Determination of the consensus sequences at transfer number 10 showed that, at high bacterial density, mutation C2011A was lost in both replicas, whereas at low bacteria density, the mutation could be preserved, although it was always accompanied by U830C  . In all 7 cfu/ml. From this point on, the increases were low (30°C) or turned into declines (43°C)  and the other one higher (43°C). It is noteworthy that, in both cases, the curves of viral yield as a function of bacterial density approached a plateau at about 3 × 10s (43°C) . In this5 cfu/ml. Under this condition, the virus was extinguished in both replicas carried out either at 30 or 43°C, probably due to the low viral production when two selective pressures  are operating together. As it happened in the experiment carried out at 37°C, another observable phenotypic feature was that in all lineages evolved at 30 and 43°C [with the only exception of the two replicas of Qβ (3 × 108)43°C] as the number of transfers increased the lysis plaques became smaller than usual. There are several theoretical and experimental studies that attempt to relate the changes in the parameters defining the viral infective cycle with the size of the lysis plaques  selected mutation C2011A in the A1 protein, similarly to Qβ evolved at 37°C and bacterial densities lower than 3 × 108 cfu/ml  at high and low bacterial densities could be maintained at this temperature (5 cfu/ml) (C2011A propagated at 43°C (non-selective temperature) at low host density showed an increase in the virus titers as the number of transfers increased  . A similncreased . Mutationcreased . SequencThe most important conclusion that can be drawn from this work is that adaptation of Qβ to reduced host availability can be addressed by at least two different mutations that increase virus entry into bacteria. The choice of one or other depends on environmental parameters such as temperature that may affect the strength of the virus-cell interaction, the ease of internalizing the virus genome or the ability to generate an infectious progeny. The balance between the advantages and the fitness costs of each mutation under the particular conditions of virus replication is what decides if it is selected or not.The original contributions presented in this study are included in the article/EL designed the study and wrote the manuscript. ML-C, AR-M, and ELl performed the experiments. EL and ML-C analyzed the data. All authors interpreted the results and reviewed the manuscript.


A series of exo‐olefin compounds with a methyl methacrylate (MMA) dimer structure as the R group and a substituted α‐methylstyrene unit as the −CH2C(=CH2)Z (Z: Ph−Y) group were synthesized and used for the radical polymerization of MMA in toluene and PhC(CF3)2OH. These compounds underwent transfer of the CH2C(=CH2)Z group via addition‐fragmentation of the propagating methacryloyl radical. More electron‐donating (Y) substituents, such as methoxy and dimethylamino groups, produced polymers with narrower molecular weight distributions. A continuous monomer addition method further improved molecular weight control and enabled the synthesis of colorless, sulfur‐free, multiblock copolymers of methacrylates in homogeneous solutions.In this work, the development of  exo‐olefin compounds (R−CH2C(=CH2)Z) as chain‐transfer agents for the sulfur‐free RAFT polymerization of methacrylates in homogeneous solution. A series of R−CH2C(=CH2)Z with the MMA‐dimer structure as R and substituted α‐methylstyrene unit as −CH2C(=CH2)Z (Z: Ph−Y) were synthesized. More electron‐donating substituents (Y) work better. Colorless sulfur‐free multiblock copolymers were prepared via sequential monomer additions.This work develops  The addition‐fragmentation reaction is common in not only organic but also polymer chemistry.3, 4 Iexo‐olefin compound such as R−CH2C(=CH2)Z can work as a RAFT agent via the addition of propagating radical species to the C=C bond followed by β‐scission of the C−C bond. In this case, the transferable group is not −SC(=S)Z but instead −CH2C(=CH2)Z and is thus free from sulfur. Such sulfur‐free RAFT agents have long been desired for the preparation of colorless and odorless polymers, particularly in view of their applications. However, the RAFT process for exo‐olefin compounds is generally slow, and the chain‐transfer constants of exo‐olefin compounds are low in comparison to those of thiocarbonylthio compounds due to less reactive C=C bonds and stronger C−C bonds.However, the term RAFT can be used in a more general sense to describe a reversible chain‐transfer reaction,exo‐olefin‐terminated poly(methacrylate) (Pn: H−(CH2C(CH3)(CO2R′))n−CH2C(=CH2)CO2R′), which was prepared by cobalt‐mediated catalytic chain‐transfer polymerization of methacrylate (CH2=C(CH3)CO2R′), was used as a “macromonomer” for the heterogeneous radical emulsion polymerization of another methacrylate (CH2=C(CH3)CO2R′′).exo‐olefin‐terminated polymer was named a macromonomer in the literature, it worked as macro chain‐transfer agent, which generates propagating poly(methacrylate) radicals via β‐scission to produce block copolymers with relatively narrow molecular weight distributions (MWDs) (Mw/Mn=1.2–1.4) and controlled molecular weights via the RAFT mechanism. This emulsion polymerization was further used for the synthesis of multiblock polymers with different methacrylates via slow continuous monomer addition.[exo‐olefin‐terminated polymers are not very high (Ctr ≈0.2),[Prior to the advent of efficient sulfur‐based RAFT agents,exo‐olefin‐based RAFT agent by designing appropriate R and Z groups in R−CH2C(=CH2)Z to control the homogeneous radical polymerization of methacrylates (Scheme 2C(CH3)(CO2Me))2−) because the chain‐transfer constants of a series of exo‐olefin‐terminated poly(MMA) (PMMA) molecules  are almost the same (Ctr ≈0.2) when n≥2 in Pn due to similar steric hindrance around the cleavable C−C bond.2C(=CH2)Ph−Y moiety can be regarded as an α‐methylstyrene unit, which should have higher reactivity toward the methacryloyl radical than the −CH2C(=CH2)CO2Me moiety in Pn. The reactivity of the exo‐olefin‐based RAFT agent can consequently be enhanced with an electron‐donating substituent analogous to the copolymerizability of MMA and substituted styrenes.Pn.[2C(CH3)(CO2Me))2−CH2C(=CH2)Ph−Y) and used them as chain‐transfer agents for the RAFT polymerization of MMA and block copolymer syntheses in homogeneous solutions.This study aimed to develop a new and efficient sulfur‐free exo‐olefin‐based chain‐transfer agents, several reactions related to controlled radical additions and polymerizations were utilized (Scheme 1P: CH3C(CH3)(CO2Me)−CH2C(=CH2)CO2Me) was first prepared by the cobalt‐mediated catalytic chain‐transfer (CCT) oligomerization of MMA1P was hydrogenated and chlorinated for conversion into an MMA dimer chloride (H−(CH2C(CH3)(CO2Me))2−Cl), which can also work as an efficient initiator for the metal‐catalyzed living radical polymerization of MMA.For the synthesis of the d Scheme . An MMA Kharasch or atom transfer radical addition (ATRA) of the MMA dimer chloride and a series of unsubstituted and substituted benzaldehyde silyl enol ethers  was conducted using CuCl and 2,2′‐bipyridine as catalysts. This reaction was similar to the previously reported end‐functionalization of PMMA chloride with benzaldehyde silyl enol ether, which resulted in a benzophenone terminus during the ruthenium‐catalyzed living radical polymerization of MMA.35, 36exo‐olefins using phosphorous ylide (Wittig reagent) or Tebbe reagent, where the latter was used for only bulky ketones with 2,4‐dimethoxy or 3,4,5‐trimethoxy groups, resulting in a series of MMA dimers with substituted α‐methylstyrene units (1–6). All peaks in the 1H  spectra were assigned to the corresponding protons and carbons of the series of target compounds, indicating their successful preparation. In particular, the methylene protons and carbons of 1–6 were all observed at significantly high magnetic fields in comparison to those of the exo‐olefin‐ended MMA trimers 2P, which were prepared by the cobalt‐mediated catalytic chain‐transfer oligomerization of MMA followed by purification as described previously,exo‐olefins are at least electron rich. However, detailed discussions on the effects of the substituents on the chemical shifts and electron densities of the C=C bonds are challenging.The ketone groups were then transformed into ne units –6. All p2 or 2P in toluene at 60 °C. In the absence of any chain‐transfer agents, high molecular weight polymers (Mn=182 300) were produced in 24 h with 92 % monomer conversion . In the presence of 2P or 2, the polymerization became slightly slower and resulted in polymers with relatively low molecular weights. More specifically, the Mn value obtained with 2 was lower than that obtained with 2P (Mn=16 700 vs. 20 500). Furthermore, the Mn values decreased along with monomer conversion and approached the calculated values assuming that one molecule of 2 generates one polymer chain. These results indicate that 2 is superior to 2P, which is among the most effective sulfur‐free RAFT agents in the radical polymerization of MMA.Mn value was still higher than the calculated value, indicating that all molecules of 2 did not participate in the polymerization.The homogeneous radical polymerization of MMA was investigated using 2,2′‐azobisisobutyronitrile (AIBN) in the absence and presence of 3)2OH, which has been reported to increase the addition rate of the PMMA radical to the styrenic moiety of 2 via the hydrogen bonding of the acidic alcohol to the carbonyl group at the chain‐end of the PMMA radical because of an enhanced effect of PhC(CF3)2OH on copolymerization of (meth)acrylic monomers and styrenic or olefinic monomers.[3)2OH without using any chain transfer agents was slightly faster and resulted in higher molecular weight polymers (Mn=274 300) . Upon the addition of 2P or 2, the molecular weights were significantly lowered. In particular, the final Mn obtained with 2 became even lower (Mn=14 300) than that with 2 in toluene (Mn=16 700). These results indicate that fluoroalcohol is a suitable solvent for enhancing the chain‐transfer ability of 2 to generate PMMA radicals.The solvent was then changed to a fluoroalcohol, PhC were then evaluated in the radical polymerization of MMA with AIBN in PhC(CF3)2OH at 60 °C. All polymerizations proceeded almost quantitatively in 60 h, although the rates changed slightly with the structures . The molecular weights of the polymers depended significantly on the substituents and decreased in the following order: 3>1≈2≈5>4>6 , indicating that almost all 4 and 6 molecules worked as chain‐transfer agents under homogeneous conditions. Therefore, MMA dimers with 3,4‐dimethoxy and 4‐dimethylamino styrene units are more effective sulfur‐free chain‐transfer agents for radical polymerization of MMA than the previously reported exo‐olefin‐terminated PMMA (Pn). However, those with bulky substituents, such as 2,4‐dimethoxy and 3,4,5‐trimethoxy styrene units, were less efficient. Similar tendencies were observed in toluene , although the obtained molecular weights were slightly higher than those obtained in PhC(CF3)2OH.The series of Ctr),[Mn‐conversion plots proposed by Müller et al. is the simplest one to estimate approximate values.[−7 M as in a similar way to the papers by Moad et al,[Ctr values were estimated for the polymerizations in PhC(CF3)2OH at 60 °C exo‐olefin‐based chain‐transfer agents were thus estimated as follows: 0.22 (3), 0.40 (1), 0.41 (2), 0.42 (5), 0.62 (4), 1.50 (6). These results indicate again that 4 and 6 are relatively efficient chain‐transfer agents.Among various methods to calculate chain‐transfer constants Ctr,[43,1H NMR spectra of all of the polymers obtained with 1–6 showed the characteristic peaks of the −CH2C(=CH2)Ph−Y groups, such as methylene, phenyl, methoxy, and dimethylamino, at the ω‐chain end ) calculated from the peak intensity ratios of the chain‐end methylene protons (e) to the side‐chain methyl ester protons (c) were in good agreement with those measured by size‐exclusion chromatography (SEC) using PMMA as the standard (Mn(SEC)). These results indicate that all of the obtained PMMA chains nearly quantitatively possess −CH2C(=CH2)Ph−Y termini, which were formed via the RAFT mechanism. In addition, the obtained polymers are new end‐functionalized PMMAs with various substituted styrene groups, which can be used as macro‐RAFT agents for the radical polymerization of methacrylates (see below) or macromonomers for radical copolymerization with other mono‐substituted vinyl monomers, such as acrylates and styrenes. The end functionality (nF) calculated from the ratio of Mn(SEC) to Mn(NMR) was thus close to unity. However, the nF obtained with 6, with a dimethylamino group, was 0.86, suggesting the lower stability. In total, 4, having a 3,4‐dimethoxystyrene terminus, is thus most effective as a sulfur‐free RAFT agent for the homogeneous radical polymerization of MMA.The d Figure . The MnMn values of the polymers obtained using 2,2′‐azobis(N‐butyl‐2‐methylpropionamide) (VAm‐110) in the presence of 2 at 100 °C in PhC(CF3)2OH were lower and closer to the calculated values than those at 60 °C . However, the chain‐end fidelity was decreased (nF(ω1)=0.91) in comparison to that obtained at 60 °C (nF(ω1)=0.98), suggesting that side reactions occurred at a higher temperature.Effects of polymerization temperature were also studied. The 4 at 60 °C was then isolated by preparative SEC  and used as a macro‐RAFT agent for the second radical polymerization of MMA in PhC(CF3)2OH at 60 °C. The polymerization occurred smoothly, and the conversion of MMA reached 91 % in 24 h , resulting in polymers with higher molecular weights  . In particular, the Mn values increased in direct proportion to monomer conversion and agreed well with the calculated values.The PMMA obtained from 4  as the macro‐RAFT agent with benzyl methacrylate (BzMA) as the second monomer under similar conditions. The conversion of BzMA reached 96 % in 27 h , resulting in polymers with similarly higher molecular weights  . A similar linear increase in the Mn values was confirmed and in good agreement with the calculated vales.Furthermore, block copolymerization was investigated using another PMMA obtained from 1H NMR spectrum of the obtained polymer showed signals from both the MMA and BzMA units and the terminal 3,4‐dimethoxystyrene moiety, such as phenyl, methoxy, and methylene groups . In particular, the chemical shifts of the terminal phenyl and methylene groups changed slightly from the shifts of these groups when attached to PMMA as the macro‐RAFT agent, indicating that the −CH2C(=CH2)Ph−Y group was efficiently transferred to the poly(BzMA) terminal via RAFT block polymerization. The end functionality of the exo‐olefin group attached to the BzMA unit was 0.99 and close to unity. In addition, the number‐average degrees of polymerization (DPn) of MMA and BzMA calculated from the peak intensity ratios of the terminal exo‐olefin groups to the side chain methyl and benzyl ester groups were 29 and 18, which were close to the theoretical values, 29 and 19, respectively. Thus, PMMA with the terminal 3,4‐dimethoxystyrene unit quantitatively operates as a macro‐RAFT agent to produce block copolymers of MMA and BzMA.The Multiblock copolymerizations of methacrylates were further investigated by sequential monomer addition using a syringe pump, by which the monomers were slowly supplied to the homogeneous solutions to maintain the monomer concentrations at a relatively low level. This method could slow propagation relative to the chain‐transfer reaction and narrow the molecular weight distributions.n‐butyl methacrylate (BMA) was first conducted using 2 and AIBN in PhC(CF3)2OH at 60 °C. As shown in Figure S14, MMA, as the first monomer, was added to a solution of 2 and AIBN in PhC(CF3)2OH with a syringe pump at a constant rate for the first 11 h. MMA monomer conversion reached 93 % 16 h after the reaction started. The obtained polymers had a relatively narrow MWD (Mw/Mn=1.50) and controlled Mn (Mn=2500), agreeing well with the calculated value . Similarly, EMA was added at a constant rate for 11 h. The polymer obtained after a total of 36 h had a narrower MWD (Mw/Mn=1.45) and controlled Mn (Mn=4200), and the monomer conversions were ≥99 % and 91 % for MMA and EMA, respectively. Finally, BMA was added for 12 h. The MWD became even narrower (Mw/Mn=1.41), and the Mn (Mn=6900) agreed well with the calculated value . The polymers obtained by continuous monomer addition thus had more controlled molecular weights and narrower MWDs.Triblock copolymerization of MMA, ethyl methacrylate (EMA), and 1H NMR spectra of the polymers obtained after the addition of each monomer showed the characteristic alkyl protons of its pendant ester moiety /Mn(NMR)) of the exo‐olefin groups were 0.95, 0.95, and 0.91 at each stage and thus close to unity. Furthermore, the DPn values obtained from the 1H NMR spectra were close to the calculated values. These results indicate the formation of MMA, EMA, and BMA triblock copolymers using the sulfur‐free RAFT agent in homogeneous solutions.The y Figure . In addi4 and AIBN in PhC(CF3)2OH at 60 °C  were 0.96, 0.96, 0.85, and 0.72, suggesting that some of the ω‐chain ends were gradually lost with the increasing number of monomer addition steps. However, more interestingly, the final products obtained by precipitation in methanol were completely colorless, as shown in the inset in Figure Similar techniques were further used for the tetrablock polymerization of MMA, EMA, BMA, and BzMA with C Figure . The MWDMn values were apparently higher than the calculated values, but they became close to those values with monomer addition. In addition, the MWDs became narrower and finally reached Mw/Mn=1.52, which was slightly broader than that obtained with continuous monomer addition. A series of 1H NMR spectra also indicated the formation of tetrablock copolymers, although the nF values gradually decreased  .For reference, the tetrablock polymerizations of MMA, EMA, BMA, and BzMA were investigated by adding each of the polymers at once without using a syringe pump . In this case, the initial exo‐olefin compounds composed of styrenic groups and MMA dimer units worked as sulfur‐free RAFT agents for the homogeneous radical polymerization of methacrylates. Their performance as chain‐transfer agents was enhanced with incorporation of electron‐donating substituents on the phenyl group and was superior to that of the exo‐olefin MMA trimer, which was previously one of the most efficient sulfur‐free RAFT agents. The obtained polymers further act as macro‐RAFT agents to prepare methacrylate block copolymers. In addition, multiblock copolymers were obtained by sequential monomer addition, whereas continuous addition using a syringe pump was efficient for producing narrow MWDs. These sulfur‐free exo‐olefin‐based RAFT agents could contribute to further developments in controlled polymerization via the RAFT mechanism and in the design of polymer materials.A series of new The authors declare no conflict of interest.1As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


Digital workflow is showing an increasing tendency in everyday dentistry. Accuracy is essential during digital dental workflows for all indication areas. The present study aimed to evaluate the effect of software updates on the accuracy of intraoral scanner (IOS) devices.3Shape Trios 3 Pod with software versions 18.1.2. (TRI3_1) and 20.1.2. (TRI3_2); 3Shape Trios 4 Move, version 19.2.2. (TRI4_1); and 3Shape Trios 4 Pod, version 20.1.1. (TRI4_2) were used to take direct optical impressions from a polymethyl methacrylate (PMMA) full arch reference model with prepared teeth  and an edentulous region (between FDI 14 and 17). The scanners were used eight times; STL files were imported into Geomagic Control X for accuracy assessment by comparing them to a reference data set created by an industrial high-precision scanner (AICON SmartScan-3D C5). The average deviation of the surface points was calculated in three locations: across a full arch (Parameter 1), the region of a four-unit bridge (Parameter 2), and a single prepared abutment (Parameter 3).In parameter 1 and 2, the newest model with the latest software (TRI4_2) reached the highest accuracy (31.06 ± 5.24 µm and 21.69 ± 7.50 µm). In parameter 3, an older generation scanner running legacy software produced the highest accuracy: TRI4_1, 11.75 ± 0.35 µm.Appropriate software updates can significantly increase the trueness and precision of intraoral scanner devices. With updated software, the older generation can match the accuracy level of latest equipment. Digital workflow is showing an increasing tendency in everyday dentistry. The beginning of digital dentistry can be placed in the early’80 s . From thThere is less information in literature about the impact of the hardware and software components on IOS performance , 30. TheNumerous studies examined the accuracy of intraoral scanners, but only a few attempted to evaluate the effect of software updates on accuracy , 30. TriThe present study aimed to investigate the effect of software updates on trueness and precision in case of two different generations of intraoral scanners with four different software versions. The null hypothesis was that there is no association between the software version of the IOSs and the accuracy of digital impressions. The alternative hypothesis was that the newer software version of the IOSs generates a more accurate digital impression.Our study examined two generations of intraoral scanners with four different software versions Table . The tesThe reference model was scanned by a highly accurate industrial scanner . According to the user guide, the reference scanner’s accuracy was a maximal 8-μm feature accuracy (and a 2-μm resolution limit) . An expeSurface deviation of the full-arch scan: Variation between the digital impressions made by the examined IOSs and the reference dataset. It represents the global accuracy of the full arch. (Parameter 1).Surface deviation of the abutment teeth 14 and 17 and the alveolar ridge between them (four-unit bridge): It presents the distortion effect of the edentulous ridge. (Parameter 2).Surface deviation of the prepared incisor : this paThe 3D analysis software exported the root mean square (RMS) values of spatial deviations for use in a statistical software package. The arithmetic means of the RMS values were used to indicate the trueness, and their standard deviation (SD) parameters were used to indicate the precision of the investigated scanner based on an article by Renne, Mennito and Vág in 2021 .Eight individual RMS data points were available for each of the three Parameters described above and each IOS, resulting in a dataset of 96 observations. Scanner performance was described in terms of mean ± SD of RMS. Device versions were compared pairwise using Student's two-sample t-test  or Wilcoxon's rank-sum test (otherwise). The statistical package Stata was used for data handling and analysis.Significant differences were found between the generations and between the software versions. The differences in tendencies were not linear, so we cannot state that newer generations and newer software always reproduce the geometry of the oral cavity in a more accurate fashion. The final result depends also on the region under investigation Table , Fig. 3.In the case of full-arch digital impressions the accuracy results from lowest to highest accuracy are the following: TRI3_1 (90.24 ± 15.35 µm), TRI4_1 (52.91 ± 7.44 µm), TRI3_2 (47.44 ± 9.17 µm), and TRI4_2 (31.06 ± 5.24 µm). The newest software version with the older hardware cannot reach the accuracy level of the newer hardware with the second-latest software. The updated software reached significantly better accuracy in both generations. We did not find a significant difference in accuracy between TRI4_1 and TRI3_2, which suggests that with the proper software update, the older generation could perform similarly to new equipment in terms of accuracy.The observed data showed a decrease in the deviation from perfect trueness and precision as hardware and software evolved.The accuracy results of teeth 14 and 17 and the edentulous ridge in decreasing order of deviation were the following: TRI3_1 (117.35 ± 20.11 µm), TRI3_2 (45.86 ± 14.84 µm), TRI4_1 (41.04 ± 16.48 µm), and TRI4_2 (21.69 ± 7.50 µm). We found statistically significant differences between the hardware generations and between the majority of software versions, indicating that the newer versions could create more accurate digital impressions, and the distortion effect of the alveolar ridge is getting reduced – the stitching mechanism of the updated software algorithm seems to work better. Although TRI3_2 and TRI4_1 did not have significant accuracy differences between them it can be seen again that the old generation with newer software can reach the accuracy level of the new IOS models.The accuracy results of the prepared tooth 11 in decreasing order of deviation were the following: TRI3_2 (14.45 ± 0.36 µm), TRI3_1 (13.26 ± 0.94 µm), TRI4_2 (12.21 ± 0.71 µm) and TRI4_1 (11.75 ± 0.35 µm). We found a significant difference between TRI3_1 and TRI3_2, but no significance was shown between TRI4_1 and TRI4_2. With this parameter, the latest software produced lower accuracy. The lack-of-precision assessment of the TRI4_2 also produced a greater value. All deviations from perfect trueness were below 20 µm, which is comfortably within the clinically acceptable range.Numerous studies investigated the different properties of IOS: scanning speed, ergonomic properties, special features, accuracy , 34–37. In our present study, best-fit alignment was performed. Two different generations of intraoral scanners were used: 3Shape Trios 3 and 3Shape Trios 4. A generation change means, that the manufacturing company upgrades an already existing intraoral scanner (previous generation) in terms of both hardware and software, creating a new version (new generation model) . A softwThis study investigated the effect of software updates on the accuracy of IOS devices. Two different generations of 3Shape Trios intraoral scanners (Trios 3 and Trios 4) and four different software versions were evaluated for their accuracy (trueness and precision). For accuracy, in the literature, the clinically acceptable range for the marginal fit of a restoration is 50 to 120 μm , 10. ExaThe null hypothesis was rejected on analysis of full arch and bridge scan data: an association between the recency of the software version of the IOSs and a greater accuracy of digital impressions was found.In case of parameter 1, there was a significant improvement in trueness and precision. The Trios 3, with the latest software version (TRI3_2) was more accurate than the Trios 4 Move with the previous software version (TRI4_1), which is the latest generation of the 3Shape company’s IOS devices. Furthermore, the accuracy of the software updated Trios 4 Pod intraoral scanner (TRI4_2) was better than that of the mentioned Trios 3 (TRI3_2). The latest generation of 3Shape products (Trios 4 Pod) with the updated software (TRI4_2) produced the best accuracy result in our study, except in case of parameter 3, where the Trios 4 Pod with the older software (TRI4_1) proved to have the highest trueness. It is important to highlight that the software run by the Trios 4 Pod was not the latest software version in the dental market. Based on the result, it could be hypothesised that the hardware version of the IOS could impact the accuracy. Ender et. al.  in 2019 In the case of parameter 2 significant differences could be seen between the results of the oldest (TRI3_1) and the latest (TRI3_2) software versions of Trios 3 intraoral scanners. The measured data of parameter 2 showed an increasing tendency in trueness and precision compared to parameter 3 (surface deviation of the prepared incisor). It has been stated in previous studies that longer captured distances result in less accurate digital impressions .An edentulous ridge between teeth 14 and 17 is an important part of our measurement because most scanners capture the surfaces with increased inaccuracy across edentulous ridges. Edentulous regions contain less information than tooth surfaces ; therefoIn the case of parameter 3, negative impact of software updates on trueness was found. The range of variation across scanner configurations was less than 5 µm, so the difference's clinical impact is negligible. In our research more accurate data were obtained than in the literature: Park et al.  examinedFew studies in the literature evaluate the effect of software updates on the accuracy of intraoral scanners . In furtThere are some limitations to this study. Our results are based on model scanning procedures (in vitro study); it could be interesting to measure these parameters in clinical circumstances. In clinical situations, numerous factors can impact the accuracy of IOS, such as the saliva flow rate in the oral cavity, the individual characteristics of the patient, and the operator's skills in digital impression-taking. Moreover, our study used only two scanners from the same manufacturer. They use confocal laser technology, and the method of recording data is a video sequence . Many tyBased on our results the null hypothesis was rejected. Within the current study's limitations, we conclude that new generations of intraoral scanner hardware and software can significantly increase the trueness and precision of the devices when it comes to full-arch scanning. The presence of an edentulous region may still adversely affect the accuracy of the results; however, software updates seem to achieve more accurate STL files. All versions create a clinically acceptable digital impression of a single abutment.