Although stretching can acutely increase joint range of motion (ROM), there are a variety of factors which could influence the extent of stretch-induced flexibility such as participant characteristics, stretching intensities, durations, type (technique), and muscle or joint tested.The objective of this systematic review and meta-analysis was to investigate the acute effects of stretching on ROM including moderating variables such as muscles tested, stretch techniques, intensity, sex, and trained state.A random-effect meta-analysis was performed from 47 eligible studies (110 effect sizes). A mixed-effect meta-analysis subgroup analysis was also performed on the moderating variables. A meta-regression was also performed between age and stretch duration. GRADE analysis was used to assess the quality of evidence obtained from this meta-analysis.p < 0.001; I2 = 33.32). While there were ROM increases with sit and reach (P  = 0.038), hamstrings (P < 0.001), and triceps surae (P  = 0.002) tests, there was no change with the hip adductor test (P = 0.403). Further subgroup analyses revealed no significant difference in stretch intensity (P  = 0.76), trained state (P  = 0.99), stretching techniques (P  = 0.72), and sex (P  = 0.89). Finally, meta-regression showed no relationship between the ROM standard mean differences to age  and stretch duration , respectively. GRADE analysis indicated that we can be moderately confident in the effect estimates.The meta-analysis revealed a small ROM standard mean difference in favor of an acute bout of stretching compared to non-active control condition  −0.677 to −0.434; A single bout of stretching can be considered effective for providing acute small magnitude ROM improvements for most ROM tests, which are not significantly affected by stretch intensity, participants’ trained state, stretching techniques, and sex. The online version contains supplementary material available at 10.1186/s40798-023-00652-x. The meta-analysis on joint range of motion (ROM) increases revealed a small effect size in favor of an acute bout of stretching compared to the control condition.Subgroup analysis revealed a significant increase in ROM with sit and reach, hamstrings, and triceps surae tests, but no improvement with the hip adductor tests. Whereas all moderating variables presented significant increases in ROM, further subgroup analyses revealed no significant difference in ROM gains with the stretch intensity, trained state of the participants, stretching techniques, and sex.A meta-regression showed no relationship between the effect sizes to age and stretch duration, respectively.The online version contains supplementary material available at 10.1186/s40798-023-00652-x. Recent commentaries , 2 and sSome of the stretching variables affecting acute changes in ROM or flexibility are the type of stretch technique, intensity, duration, as well as the sex and trained state of the individual , 15. AllA number of acute stretching studies have shown that submaximal intensity stretches provide similar ROM benefits as near maximal point of discomfort stretches –41. AposNearly, all stretching duration can ameliorate ROM . The incBaseline measures of flexibility are commonly reported to be greater for women than men –68, whicTherefore, the objective of this meta-analytical systematic review was to investigate the acute effects of stretching on ROM, with consideration of moderating variables such as stretching techniques, intensity, duration, as well as trained state or age of the participants, muscles tested, and sex.This review was conducted according to the 2020 PRISMA guidelines and the suggestions from Moher et al.  for systIn accord with PICOS  criteria, this review considered studies that investigated the intervention of an acute (single bout or session) effect of stretching on joint ROM (outcome) in healthy participants (population) compared to non-active control conditions (comparator). We included peer reviewed original studies published in English. The studies were included when they were either randomized controlled trials or controlled trials (type of studies). This implied that we excluded studies which were dealing with the training  effects of stretching, investigated any combined treatment , or had another treatment as control condition . Moreover, we excluded review papers, case reports, special communications, letters to the editor, invited commentaries, conference papers, and theses.An electronic literature search was performed in PubMed, Scopus, Web of Science, and SPORTDiscus. Papers were considered if they were published up to September 2022. Using AND and OR Boolean operators a systematic search was conducted using the following keywords: flexibility, “range of motion,” extensibility, and stretch*. In addition to the aforementioned keywords, the studies were filtered using the subsequent keywords to include controlled trials: “randomized controlled trial,” “controlled clinical trial,” randomized, placebo, randomly, and trial. Furthermore, to exclude animal studies ,we added a NOT operator with the following MeSH Term “exp animals/ not humans.” , the sample size, the characteristics of the intervention , and the results of the main variables (flexibility parameters) were extracted. For the flexibility parameters, pre- and post-intervention values plus standard deviations of the stretching and control groups were extracted. If some of the required data were missing in the included studies, the authors of the studies were contacted via email or similar channels . For studies with no available data, the corresponding authors were contacted. If no response was received from the corresponding authors, the studies were excluded.Egger’s regression intercept test and visual inspection of the funnel plot were applied to detect possible publication bias.The methodological quality of the included studies was assessed using the PEDro scale. In total, 11 methodological criteria were rated by eight independent researchers . A point was given if the study met the eligibility criteria and evidently a score of zero was assigned if the criteria was not satisfied. Hence, higher scores indicated better methodological quality of the study. In the case of conflict between the eight researchers, the methodological criteria were reassessed and discussed.Grading of Recommendations, Assessment, Development and Evaluations (GRADE) rating analysis was used to assess the quality of the outcomes by using the GRADEpro Guideline Development Tool software (gradepro.org). In general, GRADE has four levels of evidence quality: very low, low, moderate, and high. For GRADE analysis, six evaluation components were adopted .2 statistics were calculated to assess the heterogeneity among the included studies, and thresholds of 25%, 50%, and 75% were defined as having a low, moderate, and high level of heterogeneity, respectively [The meta-analysis was performed using Comprehensive Meta-Analysis software, according to the recommendations of Borenstein et al. . By applectively . An alphOverall, after removal of the duplicates, 4793 papers were screened, from which 42 papers were found to be eligible for this review Table . After cOverall, 110 effect sizes could be extracted from 47 eligible studies. In summary, 1658 participants with a mean age of 23.2 (± 3.4 years) participated in the included studies. Table Figure For the study design, we have included randomized trials for the GRADE analysis. Risk of bias, indirectness, inconsistency, and imprecision showed no serious shortcomings. However, risk of bias assessment of the eligible studies showed publication bias as well as there was no large effect, no plausible confounding, and no dose response gradient. As a consequence, the analysis showed that we can be moderately confident in the effect estimates. This implies that the true effect is likely to be close to the estimate of the effect.Z = −8.939; CI (95%) −0.677 to −0.434; p < 0.001; I2 = 33.32). Figure The meta-analysis on joint ROM revealed a small effect size in favor of stretching compared to the control condition . While there was an increase in ROM with the sit and reach test, hamstrings test, and triceps surae tests, no such change was seen in the hip adductor tests. Further subgroup analyses revealed no significant difference in the Q-statistics for the stretch intensity (P  = 0.76), trained state of the participants (P = 0.99), stretching techniques (P  = 0.72), and sex (P  = 0.89). Furthermore, meta-regression showed no relationship between the effect sizes to age  and stretch duration , respectively.Q-statistics of the subgroup analysis revealed a significant difference for the muscles tested  can increase joint ROM , 22, 79.The present review did not find any ROM differences based on the stretching technique. There are diverse reports indicating greater –85 or siWhereas the included studies with these 55 effect sizes used a wide variety of stretch durations, there was no significant difference in ROM gains based on stretch duration. ROM can be augmented with stretch durations as short as 5-s . Nine stSimilarly, using a high or low stretch intensity did not significantly modulate ROM gains. One of the difficulties in analyzing stretch intensity is the lack of consistency in the description of intensity . While Apostolpoulos et al.  reviewedAn acute bout of stretching will increase ROM in most tests , isolated hamstrings, and triceps surae ROM tests) with the exception of hip adductor , 119 andThe trained state, age, or sex of the participant did not present significant differences in ROM gains. Similarly, a recent meta-analysis comparing the effects of stretching and foam rolling on ROM reported no significant differences between participants’ age groups, activity levels, tested muscle by the ROM test , stretch or foam rolling duration, sex, stretching technique , and the study design  . FurtherAn initial thought might contend that the lower baseline levels of flexibility with untrained individuals would give them a greater training capacity for ROM improvements. However, when considering the capacity for extreme improvements in ROM seen with certain athletes , the extent of change is capacious. Thus, even with higher baseline flexibility, trained individuals still have extensive potential for increased ROM that would not differentiate them from untrained individuals with a single (acute) session of stretching .Although older individuals tend to exhibit more restricted ROM , 84, relLimitations in the research included that not all muscle groups are equally represented in the literature, and further research should expand the scope of muscles tested as for example the limited research on hip adductors. Almost every study described in this review recruited young adults, and only two studies focused solely on females; hence a wider spectrum of participants needs to be investigated.This systematic review and meta-analysis demonstrated a small magnitude increase in ROM with stretching compared to the control condition. Acute increases in ROM occurred with all muscles tested , but no improvement with the hip adductor tests, which might be attributed to more natural anatomical and functionally restricted movement patterns. There was also no significant difference in ROM gains with the stretch intensity, duration, trained state of the participants, stretching techniques, age, or sex suggesting relative acute increases in ROM possess a broad capacity for acute improvement. Consequently, it can be suggested that all types of stretching can be implemented acutely for diverse populations  with similar results expected.Additional file 1. Search Strategy.Additional file 2. Table S1: PEDro scales.


For external rotation with arm at side, there was no significant difference between planning (24° ± 26°) and post-operative clinical observation (19° ± 12°) (p = 0.38). For the angle measurements, the GMA was significantly higher , and the GH angle, significantly lower on the virtual planning , while the MH was not different (p = 0.33). Conclusions: The virtual RoM given by the planning software used in this study differs from the real post-operative passive RoM, except for external rotation. This can be explained by the lack of ST joint and soft tissues simulation. However, in focusing on the virtual GH participation, the simulation looks informative. Some modifications between the glenoid and humerus starting positions before running the motion analysis could be provided for making it more realistic and predictive of the RSA functional results. Level of evidence: III.Introduction: The purpose of this study was to analyze the real range of motion (RoM) measured in patients operated on for reverse shoulder arthroplasty (RSA) and compare it to the virtual RoM provided by the preoperative planning software. Hypothesis: There was a difference between virtual and real RoM, which can be explained by different factors, specifically the scapula-thoracic (ST) joint. Methods: Twenty patients with RSA were assessed at a minimum follow-up of 18 months. Passive RoM in forward elevation abduction, without and with manually locking the ST joint, and in external rotation with arm at side were recorded. The humerus, scapula, and implants were manually segmented on post-operative CTs. Post-operative bony structures were registered to preoperative bony elements. From this registration, a post-operative plan corresponding to the real post-operative implant positioning was generated and the corresponding virtual RoM analysis was recorded. On the post-operative anteroposterior X-rays and 2D-CT coronal planning view, the glenoid horizontal line angle (GH), the metaphyseal horizontal line angle (MH), and the gleno-metaphyseal angle (GMA) were measured to assess the extrinsic glenoid inclination, as well as the relative position of the humeral and glenoid components. Results: There were some significant differences between virtual and post-operative passive abduction and forward elevation, with (55° and 50°,  Restoring range of motion (RoM) is one of the main challenges of reverse shoulder arthroplasty (RSA). Functional outcomes are closely related to bony or prosthetic impingement, especially during adduction and external rotation with elbow at side ,2. ScapuThree-dimensional (3D) preoperative planning solutions have been developed to improve glenoid implant positioning and optimize RSA biomechanics ,5. In a ST participation is more important for RSA biomechanics than for a normal shoulder ,10,11. WThe purpose of this study was to analyze the real RoM measured in patients operated on for an RSA and compare it with the virtual RoM given by the preoperative planning software in the same implant positioning conditions . The hypThe study was approved by our institutional review board (IRB: 13B-T-SHOULDER-RM).2. The surgical indications were cuff tear arthropathy (CTA) for 11 patients, primary osteoarthritis (OA) for 7 patients, and rheumatoid arthritis and post-instability arthritis for 2 patients. This was a retrospective clinical and experimental study involving 20 patients , who were operated on for RSA by two surgeons between 1 January and 31 December 2016 (IRB: 13B-T-SHOULDER-RM). The mean age was 73.8 (63–85) years. The mean BMI was 27.1 (19.5–34.4) kg/mIn four patients, the AEQUALIS™ REVERSED II shoulder system was used, while the AEQUALIS™ ASCEND™ FLEX shoulder system was implanted in 16 patients . Patients with missing post-operative RoM data and pre- and post-operative imaging were excluded.® 3D Planning software  provides a validated method for automatic segmentation, 3D reconstruction, and accurate 3D pre-operative glenoid (version and inclination) and humeral (inclination and posterior subluxation) measurements [The BluePrinturements ,16. This® software runs a virtual RoM analysis in the different degrees of freedom: abduction/adduction, flexion/extension, and internal/external rotation arm at side. The maximum range of motion relative to bony or implant impingement between the humeral and scapular sides is recorded , the RSA procedure was performed for each patient;At a minimum follow-up of 18 months, a post-operative CT scan was performed for each patient using the acquisition characteristics required by the BluePrint® 3D Planning software, in addition to the routine clinical and radiological assessments, which were conducted by a single surgeon;For all shoulders, the planning software’s automated segmentation algorithm was used to extract the pre-operative humerus and scapula 3D models from the pre-operative CTs A;On all the post-operative CTs, the humerus, scapula, and implants were segmented manually B;Registration and superposition were performed between the pre- and post-operative bony structures using the PTC Creo® Version 6.0 software  C;A software program that takes the registration data and generates “post-operative planning” files equivalent to the real post-operative implant positioning was developed;Surgeons could open the post-operative planning file and access all BluePrint® 3D Planning software measurements, in particular, the RoM analysis D.This protocol guaranteed that the implant choice and positioning were identical between the virtual and real conditions, making it possible to compare the RoM measurements between both conditions.Passive RoM in abduction and forward elevation without and with manually locking the scapula-thoracic joint, as well as the external and internal rotation with elbow at side, were assessed clinically in each patient at a minimum follow-up of 18 months. Those measurements were compared to the virtual RoM generated by the “postoperative planning”. Three angles were measured on post-operative anteroposterior x-rays in neutral rotation, and on a planning 2D-CT view passing by the larger diameter of the sphere. The first was the glenometaphyseal angle (GMA)  between p ≤ 0.05. The statistical analysis was performed by a biostatistician using XLSTAT Life Sciences software . The two-sided Wilcoxon–Mann–Whitney test was used to compare two variables. The significance level was set at p < 0.0001). Passive real forward elevation was 141° (±24°), while the virtually planned motion was 91° (±24°). This 50° difference was significant (p < 0.0001). At a mean follow up of 26.3 months (18–38), the mean post-operative passive abduction was 136° (±28°), while the virtual abduction given by the software was 81° (±13°). There was a significant difference of 55° between the two conditions (p < 0.002 and p < 0.0001). In those conditions, the abduction ratio (GH/ST) was 0.92. The forward elevation ratio (GH/ST) was 0.81.When manually locking the scapula-thoracic joint, the mean post-operative passive abduction was 65° (±18°), and the passive forward elevation was 63° (±20°). The differences with the planned virtual motions were, respectively, 15° and 27° . Internal rotation was not interpretable because the measurement scale differs between the virtual motion given by the software (in degrees) and the real post-operative motion assessed by the surgeon   and the post-operative clinical observation  (reached) .p < 0.0001). The GH angle was significantly lower on the virtual planning , while the MH angle was not different (p = 0.33)  than on the post-operative X-ray (29.1° ± 18.2°) ( = 0.33) .Post-operative passive forward elevation and abduction were significantly different to the virtual post-operative RoM, while the features and the positioning of the components were exactly the same between planning and surgery. However, those differences were less important when the ST joint was manually neutralized during the clinical evaluation. The ST joint was not taken into account during the RoM simulation. In our study, the ratios (GH/ST) for passive forward elevation and abduction in usual clinical exam conditions (ST joint free) are estimated at 0.82 and 0.92. Those values are close to previous values observed by different authors based on kinematic studies ,12, wherST joint participation in shoulder RoM partially depends on glenoid inclination. Some authors reported that the more the glenoid is downward, the greater the action of the ST joint for carrying out this motion, especially for abduction and forward elevation ,21. For Other factors may explain the differences between virtual and post-operative real RoM. The humerus position during simulated abduction is not strictly aligned with the glenoid surface . The abdOur study has several limitations. The number of patients involved is relatively small. The method for measuring the post-operative passive mobilities with the manual neutralization of the ST joint is not so easy achieve and it is not reproducible, and consequently, not so accurate for assessing the range of motion. This could be one of the reasons, as reported above, for explaining the difference observed between virtual and real RoM results. Additionally, this clinical evaluation remains subjective and surgeon-dependent. Nevertheless, to our knowledge, this is the first study reported aiming to address the relevance of a pre-operative planning software program with a RoM simulation. Our study protocol was rigorous. The same conditions were used for implant choice and positioning between the planning and the surgery. The passive RoM with and without the ST joint was assessed. Utilizing the limited available data, an associated radiological analysis was produced.The virtual RoM given by the 3D planning software programs used in this study differs from the true post-operative RoM, except for the external rotation with elbow at side. This can likely be explained by the lack of simulation rendering for ST joint and soft tissues during the virtual analysis. However, the RoM simulation provided looks informative for GH participation. Some modifications, such as the starting scapula inclination, as well as its position relative to the humerus during abduction, could be inputted in the planning software to make it more realistic and predictive of the RSA functional results.


A. fumigatus reference strain Af293 in intermicrobial competition with Pseudomonas aeruginosa, and sensitizes A. fumigatus for antifungal effects of nikkomycin Z. We compared the sensitivity of two virus-infected (VI) and one virus-free (VF) Af293 strains to hypertonic salt. Salt stress impairs the growth of VI and VF at all times; VF control growth always exceeds VI, and VF growth in salt always exceeds VI. Since VF growth exceeds VI in the presence and absence of salt, we also examined growth in salt as a percentage of control growth. Initially, as a percentage of control, VI exceeded VF, but at 120 h VF began to exceed VI consistently even by this measure; thus, at that time the growth of VF in salt surges in relation to control growth, or, alternatively, its growth in salt persists compared to the relative inhibition of VI. In summary, virus infection impairs the response of A. fumigatus to several different stresses, including hypertonic salt.Infection with Aspergillus fumigatus polymycovirus 1 (AfuPmV-1) weakens the resistance of biofilms of common  Aspergillus viruses, and the complexity of their effects, has been recently reviewed elsewhere in detail [Fungi, similar to all living organisms, may be infected by viruses known as mycoviruses; however, there are few data on the effects of viral infections on the fungal host physiology. Most studies have failed to demonstrate any effects, contributing to the notion that mycoviruses are cryptic, while a few have shown effects on traits such as growth, sporulation, virulence and drug resistance. The current understanding of n detail .Polymycoviridae. Members of the family Polymycoviridae and its sole genus Polymycovirus have multi-segmented double-stranded (ds) RNA genomes, whereas most lack a conventional capsid structure [Aspergillus fumigatus (Af), which is the main pathogen causing aspergillosis in immunocompromised individuals, investigating the effects of AfuPmV-1 infection may provide insight into alternative approaches to combat Af.Aspergillus fumigatus polymycovirus-1 (AfuPmV-1)  is the ptructure . In the Pseudomonas aeruginosa, but also against effects of bacterial volatile organic molecules [In previous studies, we showed that AfuPmV-1 impairs Af in intermicrobial competition, largely in competition for iron with the pathogenic bacterium olecules . The forolecules . We now Fully sequenced reference strain Af293  was infe6 conidia/mL of inocula, and 0.01 mL of this was added to 6 mm paper discs on 4 study plates. RPMI 1640 agar plates were made using 100 × 15 mm Petri dishes; Bacto Agar  3.75 g and 75 mL de-ionized water were autoclaved and then allowed to cool. RPMI 1640 medium, 225 mL, prewarmed in a water bath at 50 °C was added. To produce high salt agar, NaCl 14.03 g was added to the RPMI 1640 medium. The RPMI medium, with or without NaCl, was added to the Bacto agar mixture, and 25 mL per plate poured.Agar plates were inoculated with the strains under study, and when conidiation had occurred conidia were harvested, as previously described , to prod2) of the isogenic strains on RPMI1640 agar in the presence and absence (control) of 0.8 M NaCl  was compared in several experiments , over 48–144 h. The salt concentration in this study was selected on the basis of preliminary experiments, selecting a concentration showing a definite growth inhibition of strains.Growth . Examples of this can be seen in the top half of NaCl 0.8 M impaired Aspergillus growth. In five of five experiments, this was demonstrated at 48, 72, 96, 120 and 144 h, for strains 18–95 (virus-infected) and 18–42 (virus-free) (p = 0.02–<0.0001), five of five at 120 h (p = 0.002–<0.0001) and three of three at 144 h (p = 0.002–0.0002). Examples of this can be seen in the top half of In six experiments, at every time point, the growth of the virus-free strain 18–42 was superior to the virus-infected strain (18–95). This was not as pronounced at the beginning of the time course, at 48 h, when impaired radial growth could first be reliably measured, or at 72 h. With further time of incubation, the difference in favor of the virus-free Aspergillus strain was consistently significant in five of five experiments at 96 h (p = 0.003–<0.0001), 72 h , 96 h , 120 h  and 144 h . Examples of this can be seen in the top half of In five of five experiments, at every time point studied, virus-free Aspergillus control growth was also always greater than the virus-infected strain. This was recorded at 48 h .Given the dramatic differences both in growth between the virus-free and virus-infected strains in the presence of salt, and the dramatic differences in the absence of salt, we examined the susceptibilities of the two strains to salt as a function of the relative inhibition compared to control growth. This reiterated physiologic differences between the strains that were time-dependent. At the beginning of growth, at 48, 72 and 96 h, the relative growth in salt (as a percent of control) of the virus-free strain was less than the virus-infected at all three time points. In three of five (48 h), three of five (72 h) and two of five (96 h) experiments, the lesser virus-free strain relative growth differences were also significant (p = 0.03–0.003) at 120 h. This  indicated that at about 120 h there was a transition, and small variations in inoculum, growth, temperature, or time of reading between experiments could cause this transition point to lodge on one side or the other of this 120 h break point, shifting from borderline significant differences to significant differences. We presume that recordings at less than 24 h intervals at about 120 h could have defined more precisely when the difference became consistently significantly different. By 144 h, this phenomenon was consistent in that in all three experiments the virus-free strain growth in the presence of salt, relative to control growth, was now significantly superior to the virus-infected strain (p = 0.02–0.0004).The observations at 120 h appear to indicate that approximately at that time the growth of the virus-free strain in salt consistently has a relative surge in relation to the further control growth, or, alternatively stated, its growth in salt persists compared to the relative inhibition in salt of the virus-infected strain. Now, at this time, there was no inferiority in the relative growth of the virus-free strain. In two of five experiments there was no statistically significant difference between the strains, but in three of five the virus-free growth relative to control was now also significantly superior to the virus-infected . Its growth in the absence of salt was inferior to the virus-free strain 18–42 at all times (p = 0.0002–<0.0001). Its growth in the presence of salt was impaired compared to virus-free strain 18–42 at all times (p ≤ 0.0001). Relative to control growth, at all times its salt impairment was also greater than the virus-free strain 18–42 (p = 0.002–<0.0001). In all these comparisons, the re-infected strain 19–40 was also impaired in growth more, in the presence and absence of salt, and also relative to its control growth, to infected strain 18–95, suggesting even greater impairment by the virus in the newly infected strain 19–40. As indicated previously, virus-free strain 18–42 had been re-infected with the virus, producing infected strain 19–40 . An expeAspergillus strains. Virus-free strain control growth always significantly exceeded that of the virus-infected, and virus-free strain growth in salt exceeded that of the virus-infected strains. Since virus-free strain growth exceeds that of the virus-infected in the presence and absence of salt, we also examined growth in salt as a percentage of control  growth. Initially, as a percentage of control, virus-infected strain growth exceeded virus-free, but at 120 h the virus-free strain growth began to exceed that of the virus-infected strain consistently and significantly, even by this measure as well, and persisted; thus, at that time, the growth of the virus-free strain in salt surged relative to its control growth compared to the virus-infected, or, alternatively, its growth in salt persisted compared to the relative inhibition of the virus-infected strain. The overall greater resistance to salt stress in the virus-free strain may suggest that the virus has a role in fungal survival in nature, in water and soil, where there can be such stresses.Salt stress significantly impairs the growth of the virus-free and virus-infected Polymycoviridae family, with a sequence similarity to our AfuPmV-1, has been published [Aspergillus toxins was associated with virus infection, as we also noted [A study with a closely related virus in the ublished . The virublished , whereasublished . They noublished , though so noted . Their vso noted .A. fumigatus to several different stresses, including iron restriction, exposure to P. aeruginosa volatiles [In conclusion, virus-free strain growth in several control media exceeded virus-infected strain growth, as described here on RPMI-1640 agar, and in other media studied, including liquid mineral medium M9  and Czapolatiles , inhibitolatiles  and higholatiles .We have reported elsewhere on virus-free and virus-infected strain differences in the production of siderophores  and, rec


Saint Louis encephalitis virus, Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, Dengue virus, Zika virus, Chikungunya virus, Mayaro virus and West Nile virus. The objective of the present study was to determine the number of arboviruses with neuroinvasive potential isolated in Brazil that corresponds to the collection of viral samples belonging to the Department of Arbovirology and Hemorrhagic Fevers, Evandro Chagas Institute (SAARB/IEC) of the Laboratory Network of National Reference for Arbovirus Diagnosis from 1954 to 2022. In the analyzed period, a total of 1,347 arbovirus samples with encephalitogenic potential were isolated from mice; 5,065 human samples were isolated exclusively by cell culture; and 676 viruses were isolated from mosquitoes. The emergence of new arboviruses may be responsible for diseases still unknown to humans, making the Amazon region a hotspot for infectious diseases due to its fauna and flora species characteristics. The detection of circulating arboviruses with the potential to cause neuroinvasive diseases is constant, which justifies the continuation of active epidemiological surveillance work that offers adequate support to the public health system regarding the virological diagnosis of circulating arboviruses in Brazil.Viruses with encephalitogenic potential can cause neurological conditions of clinical and epidemiological importance, such as  For more than 80 years, the Evandro Chagas Institute (IEC), through a monitoring program for the collection of virological and serological data, has isolated and classified, completely or partially, about 200 different types of arboviruses pathogenic for humans ,2. Some The term arbovirus is derived from the union of the first syllables of the English expression arthropod-borne (transmitted by arthropods) followed by the suffix virus. According to the concept established by the World Health Organization (WHO) , “ArboviArbovirus infections can result in a wide spectrum of clinical syndromes, from mild febrile illness to hemorrhagic fevers and neuroinvasive forms. However, most human arbovirus infections are asymptomatic or oligosymptomatic .The Centers for Disease Control and Prevention (CDC) defines meningitis (aseptic), encephalitis and acute flaccid paralysis as the main neurological manifestations of arbovirus infection, although many cases are not recognized as manifestations caused by these viruses, which contributes to the diagnosis remaining a challenge to be faced .Population growth, disorderly urbanization and increased human traffic and international trade have contributed to the emergence and spatial spread of arboviruses in recent decades ,7. EveryPeribunyaviridae, Togaviridae, Flaviviridae, Sedoreoviridae, Rhabdoviridae, Asfaviridae and Phenuiviridae) harbor arboviruses capable of infecting humans and animals and have become important in public health [Dengue virus (DENV), the Chikungunya virus (CHIKV), the Zika virus (ZIKV), the West Nile virus (WNV) and the Rocio virus (ROCV) as neuroinvasive viruses of the greatest epidemiological interest in Brazil [Currently, approximately seven viral families , the Eastern equine encephalitis virus (EEEV), the Chikungunya virus (CHIKV) and the Western equine encephalitis virus (WEEV), which are important in public health. Additionally, the Aura virus (AURAV), the Una virus (UNAV), the Mucambo virus (MUCV), the Triniti virus (TNTV) and the Pixuna virus (PIXV) are also included [The included . Alphaviincluded . These vincluded ,16. Vertincluded .Flavivirus genus belongs to the Flaviviridae family and is composed of 53 viral representatives, including the Japanese encephalitis virus (JEV), DENV, ZIKV, and WNV, of public health importance. The following viruses are also included: the Cacipacoré virus (CACV), the Ilhéus virus (ILHV), the Bussuquara virus (BUSV) and the Iguape virus (IGUV) [The s (IGUV) . Flavivis (IGUV) .Flavivirus transmission cycle, with no known arthropod vectors. Humans are not definitive hosts but accidental hosts when they enter the habitat of these mosquitoes , Belterra (1978), Santa Bárbara and Conceição do Araguaia (1981) ,22,23,24In a recent epidemiological investigation, MAYV was detected in human populations residing in the municipalities of Parauapebas and Canaã dos Carajás in the Pará state, evidencing the circulation of arboviruses in this area, which reinforces the importance of epidemiological surveillance in areas that are under pressure from environmental changes .Haemagogus, particularly the species Haemagogus janthinomys [Aedes aegypti and Aedes albopictus) and the increasing number of international travelers certainly contribute to the increased risk of MAYV transmission [The transmission of MAYV occurs through the bite of a female mosquito belonging to the genus thinomys , which hsmission .In the case of viral infection, variations in the habitats and behaviors of viruses have been noted in recent decades, such as the spread of MAYV by infected birds or humans, resulting in a transmission cycle that may involve humans as a reservoir .Considering the lack of licensed vaccines and proven therapeutics, prevention focuses mainly on domestic mosquito control to contain the spread of MAYV .This encephalitis manifests itself with severe neurological conditions in humans and horses and has high lethality ,31. In BEpidemiological studies carried out in the Brazilian Amazon were able to positively isolate EEEV . PreviouAedes, Culex and Mansonia have already been implicated in the transmission of EEEV among humans and horses, as they feed on birds and mammals [The EEEV transmission cycle occurs between birds and mosquitoes, and the genera  mammals . Another mammals .Aedes and Culex, and viral isolation has also been obtained in chickens, pheasants, rodents, rabbits, ungulates, turtles, snakes and blood from wild birds [Western equine encephalitis is a serious neurological disease capable of affecting humans and horses and is widespread in Canada, Latin America and the USA. The first isolation of this virus occurred in 1938  in the Uld birds .Aedes serratus and Psorophora ferox, respectively. MUCV was isolated from all four types of sources . PIXV was isolated twice from mosquitoes (Anopheles nimbus and Trichoprosopon digitatum) and once from a wild rodent (Proechimys guyannensis). AURAV, found in South America, has not been linked to any disease in humans or animals.The WEEV complex contains the following viruses: WEEV, AURAV, UNAV, TNTV, PIXV and MUCV. Some viruses originally isolated in the Amazon region have not yet been fully characterized  to justiDasyprocta aguti and Orizomys sp.) and mosquitoes (Sabethes spp.) in the Brazilian Amazon [TNTV was isolated from rodents  comprising 89 viral species [Yellow fever virus (YFV), DENV, JEV, WNV and tick-borne encephalitis viruses are pathogenic to humans. Other members cause economically important diseases in domestic or wild animals. Other viruses in this group include SLEV, ROCV, CACV, ILHV, BUSV and IGUV [The  species , of whic species . Mammalsand IGUV .Ae. aegypti and Ae. albopictus are implicated as transmitters of flaviviruses. These are able to mutate and/or adapt to new zoonotic cycles, re-emerging in places that have suffered environmental impacts as a result of human action in nature [The viral particle measures 40 to 60 nm in diameter and has a protein capsid with icosahedral symmetry, a single-stranded RNA genome, and positive polarity, approximately 11 kb in length . Blood-sn nature .Aedes mosquitoes. In urban centers, this transmission occurs between mosquitoes and humans, with Ae. aegypti as the main vector, and Ae. albopictus and Ae. polynesiensis as secondary vectors. Ae. aegypti originated from the African continent and later spread to Asia and America by sea, arriving in Brazil in the mid-18th century by slave ships. The reports mentioned in the scientific literature mention the 1920s (20th century) as the beginning of epidemics that occurred in Brazilian territory, with the first reports occurring in the state of São Paulo in 1916 and Rio de Janeiro (Niterói) in 1923 [DENV is represented by four distinct serotypes: DENV-1 to DENV-4, which are maintained in cycles involving small primates and Ae. aegypti vector in Brazil without success, causing viral circulation again in 1963 with the reemergence of DENV-2 and DENV-3 in several countries. At the end of the 1960s, the vector was already circulating in cities, such as Belém, Salvador and Rio de Janeiro [In 1955, campaigns were carried out in an attempt to eradicate the  Janeiro .In the 1980s, there was an expansion of dengue throughout Brazil, with the gradual emergence of the four serotypes, which resulted in the emergence of arbovirus epidemics that are still observed in the country . RegardiClinically, dengue manifests as an acute infectious disease characterized by fever, myalgia, headache, joint pain, retro-orbital pain, rash, thrombocytopenia, lymphadenopathy and leukopenia. Fever is sudden and high, persisting for two to seven days. Rash and pruritus are evident in the convalescent phase of the disease .The most severe clinical phase is called dengue hemorrhagic fever (DHF), which is characterized by increased vascular permeability and results in hypovolemic shock, which can lead to tissue damage and multiple organ failure .Ae. aegypti mosquitoes, there are also reports of vertical transmission and blood transfusion transmission. Studies, especially in endemic areas, such as the Amazon region, should be initiated in blood banks to determine the risk of infection for DENV and other flaviviruses for blood product recipients, especially in epidemic periods [In addition to the bite of  periods ,64.Culex genus, and birds are considered amplifying hosts found in urban and rural environments. On the other hand, men and domestic animals are hosts that show symptoms but do not develop significant viremia [SLEV is transmitted by mosquitoes of the  viremia .Sabethes belisarioi mosquitoes collected on the Belém–Brasília highway [Reservoirs of this virus mainly include birds, primates, marsupials and other wild animals. The first record of the isolation of this virus was in the USA from an insectivorous bat, and in Brazil  isolatio highway .In Brazil, only three cases of SLEV in humans were reported in the 1970s, two in the northern region and a third identified in the municipality of São Pedro/SP in 2004 . More reMost reported cases of SLEV go unnoticed because they are asymptomatic, subclinical or have symptoms similar to those of a common cold. The main signs and symptoms observed include fever, headache, myalgia, nausea/vomiting and drowsiness .The illness caused by SLEV can range from mild symptoms, including fever and headache, to severe illness, such as meningitis and encephalitis . The perWNV was identified in the West Nile district of Uganda . Over thHowever, it was only in 2018 that the virus was identified and isolated from the central nervous system (CNS) of horses with neurological disease in the state of Espírito Santo ,78. AddiSymptoms vary in each host, ranging from the asymptomatic form, common in horses and humans, to neurological manifestations in birds, such as disorientation, ataxia, tremors and head tilt, followed by death. Approximately 20% of humans who develop symptoms have nonspecific clinical signs, such as fever, body pain and myalgia, and a smaller number of those affected may have the neuroinvasive form, characterized by meningitis, encephalitis and paralysis progressing to death ,83.WNV transmission is maintained in an enzootic cycle, that is, between arthropods and wild birds, but it can also infect horses and mammals, including humans, the latter being considered accidental and final hosts because they develop a low viral load, which does not allow them to be involved in the transmission cycle. Other forms of transmission can be transovarian, which favors the permanence of the virus in mosquitoes, provided that there are favorable conditions for its maintenance and consequent transfer to progeny ,84, orgaIn 2002, the first case of suspected WNV infection after an obstetric procedure in a patient who received blood and blood products was reported . The worFormicarius analis) in 1977 in an Amazonian area in the state of Pará. The presence of antibodies against this arbovirus was detected in free-living monkeys captured in the state of Mato Grosso do Sul, with two animals showing antibodies against flaviviruses and one showing a monotypic reaction against CACV. More recent studies reported the isolation of CACV from mosquitoes collected in the Amazon region and from human blood in the state of Rondônia [CACV was first isolated from birds  and vertebrates in the Pantanal region and in the state of Pará, São Paulo, respectively [ILHV was isolated from a pool of mosquitoes of the genera  in 1947 . Birds aectively ,94.Culex genus and rodents, although birds can act as reservoirs. In 1971, the first case of a human febrile illness by BUSV was reported in Panamá [BUSV was originally isolated from primate blood near the city of Belém in 1959 [n Panamá . In 2015The IEC was created in 1936, with the name of North Institute for Experimental Pathology Institute, by the state government of Pará, and in 1942, it was federalized, becoming a scientific research body of the Special Service Foundation for Public Health. In 1954, with the support of the Rockefeller Foundation, studies on arboviruses in the Amazon region were hosted at the IEC, which brought innovations in field and laboratory procedures that have contributed in a decisive and invaluable way to public health and the scientific community during these five decades.The objective of this work is to present the encephalitogenic viruses that have been registered in the Department of Arbovirology and Hemorrhagic Fevers (SAARB) of the IEC over the past six decades.g for 10 min at 4 °C. After centrifugation, the samples remained at that temperature until inoculation into a newborn Swiss mouse at 2 to 3 days of life. The animals were inoculated intracerebrally with a volume of 0.02 mL of the suspensions with the aid of a 1 mL syringe and a 25 × 6 disposable needle. This inoculation step was also valid for the other biological samples. The cages that contained the animals that were to be used were identified with batch numbers and internal records of the sample, material type and inoculation date. They contained a lactating female and 6 newborn Swiss albino mice (two days old) [For viral isolation, samples such as blood, serum, suspensions of human or animal viscera and arthropods were used. The aforementioned samples (viscera and arthropods) were ground in 1000 μL of diluent buffer ), penicillin (100 U/mL), streptomycin (100 μg/mL) and gentamicin (0.05 mg/mL) in TissueLyser equipment  using 5-mm diameter “beads” (stainless steel microspheres) at 26,000 movements per minute for 4 min, followed by freezing at −70 °C and later centrifugation at 10,000 rpm for 10 min at 4 °C. Then, each sample was centrifuged at 15,652× ays old) .After inoculation, the animals were observed daily for up to 21 days, and records were made on a card (inoculation card).Mice that showed signs of disease were collected and stored in a freezer at −70 °C until laboratory tests were carried out, while those that did not show signs were disposed of following institutional protocols.Chlorocebus aethiops) and C6/36  cells which were constantly used during these decades.The Laboratory of Viral Isolation/SAARB/IEC has its own Vero , sodium bicarbonate (3 mM), penicillin/streptomycin (104 IU/mL), fungizone (2.5 mg/mL) and 10% FBS for cell growth, with the amount of FBS being reduced to 5% for cell maintenance. The cells were trypsinized every 7 days, and the medium was replaced every 2 days and kept at 37 °C under a humid atmosphere of 95% H2O and 5% CO2. For the C6/36 cells, L-15 medium was used, trypsinization was performed manually, and the cells remained in a room with a temperature of approximately 25 °C [Briefly, 100 µL of each diluted viral sample was inoculated into monolayers of the aforementioned cells propagated in 16 × 125-mm tubes and then incubated at 37 °C and 28 °C for a period of 1 hour (adsorption), and then 1.5 mL of overlay medium (maintenance medium 199 and L-15) was added to the cells. Cell cultures were observed daily to monitor possible cytopathogenic effects (CPEs) with the aid of an Axiovert S100 microscope . Negative controls were made from the inoculation of uninfected cell fluids. The observation of infected cultures was performed with the aid of an Axiovert S100 microscope (Zeiss).The samples isolated in mice registered by the SAARB/IEC from 1954 to 2009 correspond to 639 flaviviruses and 708 alphaviruses .The isolates are geographically distributed among locations in the State of Pará , with reThe isolation of flavivirus and alphavirus occurred in other locations in Brazilian states, such as Serra do Navio—ICOMI (Amapá State), Aripuanã (Mato Grosso State), Boca do Acre (Acre State), Uruaçu (Goias State), Tocantins State, Macapá (State of Amapá), Bom Jardim (State of Maranão), Instituto Baiano de Biologia (IBIO/UFBA) (State of Bahia), Manaus (State of Amazonas) and states of Rio Grande do Sul, Mato Grosso, Mato Grosso do Sul, Paraná and Minas Gerais, in addition to a registration abroad in the city of Iquitos (Peru). In the northern region of the country, specifically in the state of Pará, the isolation of flaviviruses in armadillos and sloths A and alpTogaviridae (9), Rhabdoviridae (3), Sedoreoviridae (4), Picornaviridae (21), Flaviviridae (4), Peribuniviridae (225) and Arenaviridae (27) with incomplete characterization; 8 are partially identified and therefore have unknown pathogenesis: Iriri virus, Uruará virus, Galibi virus, Codajás virus, Cajazeira virus, Marajó virus, Cantá virus, Tracambé virus and Naranjal virus.Flavivirus isolates was from the North region of the country, while the largest number of Alphavirus isolates came from states in the Northeast region, in addition to the absence of confirmed isolates for this genus in the Southeast and South regions  with neuroinvasive potential, distributed in 1484 DENV-1, 1360 DENV-2, 1409 DENV-3, 384 DENV-4, 02 ZIKV and 405 CHIKV. In this period, the largest number of  regions .Flavivirus and 13 isolates for Alphavirus, totaling in the period 1966 to 2022, using both techniques, 676 isolations from species of genera, such as Aedes, Anopheles, Culex, Mansonia, Psorophora and Sabethes, with records for AURAV, BUSV, DENV, EEEV, ILHV, MAYV, MUCV, PIXV, SLEV, TNTV, UNAV and WEEV.The technique of viral isolation from mice and using mosquitoes as an animal model of origin was used by the IEC from 1966 to 2012, and with it, it was possible to isolate about 647 viruses among known arboviruses with encephalitogenic potential and even new ones for science. Isolation records using mosquitoes as an animal model of origin restarted again from 2018, exclusively using C6/36 cell culture , and in the period between 2018 and 2022, 16 isolates were positive for The growing deforestation in the 20th century was associated with the challenge of eradicating arbovirus-transmitting vectors and the uncertain diagnosis of these viruses, whether due to antigenic similarity, causing cross-reactions (in arbovirus-endemic areas) in laboratory tests or similarity of symptoms among patients, constituting old and recurring problems in public health ,100. ConMany of the arboviruses isolated by the IEC in epidemiological research on the circulation of arboviruses, especially in the Amazon region, led to a vast biological collection, with approximately 10,000 arboviruses, including those unknown to science, many of which still have incomplete taxonomic characterization .Over all these years, the information generated by the SAARB/IEC has revealed ecological, environmental and epidemiological information about arboviruses. Studies of this nature are relevant, particularly in the Amazon region, as it has favorable conditions for the maintenance cycle of these agents, demonstrating an ideal environment for the emergence or re-emergence of arboviruses .Clinical manifestations may be absent at the time of infection, which causes unrecognized viral spread through the free movement of individuals, even to nonendemic regions, a situation that imposes the need for constant surveillance of arboviruses ,104. ThiIn light of the epidemiological scenario demonstrating the simultaneous circulation of arboviruses with encephalitogenic potential that are of public health importance, in 2017, the Ministry of Health changed the Sentinel Surveillance Manual for Arbovirus Neuroinvasive Diseases to expand the surveillance of neuroinvasive diseases caused by arboviruses .In light of what was observed in this study, it is possible to conclude that the isolation of ZIKV coincided with the first reports published by the Pan American Health Organization (PAHO), examining physical and emotional effects in the affected population, especially among women of childbearing age due to congenital infections that result in fetal malformations and deaths ,106.In light of the above, the need for constant epidemiological surveillance has become vital to investigate the circulation of arboviruses, many of which are still unknown. This is particularly true for the Amazon region which is a true hotspot for known viruses and for those completely new to science.With over 80 years of experience, the IEC has collaborated with the Ministry of Health in reporting outbreaks and epidemics, whether by reporting cases, implementing training programs or developing human resources that will contribute to the reduction in arbovirus cases .We reiterate the concern regarding the need for urgent actions to combat forest degradation that influences the spread of diseases transmitted by arthropods and their adaptation to the domestic environment with the possibility of emerging or re-emerging zoonoses .Regarding the data presented in this review, it is of great importance to understand that health education constitutes a mechanism through which the population is encouraged to modify behaviors associated with prevention.Currently for most arboviruses, no vaccine is available, and the cycle of sylvatic transmission of viruses cannot be prevented. Thus, preventive measures are necessary. These include avoiding contact in the areas of occurrence, particularly rural and wild forested areas and riverbanks, and minimizing exposure to vector bites, either by means of individual protection (use of repellents and full-length clothes) or collective protection (use of curtains and mosquito nets) .There are numerous arboviruses present in different environments worldwide, especially in the Amazon, due to the favorable conditions found there; more virulent forms of these viruses are constantly emerging and invading new habitats. Therefore, knowing the epidemiology of these arboviruses is of fundamental importance to predict future mechanisms of emergence and/or re-emergence, thereby implementing measures to control and prevent the spread of arboviruses in urban areas. In addition to encephalitogenic viruses, there are many arboviruses with little information available in the literature ,4,5. It The search for vaccines is a priority, and several live attenuated vaccines based on modern molecular biology techniques are in full pharmaceutical development. Some, such as the ChimeriVax candidates developed against WNV, Japanese encephalitis and dengue, have already shown good results in terms of safety and immunogenicity . Our col


Procedural video recordings were used to assess ovarian ligament fat score, smoke formation, occurrence of bleeding, and excision duration. Excised tissues were examined histopathologically and collateral thermal damage was scored in three anatomic zones: suspensory ligament, vascular pedicle, and uterine junction. Tissue sealers were used repeatedly following standardized cleaning protocol with instrument washing machine and ethylene oxide gas sterilization and the number of uses until device failure was recorded.Advanced bipolar tissue sealer/dividers provide the most reliable and efficient means of tissue dissection and blood vessel sealing in laparoscopic surgery and the techniques are continuously improved. In veterinary practice, cost-effectiveness is of major impact, leading to re-use of instruments designed and sold for single use. Two high-end devices were evaluated and compared in a highly standardized laparoscopic ovariectomy procedure in dogs: The new generation Ligasure Maryland Sealer/Divider (LMSD) with improved atraumatic curved jaw shape for delicate tissue handling and dissection and non-stick nanocoating, and the new-generation Articulating Enseal G2 (AENG2) with several proclaimed features improving surgical performance, including articulation of the forceps tip; improved tissue compression during sealing; unique offset electrode configuration; and specific nanoparticle coating minimizing thermal spread and tissue sticking. Twenty-one client-owned dogs admitted for elective laparoscopic ovariectomy were randomly assigned to one of two groups: ovariectomy using AENG2 on the left ovary and LMSD in the right ovary or Excision times were significantly increased for AENG2 (median 01:35 min) compared to LMSD (median 01:00 min). Minor hemorrhage from incomplete sealing occurred in 3 sites in 2 patients  and was not significantly different between groups. Smoke production as scored on videos and thermal tissue damage scores on histopathology also did not differ between AENG2 and LMSD. Both vessel sealers could be re-used repeatedly.AENG2 provides a good alternative to LMSD in laparoscopic ovariectomy, with only minor differences in measured variables. Subjectively, the articulating feature of AENG2 did not improve surgical performance in laparoscopic ovariectomy and the use of LMSD appeared more straight-forward for this specific procedure. However, differences in operating these devices may be subject to personal preference. Advanced tissue sealers have been vital in the development of minimally invasive surgery, improving surgical performance, reducing complications and procedural duration. LigaSure tissue sealer/divider has been considered to set a gold standard in laparoscopic surgery: the device-controlled tissue sealing technique is reliable, fast, easy to use, and has been shown effective to seal blood vessels up to 7 mm in diameter –10. EnseTo date, direct comparison of LMSD and AENG2 have only been performed in total laparoscopic hysterectomy in humans. Compared to LMSD, AENG2 was associated with an increase in operation time , 14, an versus (LMSD). Furthermore, the possibility of repeated use of each device was assessed after repeated cleaning and re-sterilization, to evaluate if devices would be of similar interest for veterinary practice. Based on published data in human hysterectomy, we hypothesized that there would be a difference in ovariectomy duration between LMSD and AENG2. Based on published data of older versions of both devices, we would not expect significant differences in bleeding, smoke formation, and thermal tissue damage extent.The aim of the present study was to evaluate and compare surgical performance, complications and thermal tissue damage in laparoscopic ovariectomy in dogs using (AENG2) Client-owned healthy dogs presented for elective laparoscopic ovariectomy at the Faculty of Veterinary Medicine, Utrecht University were selected for the study. Selection criteria were: a weight range of 10–50 kg, undergone at least one heat, and surgical date 3 months after the last heat. No restriction on age or body condition score was set. Owners completed a questionnaire regarding current health status, last heat cycle, number of previous heat cycles, litters, pseudopregnancies and anti-heat medication prior to the surgery. The study was approved by the institutional ethical committee and all owners provided informed consent for the study.The new generation LMSD was connected to the ForceTriad energy generator  and was used as prescribed: tissue was grabbed, sealed once ensuring that the audible signal of the generator indicated proper seal function, and transected by moving the index finger-controlled cutting blade.The new-generation AENG2 was connected to the GEN11 power generator  and used as prescribed: tissue was approached via the most ideal orientation using standard rotation function and novel articulation function of the shaft, aiming for orientation of the jaw in line with the desired plane of dissection, perpendicular to the mesovarial blood vessels. Tissue was grabbed and compressed by closing the forceps until the automatic stop, with the handles halfway closed; sealing was commenced by pushing the index finger-controlled button; cutting and continued sealing was performed after the audible signal of the generator changed in frequency by continued closing of the device handles by which the cutting blade was pushed through the tissue; electrical sealing stops when the I-Blade is fully advanced and handles fully closed followed by an audible tone that signals end of sealing event. The AENG2 instruction manual advises to advance the I-blade slowly after the change in beeping tone, especially when transecting larger vessels  was inserted through the incision. Camera guidance with a 5 mm 0° telescope  was used to confirm entrance into the abdominal cavity and the abdomen was insufflated with 2-5 L/min CO2 and limited at 8 mmHg intra-abdominal pressure . A routine visual exploration of the abdominal cavity was performed. If no abnormalities were noted, the second and third portal were placed in the midline 2-3 cm cranial and caudal to the umbilicus. Both were placed with a 6 mm skin incision, followed by stab incision with an 11-blade under intra-abdominal visual guidance of the camera telescope. Both ovaries were approached and excised in the same standardized manner ) and right ovaries (01:19 min [00:42 − 04:22]) were not significantly different (WSR P = 0.528). Excision times for AENG2 (01:35 min [00:42 − 04:22]) were significantly longer compared to LMSD (01:00 min [00:39 − 02:24]) (WSR P = 0.001). Excision times with minor bleeding were over median excision times . Excision times for both AENG2 (KW P = 0.023) and LMSD (KW P = 0.047) were longer for dogs with a mesovarian fat score of 3 compared to fat score 1. There were no significant differences for both vessel sealers comparing fat score 1 to 2 and 2 to 3 . Therefore, added articulation of the shaft was ultimately not applied in any case.Re-sterilization of both LMSD and AENG2 was performed as per protocol during the study. Eleven AENG2 vessel sealers were used during this study, with successful re-use after a median of 5 cleaning cycles (range 3–12), device failure occurred one cycle after this. Nine AENG2 broke down due to mechanical failure: falling apart of the body (n = 7) and failure to open jaw (n = 2). Two AENG2 were taken out of use because of electrical failure. Twelve LMSD sealers broke down during the study period. These LMSD devices were re-used after cleaning and re-sterilization up to 9 times, with a mean of 5.3 ± 1.3 cleaning cycles.Thirty-eight ovaries of 19 dogs were available for histopathology. Ovaries of patient number 6 were already submitted for histopathology due to a cystic structure on the left ovary that was diagnosed as a teratoma. Ovaries of patient 21 got lost and were consequently not available for histopathology.On quantitative and qualitative scores, there were no significant difference in collateral thermal tissue damage extent between left and right ovariectomy seals.Quantitative tissue damage extent was not significantly different between AENG2 1.3-2.1 mm) and LMSD (1.0-1.7 mm) for the three sites  during ovariectomy. These three incomplete sealing events all occurred in patients with high fat score, and two happened in the same patient (AENG2 and LMSD), indicating that individual patient characteristics may be a risk factor for incomplete tissue sealing rather than bipolar sealing technique. Blood loss was similar between AENG2 and LMSD in total laparoscopic hysterectomy in humans, although a ‘mesovarial fat score’ was not evaluated in humans .Subjectively, the articulating function of the ENG2 was not found to be of added value in 3-portal ovariectomy in this study. The plane of articulation is perpendicular to the opening of the jaw, which does not improve the approach to the uterine tip and mesovarium. Theoretically, the articulation would possibly have been more useful if the articulation had been in the same plane as the opening of the jaw to further improve alignment with the mesovarium. Benefits of the current articulation feature are therefore also questionable for veterinary single-portal ovariectomy, although it has been published to be beneficial in human single site total laparoscopic hysterectomy , 27. A cCollateral thermal tissue damage was not significantly different between AENG2 and LMSD on quantitative and qualitative analysis in our study. Collateral damage as measured in our study was 1.0-2.2 mm for all sites. These measurements seem comparable to previous studies in which older types of LigaSure and Enseal were compared , 6, 15. Both vessel sealers kept functioning after repeatable cleaning and sterilisation cycles as per protocol described in this paper without any negative effects for the patients. However, continuous re-use until failure was not feasible in the present study due to sterilization flow. Multiple AENG2 devices were in rotation to ensure patient flow because gas sterilization by an external company being the limiting factor in turnover time. Consequently, only three ENG2 vessel sealers stopped working by the termination of the patient study. Therefore, remaining AENG2 devices were sham tested until failure after study closure using only our routine cleaning procedure (without Ethylene Oxide gas sterilization). LMSD is a standard tissue sealer in our hospital and multiple devices are in use concurrently, for this study as well as other procedures. Therefore, several of the LMSD devices in the present study may have also been used for other procedures, such as pericardectomy and adrenalectomy. LMSD device failure often showed as an error message in the Force Triad display or loss of the electrical sealing activation knob in the handle, resulting in failure to activate the device; AENG2 device failure presented most often as the breaking apart of the welds of the handpiece after the re-sterilization process. With a median of 5.0 re-uses for AENG2 and a mean of 5.3 re-uses for LMSD, the longevity seems comparable. A previous study by Valenzano et al.  mentioneLigaSure Maryland and Articulating Enseal G2 advanced bipolar vessel sealers were similarly effective in laparoscopic ovariectomy in dogs for most tested features. Although AENG2 was significantly slower for ovary excision compared to LMSD, the clinical relevance of this finding in respect of total procedure duration may be debatable. Subjectively, the articulating feature of AENG2 was not of added value in 3-portal ovariectomy in this study and the use of LMSD appeared much more straight-forward and easy because of its completely device-controlled sealing process. The choice of either technique may vary between surgeons, based on individual preference and specific intended use. Both devices were suitable for re-use after repeated cleaning and sterilization cycles, which makes them both interesting for veterinary use.


The economic costs of distributing HIVST kits were assessed in 37 health facilities between July 2019 and March 2021 (21 months). Sensitivity analyses were also performed on unit costs to examine the robustness of our estimates related to key assumptions. In total, 16,001 HIVST kits were dispensed for 32,194 HTS sessions carried out. Program level HIVST average costs ranged $12–286, whereas site level costs ranged $4–26 across distribution channels and countries. Site level HTS costs ranged $7–8 per testing session, and ranged $72–705 per HIV diagnosis. Across countries and channels, HIVST costs were driven by personnel (27–68%) and HIVST kits (32–73%) costs. The drivers of HTS costs were personnel costs ranging between 65 and 71% of total costs across distribution channels and countries, followed by supplies costs between 21 and 30%. While program level HIVST average costs were high, site level HIVST average costs remained comparable to HTS costs in all countries. Health facility-based distribution channels operating at low volume exhibit high proportion of central costs which should be considered carefully for financial planning when run alongside high volumes mobile outreach distribution channels. HIVST can diversify the HIV testing offer at health facilities, thus improving access to screening for target populations not reached by HTS services.Achieving the first 95 of the UNAIDS targets requires the implementation of innovative approaches to knowing one's HIV status. Among these innovations is the provision of HIV self-testing (HIVST) kits in west Africa by the international partner organization Solthis (IPO). In order to provide guidance for the optimal use of financial resources, this study aims to estimate the program and site level costs of dispensing HIVST as well as HIV testing services (HTS)-excluding HIVST-in health facilities in Côte d'Ivoire, Mali and Senegal as part of the ATLAS project. We estimated from the provider's perspective, HIVST and HTS incremental costs using top-down and bottom-up costing approaches and conducted a time and motion study. We identified costs at the  The ambitious 95-95-95 strategy was announced by UNAIDS in 2020 aiming to end the AIDS epidemic by 2030  by achieIn order to reach the first 95 in west Africa, it is necessary to implement approaches that reach those in need of HIV testing and who are being missed . One of The ATLAS project, implemented by the international partner organization (IPO) Solthis (Solidarité thérapeutique et initiative pour la santé) in Côte d'Ivoire, Mali and Senegal, promotes the distribution of HIVST to hard-to-reach/hidden populations as a complementary approach to traditional HIV testing services (HTS). In the context of this study, HTS definition excludes HIVST kits provision. Two main strategies are adopted: a large-scale strategy using mobile outreaches (mobile strategy) and a small-scale strategy in health facilities. The health facilities strategy aims to reach female sex workers (FSW), men who have sex with men (MSM), people who use drugs (PWUD), partners of sexually transmitted infections patients (STI) and sexual partners of PLHIV (Index Testing) in health facilities . RegardiTo establish the merits of the use of HIVST and therefore the adaptation of programmatic strategies and optimal financing in the fight against HIV in health facilities, data on costs at the level of programs and intervention sites prove necessary . TherefoThe objective of this study is to estimate and analyse the program and site level costs of dispensing HIVST and providing HTS in health facilities using real-word data collected in Côte d'Ivoire, Senegal and Mali as part of the ATLAS project. The results of this study will inform HIV funding programs while providing key information for guiding actions tailored to low-volume health facilities.This study focused on HIVST distribution channels implemented in health facilities and targeting, FSW, MSM, PWUD, partners of STI patients and partners of PLHIV . There wreal-word, we used a top-down costing approach to collate the IPO's financial expenditures and categorize each cost item by input type and distribution channel ((bottom-up costing) to estimate economic costs . A site is defined either as a primary health care facility for general population and/or key populations, a sexual health clinic, or a community health centre recognized by government authorities. Overall, HIVST kits were distributed by 208 agents  of whom 183 were counselors  also providing HTS. The study distinguishes providers that are the health care workers paid by the state budget  and financial and technical partners that are peer educators involved in project activities.provider's perspective: HIVST and HTS. We distinguished between program and site level costs , MSM , PWUD , STI  and Index Testing  and we used standardized costing guidelines and analyzed data, ensuring consistency of methods across countries , 21. We el costs .Program costs were estimated for HIVST only, and include all costs incurred during the project phases and at all intervention levels  and provide costs of the entire program. These costs are distributed according to the three intervention phases of the project , Implementation) (development phase (June 2018-March 2019) identified sustainable distribution models in each country; the start-up phase  consisted of the validation, adaptation and implementation of the training strategy for HIVST providers; and the implementation phase (until March 2021) corresponded to the effective distribution of HIVST kits, supervision and data validation activities. For HIVST, we also estimated site level costs at health facilities, therefore excluding central costs (IPO costs). For HTS, above site-level costs were not estimated because we did not have data access, therefore, only site level costs were estimated.ntation) . The devThe HIVST kit purchase cost was provided by the IPO at $2.63 for Côte d'Ivoire and $3.08 for Senegal and Mali. Start-up, training, and all other capital costs were annualized using a 3% discount rate. All costs were estimated in 2021 US dollars (US$) using annual exchange rates . Inputs Financial costs were allocated to sites based on allocation factors presented in sensitivity analyses to examine the robustness of the results to different assumptions used in the cost analysis as follows  WHO Ethic Research Committee , and by three national ethic committees: Comité National d'Ethique des Sciences de la vie et de la Santé de Côte d'Ivoire , ComitéNational d'Ethique pour la Recherche en santé du Sénégal , and Comité d'Ethique de la Faculté de Médecine de Pharmacie et d'Odonto-Stomatologie de l'Université des Sciences et des Techniques de Bamako au Mali . The patients/participants provided their written informed consent to participate in this study.Md'E and FT-P designed the study. Md'E coordinated the study. MT and KB conducted data collection and analysis and wrote the article. AV, AS, OK, NR, MM-G, M-CB, and JL provided logistical support and intellectual inputs. All authors revised the manuscript and agreed for publication.The study was funded by UNITAID through Solthis. This work was supported by UNITAID (Grant Number 2018-23-ATLAS) with additional funding from Agence Française pour le Développement (AFD). The funders played no role in study design, data collection and analysis, the decision to publish or preparation of the manuscript.The content is solely the responsibility of the authors and does not necessarily represent the official views of UNITAID, Solthis, or UNAIDS.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Aedes aegypti Aag2 cells were naturally infected with CFAV. However, the ability of this virus to stably colonize an Ae. aegypti population via artificial infection and how it influences the vector competence of this mosquito have yet to be demonstrated.Cell fusing agent virus (CFAV) was the first insect-specific virus to be characterized, and has been reported to negatively influence the growth of arboviruses such as dengue, Zika, and La Cross, making it a promising biocontrol agent for mosquito-borne disease prevention. Ae. aegypti by intrathoracic injection, the survival of parental mosquitoes was monitored and CFAV copies in the whole bodies, ovaries, and carcasses of the injected F0 generation and in the whole bodies of the F1 generation on different days were examined by reverse transcription-quantitative polymerase chain reaction.CFAV used in this study was harvested from Aag2 cells and its complete genome sequence was obtained by polymerase chain reaction and rapid amplification of complementary DNA ends, followed by Sanger sequencing. Phylogenetic analysis of newly identified CFAV sequences and other sequences retrieved from GenBank was performed. CFAV stock was inoculated into Ae. aegypti. Aag2-derived CFAV efficiently replicated in the mosquito body and did not attenuate the survival of Ae. aegypti. However, the viral load in the ovarian tissues was much lower than that in other tissues and the virus could not passage to the offspring by vertical transmission.The virus harvested from Aag2 cells comprised a mixture of three CFAV strains. All genome sequences of CFAV derived from Aag2 cells clustered into one clade but were far from those isolated or identified from Ae. aegypti and provide valuable information on the colonization of mosquitoes by this virus.The results of this study demonstrate that Aag2-derived CFAV was not vertically transmitted in The online version contains supplementary material, which is available at 10.1186/s13071-023-06033-3. Mosquitoes can transmit various pathogenic viruses, such as dengue virus (DENV), Zika virus (ZIKV) and chikungunya virus, which can cause serious disease in humans . VirusesAedes aegypti cell line in 1975, caused cell fusion when inoculated into Aedes albopictus cells, and was subsequently named cell fusing agent virus , 13–16 dpi (GC2) and 20–23 dpi (GC3)  test. Normality and heteroscedasticity of residuals were evaluated by Shapiro–Wilk (W) test and Spearman’s test, respectively. The viral loads in the ovary and carcass were compared by two-tailed Mann–Whitney test. P < 0.05 was considered to indicate statistical significance.The data were visualized and analyzed using GraphPad Prism version 9.4 . The survival curves of The CFAV used in this study was directly harvested from the Aag2 cells. The complete genome was amplified by using 16 pairs of primers and RACE, and the products were sequenced by Sanger sequencing. There were mutations at sites 3429 and 3444 of the open reading frame (ORF). These may have been T or C at ORF3429 and A or C at ORF3444, leading to four possible combinations  increasing from -5.19 to -1.12 and finally reaching -0.86 on 10 dpi  were examined for CFAV infection on different days post-emergence. Twenty females and 20 males from both GC1 and GC2 were tested by RT-qPCR on 3 dpe, and 20 females from GC1 and GC2 were tested by RT-qPCR on 10 dpe, but none of them were virus positive. To examine whether blood-feeding would promote virus replication, the mosquitoes were fed with blood on 10 dpe, and 20 females from GC1 and GC2 were examined, respectively, 3 days later. However, the IR remained at zero (Table The progeny (F1) in two of the GCs of CFAV-infected parental Ae. aegypti may limit CFAV replication in the ovary through the PIWI-interacting RNA pathway [Ae. aegypti Haikou strain, which does not carry CFAV-EVE in its genome  derived from CFAV (CFAV-EVE) in the genome of  pathway . The MenAe. aegypti Menghai and Haikou strains were inoculated with the virus stock. CFAV copies were detected in the ovary and ovariectomized body (carcass) within 21 days of the injection. The IR of CFAV in the ovary and carcass of the Menghai strain remained at 0% for the first 3 days, similar to the previous result  at 6–21 dpi  at 3–21 dpi  used by Contreras-Gutierrez et al.  was isolis virus , 24. The in 1968 . The tra in 1968 . This hy in 1968 –29. ThusAe. aegypti-derived CFAV is that the latter encodes an additional fairly interesting Flavivirus ORF (FIFO) protein through a -1 programmed ribosomal frameshift (PRF), whereas the FIFO ORF in the former is interrupted by premature stop codons [14-14–2 strain, which had lost NS1’ expression, showed slower replication kinetics in cells and was essentially non-neuroinvasive and non-neurovirulent in weanling ICR mice [Ae. aegypti mosquitoes by CFAV, and the effects of this protein on other arboviruses that co-infect mosquitoes.An important genomic difference between Aag2-derived and p codons . PRF is p codons , while tICR mice . A transICR mice  and atteICR mice –36. A siICR mice , influenICR mice  and coroICR mice , 40. In Ae. aegypti by artificial infection could aid the establishment of Ae. aegypti colonies that carry exogenous CFAV and also provide a deeper understanding of antiviral mechanisms in mosquitoes, both of which could be useful for the development of mosquito-borne disease prevention and control programs.Elucidating what prevents CFAV from colonizing Ae. aegypti mosquitoes to establish a model that stably carries exogenous CFAV. Aag2-derived CFAV replicated efficiently in the carcass of Ae. aegypti, but the viral load was much lower in the ovary and the virus failed to infect the filial generation. We speculate that the Aag2-derived CFAV had lost the ability to invade the ovary due to FIFO protein deficiency and thus could not be vertically transmitted in Ae. aegypti. These results provide new insights for future studies on how CFAV and other ISVs colonize and are vertically transmitted in mosquitoes.We isolated three new strains of CFAV from Aag2 cells but were unsuccessful in our attempt to introduce them into Additional file 1: Table S1. Primers used in this study.Additional file 2: Figure S1. Two mutation sites in the 6th polymerase chain reaction (PCR) segment.Additional file 3: Figure S2. PCR detection of cell fusing agent virus-endogenous viral element in Aedes aegypti Menghai and Haikou strains.


Virus Evolution, Volume 9, Issue 1, 2023, vead007, https://doi.org/10.1093/ve/vead007This is a correction to: Alexis Robert, Joseph Tsui Lok Hei, Conall H Watson, Pierre-Stéphane Gsell, Yper Hall, Andrew Rambaut, Ira M Longini, Jr., Keïta Sakoba, Adam J Kucharski, Alhassane Touré, Sévérine Danmadji Nadlaou, Mamadou Saidou Barry, Thierno Oumar Fofana, Ibrahima Lansana Kaba, Lansana Sylla, Mohamed Lamine Diaby, Ousmane Soumah, Abdourahime Diallo, Amadou Niare, Abdourahamane Diallo, Rosalind M Eggo, Miles W Caroll, Ana Maria Henao-Restrepo, W John Edmunds, Stéphane Hué, Quantifying the value of viral genomics when inferring who infected whom in the 2014–16 Ebola virus outbreak in Guinea, Keywords, which should read: ‘ebola virus (EBV)’ instead of: ‘ebola virus (RSV)’.In the originally published version of this manuscript, the images for Figure 5 and Figure 6 were transposed: these are now correctly positioned above their respective legends. There was also an error in an element within section The publisher apologises for these errors which have been emended within the article.


Proper signaling of calcium and calcineurin is important in yeast, and the calcineurin pathway has emerged as a valuable target for developing novel antifungal drugs. Here, we report a role of YDL206W in calcium and calcineurin signaling in yeast. YDL206W is an uncharacterized gene in yeast, encoding a protein with two sodium/calcium exchange domains. Disrupting the YDL206W gene leads to a diminished level of calcium-induced activation of calcineurin and a reduced accumulation of cytosolic calcium. Consistent with a role of calcineurin in regulating pheromone and cell wall integrity signaling, the ydl206wΔ mutants display an enhanced growth arrest induced by pheromone treatment and poor growth at elevated temperature. Subcellular localization studies indicate that YDL206W is localized in endoplasmic reticulum and Golgi. Together, our results reveal YDL206W as a new regulator for calcineurin signaling in yeast and suggest a role of the endoplasmic reticulum and Golgi in regulating cytosolic calcium in yeast.Calcium is ubiquitously present in all living cells and plays important regulatory roles in a wide variety of biological processes. In yeast, many effects of calcium are mediated  Saccharomyces cerevisiae, is one of the most widely used model organisms in biology. Because of the remarkable efficiency of homologous recombination in yeast and the fact that it can exist as stable haploids, resources have been developed to allow the investigation of every yeast gene in a systematic way. These include the systematic deletion library for nonessential genes  , suggest2+ is a key factor that regulates many biological processes in all eukaryotes especially for the processes that require a rapid onset and/or adaptation to environmental stimulus . Calcineurin is a calcium–calmodulin-activated phosphatase that dephosphorylates and activates Crz1, a transcription factor that promotes the expression of genes containing the calcineurin-dependent response element (CDRE) (B). To examine if YDL206W is involved in calcium signaling, we first used a reporter gene (LacZ) under the control of four tandem repeats of the CDRE (4XCDRE-LacZ) . To ensure the effect we saw in the ydl206wΔ mutant is caused by a loss of YDL206W, we cloned the YDL206W gene into a single-copy yeast expression plasmid and transformed the plasmid into the ydl206wΔ mutant. As shown in Figure 1E, expressing YDL206W from the plasmid was able to restore CaCl2-induced increase of the 4XCDRE activity in the ydl206wΔ mutant to the same level as in wildtype, indicating the effect displayed in the ydl206wΔ mutant is indeed because of the loss of YDL206W.YDL206W is an uncharacterized ORF in the yeast genome. Pfam analysis of its protein sequence indicates the presence of two sodium/calcium exchanger (NCX) domains, suggesting a potential role of this protein in regulating calcium signaling A. Calcint (CDRE)  Fig. 1BBA. CalciRE-LacZ) , 26 and ydl206wΔ mutants. In this assay, cells were transformed with a plasmid that expresses apoaequorin. Transformed cells were grown to midlog phase and then incubated with coelenterazine for a short period to produce functional aequorins, which generate a bioluminescence signal upon binding to cytosolic calcium ions .The effect of disrupting YDL206W gene on calcineurin-dependent gene expression is consistent with a role of YDL206W in the control of cytosolic level of calcium. To test this, we used a well-established apoaequorin-based bioluminescence assay and compared the cytosolic calcium levels in the wildtype and the ium ions . It has ium ions , 28. Thu YDL206W B and S1Bversus the ydl206wΔ mutants in their responses to hyperosmotic stress, mating pheromone, and cell wall stress, which are all processes that have been documented or suggested to involve calcium and calcineurin . Notably, the level of increase was clearly less in the ydl206wΔ mutants. Finally, we compared the responses of wildtype and the ydl206wΔ mutants to cell wall stress. To this end, we examined the effects of raising the temperature from 30 to 37 °C, which is one of the most commonly used and physiologically relevant cell wall stresses. As shown in Figure 3E, raising the temperature led to an increase in the activity of calcineurin, as measured by 4XCDRE-LacZ reporter .Next, we sought to identify any physiological roles YDL206W might have. In particular, we wanted to investigate if YDL206W plays any role in regulating signaling pathways that involve calcium. To this end, we compared the behaviors of wildtype cineurin , 30, 31.observed . Thus, is report , pheromos report D. Notablreporter , and oncperature , the ydlat 37 °C F.Figure via confocal microscopy. Initially, we thought to use the AlphaFold predicted structure model of YDL206W to guide our tagging strategy. The structural model  shows distinct localization inside the cells. To discern the identity of the localization, we conducted colocalization studies using three markers: Erg6-red fluorescent protein (RFP) (lipid particle), Anp1-RFP (Golgi apparatus), and Sec13-RFP (ER to Golgi). As shown in Figure 4A, there was substantial colocalization between YDL206W-GFP and Anp1-RFP as well as between YDL206W-GFP and Sec13-RFP, indicating that a substantial amount of the C-terminally tagged YDL206W is present in both ER and Golgi. We then analyzed the localization of the N-terminally tagged YDL206W (GFP-YDL206W), which is also present inside the cells (B). Similar to C-terminally tagged YDL206W (YDL206W-GFP), GFP-YDL206W showed substantial colocalization with both Anp1-RFP and Sec13-RFP (B). While both GFP-YDL206W and YDL206W-GFP showed colocalizations with both Anp1-RFP and Sec13-RFP, the exact localizations of GFP-YDL206W and YDL206W-GFP did not appear to be identical.We then decided to add GFP tag to either the C terminus or the N terminus of the protein. As shown in Figure 4he cells B. Similaec13-RFP B. While C, we were not able to detect colocalization between GFP-YDL206W and MitoTracker Red. Taken together, we suggest that YDL206W is localized to the ER and Golgi.Discrepancies in localizations between the N-terminally GFP-tagged and the C-terminally GFP-tagged proteins have been previously reported with Vcx1 and Vnx1 as good examples , 35. In Figure 4CHS5   , in eukaX family . To bettX family . A featuX family . As expetococcus . Thus, i2+, which contributes to an optimal rise in cytosolic calcium and calcineurin activity. How would these stimuli increase the calcium release from ER and/or Golgi to cytosol? For exogenous calcium, it is possible that the influx of calcium from extracellular space into cytosol may enhance the membrane potential of these organelles, which facilitates the release of calcium into cytosol. This is somewhat analogous to cardiac cells, in which calcium stored in ER is released by trigger calcium, that is, the calcium entered the cells from extracellular space. In mammals, mitochondrial NCXs often form a complex with other proteins such as A-kinase anchoring protein 121 , has been identified, whereas in invertebrates, 11 have been identified . An inittein 121 , 48 and tein 121 , and theIn summary, our study indicates a clear role of YDL206W, a previously uncharacterized protein with two NCX domains, in maintaining the homeostasis of calcium and regulating signaling by calcineurin. Our work also suggests the possibility of ER and/or Golgi in regulating calcium signaling in yeast. Given the importance of calcium exchangers in health and diseases, future studies on how YDL206W is retained in ER and Golgi and how its activity is regulated would provide insights into the regulation of calcium exchangers in general.S. cerevisiae strains used in this study are BY4741  and BY4741-derived mutants lacking YDL206W (Research Genetics), BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0)-derived strains expressing RFP-tagged Anp1, Sec13, or Erg6.Standard methods for the growth, maintenance, and transformation of yeast and bacteria and for the manipulation of DNA were used throughout. The yeast pAMS366-4xCDRE-lacZ    and pVTUpVTU-AEQ . The plapYES-YDL206W-GFP that expresses C-terminal GFP-tagged YDL206W under the control of a GAL1 promoter was constructed by two steps. In the first step, a fragment containing sites for restriction enzymes BamHI, SacI, XhoI, and SalI and followed by the GFP coding sequence was amplified using the primers 5′-GGA TCC GAG CTC GAG GTC GAC ATG AGT AAA GGA GAA GAA C-3′ and 5′-CTA CTA TTT GTA TAG TTC ATC CAT GCC ATG-3′. The amplified fragment was TOPO-cloned into pYES2.1, resulting in pYES2.1-MCS-GFP. The fragment containing YDL206W coding sequences flanked with BamHI and XhoI sites was amplified using the primers 5′-CGC GGA TCC AGA ATG CAT AAA CCT TTA AGA TGG C-3′ and 5′-CCG CTC GAG CAC TTG GTG AAC TTC TAA-3′ and cloned into the pYES2.1-MCS-GFP, resulting in pYES-YDL206W-GFP. The plasmid expressing N-terminally GFP-tagged YDL206W was similarly constructed. The GFP coding sequence followed by multiple cloning sites was amplified using the primers 5′-CCA GAA TGA GTA AAG GAG AAG AAC-3′ and 5′-GGA TCC GAG CTC GAG GTC GAC GTC GTG GTC CTT GTA GTC AC-3′ and TOPO-cloned into pYES2.1, resulting in pYES2.1-GFP-MCS. The fragment containing the YDL206W coding sequence flanked with XhoI and BamHI sites was amplified using the primers 5′-CCG CTC GAG ATG CAT AAA CCT TTA AGA TG-3′ and 5′-CGC GGA TCC TTA CAC TTG GTG AAC TTC TA-3′.The plasmid ++ was measured as described previously  of coelenterazine  in the dark for 20 min. After three washes to get rid of excess coelenterazine, 100 μl of cell culture was used for the measurement of bioluminescence using the Synergy H1 plate reader. Calcium stimulation was achieved by adding 100 mM  of CaCl2. Bioluminescence was measured every 14 s, the highest frequency possible by the Synergy H1 plate reader. To measure pheromone-induced calcium rise, we followed the procedure developed previously . The dot bar graphs were generated using Interactive Dotplot (https://pubmed.ncbi.nlm.nih.gov/28974579/)  , and theCells expressing GFP-tagged YDL206W were grown to midlog phase. Cells were concentrated, and 10 μl of concentrated cell suspension was placed on a slide with a thin layer of 0.5% agar and visualized by fluorescence microscopy using an Olympus FV1000 laser-scanning confocal microscope. For Calcofluor white staining, 10 ml of midlog phase cells grown in SCD media was concentrated to 200 μl. After addition of 2 μl of Calcofluor white solution  and 2 μl of 1 M KOH, the cells were imaged using a confocal microscope. The same exposure settings were used for capturing the images to ensure consistency.Saccharomyces Genome Database, and the Arabidopsis Information Resource according to their accession ID. Multiple sequence alignments were performed by MAFFT (https://pubmed.ncbi.nlm.nih.gov/28968734/), version 7 online service  with the maximum likelihood method, and the bootstrap value was indicated at the node of the branches (Protein sequences were acquired from the National Center for Biotechnology Information,  service , 53. Thebranches .All data are available in the main text or the This article contains The authors declare that they have no conflicts of interest with the contents of this article.