In-vitro cultivation is a widely used technique for plant cultivation, not only in the field of basic research but also for the rapid propagation of valuable horticultural and pharmaceutical plants. However, the systemic effects of in-vitro cultivation on overall plant metabolism could easily be overlooked and are still poorly understood. We found that in-vitro tobacco showed slower growth, less biomass and suppressed photosynthesis than soil-grown tobacco. Many changes of metabolites and metabolic pathways between in-vitro and soil-grown tobacco plants were identified, which notably revealed a significant increase of the amino acids content under in-vitro condition. The in silico investigation showed that in-vitro tobacco downregulated photosynthesis and primary carbon metabolism, while significantly upregulated the GS/GOGAT cycle, as well as producing more energy and less NADH/NADPH to acclimate in-vitro growth demands. Altogether, the combination of experimental and in silico analyses offers an unprecedented view of tobacco metabolism, with valuable insights into the impact of in-vitro cultivation, enabling more efficient utilization of in-vitro techniques for plant propagation and metabolic engineering.The systematical characterization and understanding of the metabolic behaviors are the basis of the efficient plant metabolic engineering and synthetic biology. Genome-scale metabolic networks (GSMNs) are indispensable tools for the comprehensive characterization of overall metabolic profile. Here we first constructed a GSMN of tobacco, which is one of the most widely used plant chassis, and then combined the tobacco GSMN and multiomics analysis to systematically elucidate the impact of  Arabidopsis  are indispensable tools for the systematic characterization of metabolic behaviors. Metabolome and transcriptome analyses could globally elucidate overall metabolic changes and the underlying regulatory mechanisms. Therefore, combination of genome-scale metabolic networks and omics analysis is an excellent method for systematically elucidating the impact of in vitro cultivation on plant metabolism. In plants, omics data were successfully integrated with the Arabidopsis GSMN and rice GSMN to illustrate their metabolite acclimation profiles under different conditions , Kyoto Encyclopedia of Genes and Genomes (KEGG), and other literature sources. Our tobacco GEM, named iJTC6240, accounts for 6,240 genes, 2,887 reactions, and 2,905 metabolites localized across five intracellular compartments, including cytosol, mitochondrion, plastid, vacuole, and peroxisome  for tobacco. To our best knowledge, this is the first reported tobacco model. The metabolic network was based on information from Ntabacum_Tn90Cyc P2), which is essential for maintaining vacuolar morphology and function  that are specialized metabolites produced by Solanaceae species.The model properties of iJTC6240 were compared with those of two plant GEMs, iHY3410  and Arabpsis GEM . Figurefunction . Consistfunction . A greatAfter the GEM reconstruction, we performed several tests to validate its predictive capabilities. Firstly, the model was evaluated by simulating known metabolic functions, including absorption of PSI and PSII photons during photosynthesis, as well as the biosynthesis and degradation of secondary metabolites, such as alkaloids and terpenoids. Secondly, the ability of iJTC6240 to predict tobacco growth rates was tested. We utilized experimental photosynthetic capacity  to constin-vitro cultivations on tobacco, we selected the most common conditions to cultivate tobacco plants in vitro and in soil (as described in the methods section). The seeds germinated approximately 1 week after sowing (WAS), and the germinated seedlings were transplanted into soil or in vitro. The in-vitro plants grew much slower than that in soil, but the true leaf number of both tobacco plants was almost the same during all stages of development . To further assess the differences in the photosynthetic efficiency, the effective quantum yield of photochemical energy conversion in PS II (Y(II)) and relative electron transport rate (ETR) were calculated for different light intensities (PAR). in-vitro tobacco plants than that in in-soi ones, and gradually decreased to nearly 0 µmol at 610 µmol photons m-2s-1 following light intensity increased. The ETR curve under in-vitro condition was also lower than that under soil-grown condition (in-vitro at 230 µmol photons m-2s-1 (PAR), once reached this level, ETR decreased. Collectively, these results indicated the suppressed photosynthetic capacity under in-vitro condition.We measured the leaf chlorophyll fluorescence parameters to explore the photosynthetic system of in-vitro cultivation inhibited the growth of tobacco compared to soil-grown cultivation. To study the impact of in-vitro cultivation on tobacco metabolism and globally detect the metabolic variations between in-vitro and soil-grown tobacco plants, we performed metabolome and transcriptome analyses for 10-WAS in-vitro and soil-grown tobacco plants.The above results showed that in-vitro tobacco and soil-grown tobacco plants, each with three biological replicates. After filtering out low-quality reads from the raw data, a total of 37.77 GB clean data was generated, and 5.64−6.74 GB for each sample was obtained. The GC content was ranging from 43.20% to 43.50%, and the percentages of Q30 bases were over 92.75%. The mapped efficiency of different samples with reference genome ranged from 94.64% to 95.12%  were identified from transcriptomic data, and their GO functional annotation is shown in We generated transcriptome sequencing data for o 95.12% Table S1in-vitro and soil-grown tobacco plants, a widely targeted metabolomics analysis was conducted in triplicates. From the overlapping map of total ion current (TIC) of the quality control (QC) sample, it could be observed that there was a high overlap in the curve of the total ion flow, and the retention time and peak intensity remained consistent. This suggested that the signal stability for the same sample was good when the mass spectrometry was detected at different times  and TCA cycle, as well as its closely related pathways, such as the metabolism of starch, sucrose, fructose, mannose, pentose, galactose and pyruvate were all downregulated. At the same time, many downregulated plant secondary metabolic pathways were enriched under in-vitro condition, including the phosphatidylinositol signaling system, flavonoid biosynthesis, and phenylpropanoid biosynthesis. Collectively, enrichment analysis of DEGs suggested that in in-vitro tobacco plants amino acid metabolism was up regulated, while lipid metabolism was attenuated, and the growth inhibition was likely due to the lower activity of primary carbon metabolism and related pathways. In addition, the secondary metabolism of in-vitro tobacco plants was also weakened compared to soil-grown ones.To further identify cultivation-condition-specific metabolic pathways, KEGG pathway enrichment analysis of DEGs was performed Figure 3DAMs were utilized for the enrichment analysis based on the metabolic classification. The upregulated amino acids and derivatives were enriched among the DAMs with the greatest enrichment degree , Asn (1.92-fold), Ser (1.21-fold), and Val (1.19-fold), but not Pro (0.82-fold). Among these, Ser, Val and Pro were also detected in the metabolomic data, and their metabolic peak areas were consistent with the measured absolute concentrations. Notably, the concentration of Gln, which was not detected in the metabolomic data, was much higher than that of the other amino acids and was primarily responsible for the significant upregulation of amino acids in in-vitro tobacco plants.To further quantitatively compare the common amino acids in plants from different culture conditions, automatic amino acid analyzer was utilized to determine the absolute concentrations of free common amino acids. The overall level of free amino acids under o plants Figure 4in-vitro condition, the pattern was consistent with the results of 10-WAS plants. These results indicated the metabolic variations we detected were mainly due to the different cultivation systems.In order to further exclude the influence of developmental status (such as flowering transition) on plant metabolism, 7-WAS tobacco metabolic variations between two cultivations were also tested except for 10-WAS tobacco. We measured the concentrations of amino acid, one of the most characteristic metabolic variations, for 7-WAS tobacco  and the glutamine synthetase (GS)/glutamine-2-oxoglutarate aminotransferase  cycle were upregulated under in-vitro condition, and the metabolomic data also showed that the concentration of Gln was higher than that under soil-grown. After normalized by the biomass rate, more ATP was produced by proton pumps (1.5-fold), while less NADH/NADPH  was produced under in-vitro condition.To further explore how different culture conditions influence growth, we examined the internal flux distributions under soil-grown and id cycle Figure 5+-oxidoreductase, is the most extensively used pathway (Photosynthetic organisms use various electron flow pathways to generate redox power from light energy in the form of NADPH. The linear electron flow (LEF) pathway, where the electrons flow from PSII to PSI through Cytochrome b6f complex and ferredoxin NADP pathway . Additio pathway .in-vitro conditions, suggesting that the CEF pathway is possibly always operational. Interestingly, the predicted rate of photosynthesis under in-vitro condition was much more dependent on the CEF pathway than under soil-grown condition. In fact, the corresponding energy generated by the CEF pathway under in-vitro condition was 42.9-fold higher than in soil-grown plants. The CEF pathway under in-vitro condition, rather than LEF, was used increasingly to generate more energy, that may response to the lower reducing power demand in the Calvin cycle because of low CO2 uptake, as well as the need to scavenge excess reducing power, which is cytotoxic. Additionally, considerable ATP was needed in a multitude of metabolic pathways, including Gln biosynthesis by glutamine synthetase and sucrose import via transmembrane transporters. Generally speaking, the CEF pathway carried the higher fluxes, highlighting its flexibility and also suggesting that the CEF pathway may contribute to the adaptation of energy demand under in-vitro condition.The simulations revealed that both the LEF and CEF pathways carried noticeable fluxes under soil-grown and in-vitro plants compared to soil-grown plants, which was attibuted to the lower CO2 uptake. In both simulations, the Calvin cycle was driven by the supply of CO2 from cytosol to plastid and was subsequently withdrawn in the form of 3-phosphoglycerate (3PG), that can be transported to cytosol. The 3PG in the cytosol was utilized for energy production through cytosolic glycolysis and the TCA cycle, in agreement with previous reports  instead of energy generation, with a decreased flux (by 45%). 13DPG was converted into glyceraldehyde 3-phosphate (G3P) that participated in PPP and gluconeogenesis. Under in-vitro condition, the plastidic dihydroxyacetone phosphate (DHAP) produced from G3P by fructose-bisphosphate aldolase was transported to cytosol to synthesize Fru-1,6-bisP, while under soil-grown condition it was utilized to synthesize plastidic G6P, which was transported to cytosol to enter Embden-Meyerhof-Parnas (EMP) pathway.The Calvin cycle exhibited appreciable lower fluxes (by 43%) in  reports . In thisPhaseolus vulgaris), which indicated that the TCA cycle was almost completely inhibited under light  acetyl-CoA from pyruvate and (2) succinate from cytosol. The TCA cycle of in-vitro tobacco plants was impaired (flux reduction by 77%), with an increase of malate production from fumarate (2.9-fold) and from oxaloacetate (by 68%). The malate highly produced by TCA cycle in mitochondria was then transported to plastids, where it was converted into oxaloacetate by malate dehydrogenase (NAD) to provide reducing power for the GS/GOGAT cycle, while the oxaloacetate was utilized to form Asp.In the simulation of soil-grown tobacco, no flux was observed from citrate to succinate, while under the in-vitro condition (by 92%) with a 90% flux of oxidative NADH as the first step of the respiratory chain. Additionally, succinate dehydrogenase  has a dual function as a component of the TCA cycle and the electron transport chain (ETC). The flux through SDH was comparable with its TCA cycle flux (76.7%).The fluxes within mitochondrial respiration were relative mild reduced under in-vitro condition, noticeable levels of ATP were consumed for this active transmembrane transport process. The sucrose invertase (INV) was predicted to degrade Suc into fructose and glucose, while that is not an energetically efficient way to break down Suc when compared to the cleavage of Suc by sucrose synthase (SUS) (Since sucrose (Suc) was imported from the surrounding medium under se (SUS) . Subsequ4+ and/or NO3- as nitrogen sources. After uptake, nitrate is firstly reduced into nitrite by nitrate reductase (NR) in cytoplasm, after which nitrite is transported into plastid and reduced to ammonium by plastidic nitrite reductase (NiR). In this regard, the flux analysis results indicated that this route exhibited reduced fluxes (by 75%) under in-vitro condition, and the resulting lower consumption of reducing power may be related to its lower supply from photosynthetic light reactions, and a greater proportion were shifted toward energy generation.In our model, we considered that tobacco can take up NHin-vitro condition. The ammonium assimilation in plastid is accomplished by the GS/GOGAT cycle, where GS catalyzes glutamine (Gln) synthesis by incorporating a molecule of ammonium into glutamate (Glu) in an ATP-dependent manner, while GOGAT produces two molecules of Glu by transferring the amide from Gln to 2-oxoglutarate (2-OG). Plants possess two isoforms of GOGAT that differ in their electron donor for catalysis, NADH-GOGAT (EC 1.4.1.1) and ferredoxin (Fd)-GOGAT (EC 1.4.7.1), both of which are located in the plastids (in-vitro than in soil-grown plants (4.91-fold). Collectively, it can therefore be seen that the higher activity of the GS/GOGAT cycle under in-vitro condition results in increased Gln synthesis at the expense of Glu, since the flux through GS was higher than through GOGAT. These findings may offer a reasonable explanation for the higher Gln but lower Glu content under in-vitro condition compared to soil-grown plants.Interestingly, ammonium assimilation, by which inorganic N is converted into organic N, was higher under plastids . Our modin-vitro cultivation. MS medium was originally designed for tobacco callus and is widely used for many plant species nowadays. MS medium contains macronutrients that include high levels of nitrate and organic additives, such as sugar, and vitamins. Sugar provides metabolic energy and carbon skeletons for the biosynthesis of the organic compounds required for cell growth and also act as an osmotic component in the culture medium. in-vitro plants is positively correlated with the sucrose content in the culture medium. However, high sucrose concentration added to the culture medium can cause starch and sucrose accumulation in the leaves, which can reduce the photosynthetic activity ] in the culture medium. The increasing nitrogen supplement could lead to an increase in the total nitrogen content in tissues. in-vitro tobacco. Furthermore, in silico simulations showed in-vitro tobacco had a higher activity of the GS/GOGAT cycle fixing the substantial amounts of ammonium at the expense of Glu to produce Gln, which may be attributed to an abundance supplement of ammonium salts in the culture medium.The composition of the culture medium is crucial for activity . In our in silico investigation, that resulted in lower growth rate and less biomass under in-vitro tobacco. The Calvin cycle was subjected to energy and reducing power. Although ATP production was abundant, the generation of reducing power (NADH/NADPH) is markedly limited indicated by our systematical combined omics-based analysis. Here we gave three suggestions on how to improve the situation under in-vitro condition. Firstly, our model prediction showed a surprisingly high level of cyclic photophosphorylation, resulting in the reduction power being utilized for the generation of abundance energy. We proposed suppressing cyclic photophosphorylation and boosting non-cyclic photophosphorylation activity for this purpose. Additionally, our model survey also predicted increased flux from fumarate to malate but decreased subsequently in the TCA cycle. To improve the flux towards oxaloacetate and consequently the overall TCA cycle, we suggested that supplement the culture medium with appropriate pyruvate or organic acid  salts, which could potentially elevate the concentration of TCA metabolites intermediates and thereby drive the TCA cycle to generate reducing power. Finally, addition of glycerin into medium was supposed to help to alleviate the deficiency in reductive power.Compared to soil-grown tobacco, although there was additional carbon (sucrose) uptake, we noticed diminished carbon fixation in the Calvin cycle in our in-vitro tobacco than soil-grown tobacco, we proposed: Firstly, since we provided sufficient detail in iJTC6240 to define all individual electron transport reactions including photosystem II (PSII), photosystem I (PSI), and the cyclic/non-cyclic electron transfer, both photons utilized in PSII and PSI exhibited lower level of flux  under in-vitro compared to soil-grown tobacco in our observations. We speculated that maybe an appropriately elevation of effective photons utilization that not inducing photoinhibition would confer improvements. Secondly, light quality serves as a pivotal factor affects various morphological processes in plants, it was reported that in blue light, photosynthesis was up-regulated (in-vitro.To improve the suppressed photosynthesis in egulated . In thatNicotiana tabacum cv. Petit Havana) seeds were sterilized with 75% ethanol for 1 min, and then sterilized with 10% sodium hypochlorite for 20 min, rinsed with sterile water three times, and finally germinated on Murashige and Skoog (MS) agar plates containing 3% (w/v) sucrose in a climate chamber at temperature of 25°C with a light/dark cycle of 16 h/8 h and photon fluence of 186 μmol m −2 s −1. The germinated seedling was transplanted into pots containing soil/vermiculite mixture (1:1) or into 500 mL glass culture vessels (covered with gas-permeable caps) containing solidified MS medium (100 mL per vessel) with 3% sucrose. The fresh weight of the tobacco aboveground part was measured using an electronic balance. The dry weight was recorded after drying the samples in an oven at 65°C to constant weight.Tobacco  and the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system , respectively. RNA concentration was measured using the Qubit® RNA Assay Kit in the Qubit®2.0 Flurometer .Genomic DNA was isolated using a Super Plant Genomic DNA Kit . Total RNA was extracted from 10-WAS plants using an RNAprep Pure Plant Plus Kit . DNA and RNA degradation and contamination were monitored on a 1% (w/v) agarose gel. RNA purity and integrity were assessed using the NanoPhotometerin-vitro and soil-grown tobacco plants. Qualified RNA from each samples were shipped on dry ice to the Biomarker Biotechnology Corporation  for cDNA library construction and RNA sequencing. The library was prepared using the VAHTS® Universal V8 RNA-seq Library Prep Kit for Illumina . Subsequently, the cDNA libraries were paired-end sequenced on an Illumina NovaSeq 6000 platform . Clean reads were obtained by removing reads containing adapters or ploy-N and low-quality reads from the original data. At the same time, the percentage of Q30 bases and GC-content of the clean data were calculated. All downstream analyses were based on clean data of high quality. After performing quality control, we obtained clean reads which were then mapped to the tobacco reference genome Ntab_TN90  and the public databases (Metlin and Massbank). The characteristic ions of each metabolite were obtained by triple four-stage rod screening, the signal intensity of the characteristic ions was obtained in the detector, and MultiQuant software was used to integrate and correct the chromatographic peaks. All integration data for the chromatographic peak areas were exported and saved. To detect the repeatability of the samples, QC samples (mixed sample extracts) were inserted in every 10 test samples during the analysis. The accuracy and reproducibility of metabolite detection could be determined by using the overlapping display analysis of mass spectrometry TIC of different QC samples. Combined FC of metabolite difference and the variable importance in projection (VIP) value of the orthogonal partial least squares discriminant analysis (OPLS-DA) were used to screen DAMs. The FC ≥ 2 or FC ≤ 0.5, and VIP ≥ 1 were considered as the standard of screening. KEGG pathway database was used to annotate the DAMs.Metabolite profiling was carried out using a widely targeted metabolomic method by Metware Biotechnology  for the same samples as transcriptome. Briefly, the freeze-dried plant samples were crushed using a mixer mill  and extracted with 70% methanol extraction solution. After centrifugation, the supernatant was filtrated and then analyzed using a UPLC-ESI-MS/MS system in conjunction with UPLC (Shim-pack UFLC Shimadzu CBM30A system) and MS (Applied Biosystems 4500 Q TRAP). The analytical parameters were set as the same as An automatic amino acid analyzer  was used for the determination of free amino acids. The samples were diluted with 10 mL of boiling distilled water and incubated in a water bath at 95°C for 10 min. The extraction was repeated three times and the supernatant was obtained after centrifugation. After adding 10% sulfosalicylic acid, the mixture was centrifuged at 13400 g for 10 min, then dissolved in 0.02 M HCl, and passed through a 0.22-µm pore-size filter membrane into a brown sample bottle for further testing. An autoanalyzer based on the ninhydrin method was used to quantify the free amino acids according to the method described by v/FmF) was calculated as (Fm – Fo)/Fm, where Fm is the maximum chlorophyll fluorescence, measured immediately after a saturating light pulse, while Fo, the steady-state chlorophyll fluorescence, was measured after dark acclimation before a saturating light pulse. The effective PS II quantum yield of the illuminated samples was calculated using the formula Y(II) = (Fm’-F)/Fm’. The trend of Y(II) and the chloroplast electron transport rate (ETR) parameter was determined on the basis of a rapid light curve (RLC) at PAR of 0, 10, 35, 80, 145, 230, 335, 460 and 610 µmol photons m−2 s−1.Chlorophyll fluorescence parameters were measured at room temperature using a pulse-amplitude-modulated (PAM) fluorometer . The expanded leaves were dark-adapted for 20 min before measurement. Six different areas of interest (AOI) were randomly selected on each leaf. The maximal quantum yield of photosystem II (Nicotiana tabacum) metabolism was obtained from Ntabacum_Tn90Cyc and KEGG  as inputs along with the exchange of CO2, oxygen, and water with the environment.An initial reaction set for tobacco  associations was utilized to localize the individual reactions in the draft model to appropriate subcellular compartments by querying tobacco gene sequences against UniProt database. If it was not available in UniProt, the compartmental information from the GEM  of a phycuration .Arabidopsis  genome-scale metabolic model   using thij is the stoichiometric coefficient of metabolite i in reaction j, n is the set of all reactions in the model, V is a vector of reaction flux, m is the transporter reaction of each biomass component, and t is the corresponding transporter flux of the biomass component.where Sin-vitro/soil-grown plants. E‐Flux utilizes gene expression data to set the upper and lower bounds of reactions using gene–protein–reaction (GPR) associations from the model. In detail, the transcriptome data were transformed by log2(FPKM+1) using the mean values of three duplicates. The mathematical operations utilized to calculate the numerical values were the sum of “OR” expressions. Finally, the GPR rule values were normalized, divided by the maximum value in each condition. The normalized values were used to establish the new lower and upper reaction bounds. Exchange reactions corresponding to each growth environment were set as −1/1. After that, the condition‐specific models were obtained with specific reaction bounds.The E‐Flux algorithm  was usedin-vitro condition was modeled by allowing photon influx with active photosynthesis and sucrose as the carbon and energy input (Sucrose_Cyto_tx), with all other constraints the same as the soil-grown one. Constraint-based FBA was run with the objective function of biomass maximization.The free amino acid data was incorporated into the model as the biomass component. Under soil-grown condition, photon influxes were allowed from the photon input reaction with active photosynthesis and with all organic sources of energy set to zero. The t-test in Microsoft Excel. A value of p< 0.05 was statistically significant.For quantitative analysis, values were expressed as the means ± standard errors of three independent biological replicates. Statistical significance analysis was performed using a Student’s https://www.ncbi.nlm.nih.gov/bioproject, PRJNA1014250; https://www.ebi.ac.uk/metabolights, MTBLS8557.The original contributions presented in the study are publicly available. This data can be found here: JY: Formal Analysis, Writing – original draft, Writing – review & editing. XW: Formal Analysis, Writing – original draft, Writing – review & editing. QY: Formal Analysis, Writing – original draft. JS: Formal Analysis. JC: Formal Analysis. ZL: Conceptualization, Writing – original draft, Writing – review & editing, Supervision, Funding acquisition. HM: Conceptualization, Writing – review & editing, Supervision, Funding acquisition.


The coronavirus disease 2019 (COVID-19) pneumonia may cause cystic features of lung parenchyma which can resolve or progress to larger blebs. Pneumothorax was more likely in patients with neutrophilia, severe lung injury and a prolonged clinical course. The timely diagnosis and management will reduce COVID-19-associated morbidity and mortality.We present 11 cases of spontaneous pneumothorax managed with chest tube thoracostomy or high-dose oxygen therapy. Isolated spontaneous pneumothorax was detected in all cases.Eight cases were male and 3 cases were female. There were bilateral ground-glass opacities or pulmonary infiltrates in the parenchyma of the 10 cases. We detected neutrophilia, lymphopaenia and increased C-reactive protein, Ferritin, lactate dehydrogenase, D-Dimer, interleukin-6 levels in almost all cases. Chest tube thoracostomy was sufficient to treat pneumothorax in our 9 of case. In 2 cases, pneumothorax healed with high-dose oxygen therapy. Favipiravir and antibiotic treatment were given to different 10 patients. In our institution, all patients with COVID-19 infection were placed on prophylactic or therapeutic anticoagulation, unless contraindicated. The treatments of patients diagnosed with secondary spontaneous pneumothorax during the pandemic period and those diagnosed with secondary spontaneous pneumothorax in the previous 3 years were compared with the durations of tube thoracostomy performed in both groups.The increased number of cases of pneumothorax suggests that pneumothorax may be a complication of COVID-19 infection. During medical treatment of COVID-19, pneumothorax may be the only reason for hospitalization. Although tube thoracostomy is a sufficient treatment option in most cases, clinicians should be aware of the difficulties that may arise in diagnosis and treatment. Coronavirus disease 2019 (COVID-19) pneumonia may cause cystic features in the lung parenchyma, which can resolve or progress to larger blebs . Coronavirus disease 2019 (COVID-19) pneumonia may cause cystic features in the lung parenchyma, which can resolve or progress to larger blebs , 3% in pHere, we report the management of 11 cases of SSP detected among COVID-19 patients in our hospital between March 2020 and November 2020. Patients in whom the pneumothorax developed as a consequence of trauma, iatrogenic trauma, barotrauma or other secondary causes were excluded. COVID-19 diagnoses were confirmed using the reverse transcription-polymerase chain reaction technique based on severe acute respiratory syndrome coronavirus 2 antigen detection in nasopharyngeal swabs. The treatments of patients diagnosed with SSP during the pandemic period and those diagnosed with SSP in the previous 3 years were compared and the durations of tube thoracostomy performed in both groups, as well.P < 0.05). The demographic, clinical, laboratory and imaging data of those patients who developed an SP are shown in Tables All analyses were performed using the Mann–Whitney non-parametric test if non-normally distributed  was given in addition to medical treatment. The worsening of the lesions continued on the third day and the patient, who developed sudden respiratory distress due to decreased oxygen saturation on the fourth day, was intubated. Since the oxygen-saturation values did not improve after intubation, chest radiography was performed and a pneumothorax was found in the right hemithorax. The general condition of the patient deteriorated due to sepsis and he died on the sixth day from sepsis and respiratory failure.An 86-year-old male patient was admitted to the emergency department with complaints of shortness of breath, fever and weakness. A TCT scan showed bilateral ground-glass opacities and hydropneumothorax in the right hemithorax. A right chest tube was inserted into the intrapleural space. A nasopharyngeal swab sample studied from the patient was positive for COVID-19 RNA. The medical history of the patient included coronary artery disease, chronic atrial fibrillation and hypertension. The patient, who received NIMV in addition to medical treatment, did not respond to the interventions. The patient died of acute respiratory distress syndrome (ARDS) after worsening pulmonary infiltrations on the second day.A 76-year-old female patient had been hospitalized in our institution for 10 days due to COVID-19 pneumonia. Bilateral ground-glass opacities and a right pneumothorax were detected by TCT after sudden shortness of breath. A right chest tube was inserted. The pneumothorax recovered and the chest tube was terminated on the third day. The patient was on NIMV in combination with COVID-19-pneumonia treatment. She was discharged after 25 days of hospitalization due to accompanying liver and urinary system pathologies.A 72-year-old male patient had previously been hospitalized for 1 week in an external hospital for COVID-19-induced pneumonia 45 days ago. He was admitted to our clinic after suffering a chest pain for 5 days. His medical history was uneventful. A TCT scan showed bilateral ground-glass opacities and a left pneumothorax. A left chest tube was inserted. The nasopharyngeal swab sample of the patient was negative for COVID-19 RNA. The chest tube was terminated on the fourth day and the patient was discharged on the fifth day.An 88-year-old male patient was admitted to the emergency department with complaints of loss of appetite, weakness and general condition disorder that had continued for 15 days. His medical history included hypertension and DM. TCT showed diffuse microcystic changes on the left lung accompanying to a minimal pneumothorax, in addition to bilateral ground-glass opacities. As well as standard COVID-19 medical treatment, high-dose oxygen therapy was applied. The pneumothorax healed on the 4th day and the patient was discharged on the 13th day after the medical treatment was completed.A 91-year-old male patient was admitted to the emergency department with complaints of dyspnoea. He was shown to have chronic obstructive pulmonary disease with bilateral diffuse emphysema and infiltrates with a minimal right anterior pneumothorax on TCT. High-dose oxygen therapy was applied. The pneumothorax healed after 2 days. The patient was discharged after recovery on the seventh day.A 69-year-old male patient was admitted to the emergency room with sudden shortness of breath. Bilateral pulmonary infiltrates and a pneumothorax on the right lung were detected by TCT. Right chest tube thoracostomy was applied. The patient was discharged immediately after chest tube thoracostomy was terminated on the fifth day.This study included only the patients with isolated SP detected radiological and, in this study, isolated SP was detected in all the cases and no pneumomediastinum was present Table .P = 0.267). Eleven of the 19 cases with SSP (57.89%) were associated with COVID-19 infection. No statistically significant difference was found between the tube thoracostomy treatment duration in the pre-COVID-19 period and that during the COVID-19 pandemic . Although the result was not statistically significant due to the limited number of patients, the risk of SSP showed a tendency to increase with COVID-19 infection  of the 11 cases. In the same period (between March and December) over the 3 years prior to the COVID-19 pandemic, in total, 40 patients were hospitalized with a diagnosis of SSP . The annual number of SSP cases was found to have increased to 19 during the COVID-19 period, although this difference was not statistically significant (n = 4/5) and NIMV was applied in 20% (n = 1/5). Mortality was not observed in these cases. By contrast, MV/NIMV was applied in 66.6% of the smoker/ex-smoker cases (n = 4/6) and mortality was observed in 50% (n = 3). Although the clinical course of COVID-19 SP in smokers was worse and mortality was observed in 3 cases, there was no statistically significant difference in terms of the smoking and MV/NIMV relationship and the mortality and smoking relationship . The small number of cases limited the statistical evaluation. The average pneumothorax recovery and tube thoracostomy termination time were 4.8 (2–11) days in smoker/ex-smoker cases and 4.75 (3–6) days in non-smoker cases. This difference was not statistically significant . Patients who needed MV and/or NIMV after pneumothorax had a poor clinical course. These patients had a longer hospitalization period and higher morbidity and mortality compared to others Table . Three o 20% n = /5. MortaOne notable finding of this study was the presence of lymphopaenia and elevated inflammatory markers including human C-reactive protein, lactate dehydrogenase, ferritin, D-dimer and interleukin-6 levels in most patients who developed SP Table . NeutropPneumothorax is the accumulation of air in the space between the parietal pleura and the visceral pleura lining the lungs. A primary spontaneous pneumothorax can occur with no precipitating event, while an SSP is a complication of underlying lung disease. The mechanism of the injury, although not completely understood, may therefore be secondary to alveolar damage from the infection and a rupture of the alveolar wall due to increased pressure from the pronounced coughing that occurs in response to the virus . In addiet al. [et al. [et al. [Since the first cases of COVID-19 were described, pneumothorax has been characterized as a potential, though uncommon, complication. Chen et al.  describe [et al. , in an a [et al.  reviewedData from autopsy studies of COVID-19 patients have shown that the predominant pulmonary pathological findings are diffuse alveolar damage, similar to in severe acute respiratory syndrome patients, and the development of a pneumothorax, which supports the pulmonary parenchymal damage hypothesis , 16. OthLate sequelae due to COVID-19 pneumonia have been reported and can cause pneumothorax . In our COVID-19 induced pneumothorax and pneumomediastinum have been commonly reported together in the literature . All of It has previously been suggested that the development of pneumothorax during coronavirus infection is an important prognostic marker , 19. HowLymphopaenia and elevated inflammatory markers, including C-reactive protein, lactate dehydrogenase, ferritin, D-dimer and interleukin-6 levels, were found in most of the patients who developed SP in the present study Table . This waNeutrophilia and lymphopaenia can cause acute lung injury, leading to pneumothorax . NeutropDelay in admission to hospital and severe pneumonia progression was detected in 3 patients who died due to sepsis, ARDS and multi-organ failure. Chest tube thoracostomy was sufficient in the treatment of pneumothorax in 9 cases, whereas high-dose oxygen therapy was sufficient in 2 cases. The lungs were expanded in all our cases and surgery was not required.The risk of pneumothorax was increased in COVID-19 patients treated with NIMV and MV . Our stuet al. [Smoking is a poor prognostic factor in COVID-19 infection and pneumothorax. Mallick et al.  reportedThe rate of hospitalization has been reported to increase with the incidence of SP due to COVID-19 . The maiIn this study, compared to the previous years, the coexistence of COVID-19 infection with pneumothorax increased the number of SP cases and the rate of hospitalization and length of stay. Pneumothorax can therefore be considered as a poor prognostic factor in COVID-19 infection.Inflammation is common in COVID-19 pneumonia. It is possible that the developing inflammation poses a risk for SP and increases the incidence of pneumothorax. In such cases, early anti-inflammatory treatment, such as corticosteroid therapy, might decrease the frequency. In addition, during the follow-up of patients with COVID-19, new symptoms and findings such as novel chest pain, onset of shortness of breath, increase in shortness of breath or deepening of hypoxaemia should be considered as warning signs for pneumothorax.The increased number of cases of pneumothorax suggests that it may be a complication of COVID-19 infection. During medical treatment for COVID-19, pneumothorax may be the only reason for hospitalization. Although tube thoracostomy is a sufficient treatment option in most cases, clinicians should be aware of the difficulties that may arise in diagnosis and treatment.Conflict of interest: none declared.Hakki Ulutas: Data curation; Formal analysis; Project administration; Resources; Writing – original draft; Writing—review & editing). Muhammet Reha Celik: . Ilham Gulcek: Data curation; Formal analysis; Project administration; Resources. Muhammed Kalkan: Investigation; Visualization. Mehmet Ağar: Data curation; Investigation; Visualization; Writing—review & editing. Talat Kılıç: Data curation; Methodology; Supervision; Writing—original draft; Writing—review & editing. Emine Gulcek: Investigation; Resources; Writing—original draft.Interactive CardioVascular and Thoracic Surgery thanks Vanessa Diaz-Ravetllat, Katrin Welcker, Federico Tacconi and the other anonymous reviewers for their contribution to the peer review process of this article.


Experimental data show that drug-resistance-conferring mutations are often associated with a decrease in the replicative fitness of bacteria in vitro, and that this fitness cost can be mitigated by compensatory mutations; however, the role of compensatory evolution in clinical settings is less clear. We assessed whether compensatory evolution was associated with increased transmission of rifampicin-resistant tuberculosis in Khayelitsha, Cape Town, South Africa.M tuberculosis isolates and their associated clinical data from individuals routinely diagnosed with rifampicin-resistant tuberculosis in primary care and hospitals in Khayelitsha, Cape Town, South Africa. Isolates were collected as part of a previous study. All individuals diagnosed with rifampicin-resistant tuberculosis and with linked biobanked specimens were included in this study. We applied whole-genome sequencing, Bayesian reconstruction of transmission trees, and phylogenetic multivariable regression analysis to identify individual and bacterial factors associated with the transmission of rifampicin-resistant M tuberculosis strains.We did a genomic epidemiological study by analysing available M tuberculosis isolates. Compensatory evolution was associated with smear-positive pulmonary disease  and a higher number of drug-resistance-conferring mutations . Compensatory evolution was also associated with increased transmission of rifampicin-resistant disease between individuals , independent of other patient and bacterial factors.Between Jan 1, 2008, and Dec 31, 2017, 2161 individuals were diagnosed with multidrug-resistant or rifampicin-resistant tuberculosis in Khayelitsha, Cape Town, South Africa. Whole-genome sequences were available for 1168 (54%) unique individual M tuberculosis genotypes, both within and between patients, and that the in vitro replicative fitness of rifampicin-resistant M tuberculosis measured in the laboratory correlates with the bacterial fitness measured in clinical settings. These results emphasise the importance of enhancing surveillance and monitoring efforts to prevent the emergence of highly transmissible clones capable of rapidly accumulating new drug resistance mutations. This concern becomes especially crucial at present, because treatment regimens incorporating novel drugs are being implemented.Our findings suggest that compensatory evolution enhances the in vivo fitness of drug-resistant Funding for this study was provided by a Swiss and South Africa joint research award , the European Research Council (grant number 883582), and a Wellcome Trust fellowship . ZS-D was funded through a PhD scholarship from the South African National Research Foundation and RMW was funded through the South African Medical Research Council. Mycobacterium tuberculosis is threatening global tuberculosis control. Drug-resistant tuberculosis can emerge de novo through the selection of drug-resistant bacteria during patient treatment, or can reflect the patient-to-patient transmission of drug-resistant strains. In high-incidence settings, transmission, as opposed to de novo evolution, has been suggested to be the main cause of incident drug-resistant tuberculosis.The emergence and transmission of drug-resistant Evidence before this studyMycobacterium tuberculosis, the fitness cost in vitro of different mutations conferring rifampicin resistance correlates with their relative clinical prevalence, suggesting an effect in the transmission of the disease. By contrast, the role of compensatory evolution in the transmission of rifampicin-resistant tuberculosis is controversial. We searched PubMed for published studies in English from database inception to Oct 24, 2022, with the keywords “tuberculosis”, “transmission”, and “compensatory”. Although five studies have suggested that rifampicin-resistant strains carrying compensatory mutations are more transmissible, others found no such relationship. However, previous studies have been limited to the use of genetic clustering or similar approaches as a surrogate of transmissibility. Moreover, previous studies focused on the role of bacterial genetic factors, independently from host-related factors.Mutations conferring drug resistance in bacteria often cause a fitness defect, measured in terms of a reduced replicative capacity in culture. The acquisition of additional compensatory mutations can restore the replicative capacity of drug-resistant bacteria in vitro. In Added value of this studyM tuberculosis genomes of individuals who were infected, along with the associated clinical data, to identify bacterial and host-related factors that increased the risk of rifampicin-resistant tuberculosis transmission. We identified several host and bacterial factors, including compensatory evolution, that were associated with an increased risk of drug resistance amplification and transmission.We used a Bayesian reconstruction of transmission trees to delineate the transmission of rifampicin-resistant tuberculosis in Khayelitsha, Cape Town, South Africa over a period of 10 years. We analysed the Implications of all the available evidenceM tuberculosis strains carrying drug-resistance-conferring mutations, causing a low fitness cost and compensatory mutations, are more transmissible than those carrying mutations with a high fitness cost and without compensatory mutations in the high-incidence setting of Khayelitsha, South Africa. One question that has yet to be answered is to what extent the relationship between the different genetic determinants of resistance in M tuberculosis and its transmission depends on the epidemiological setting. Future research in different settings using methods capable of delineating transmission events is warranted.Mycobacterium tuberculosis, have shown that mutations conferring antibiotic resistance often confer a fitness cost in the absence of drugs, defined as a deficit in bacterial growth .M tuberculosis, several studies have shown that the mutation rpoB S450L, which causes resistance to rifampicin, is associated with a smaller loss in replicative fitness compared with other rpoB mutations.rpoB S450L among patient populations are few.Experimental studies in various bacterial species, including rpoA, rpoB, and rpoC have been identified in rifampicin-resistant strains of multiple bacterial species, including M tuberculosis.M tuberculosis, some studies have reported an increased transmission fitness of drug-resistant strains carrying compensatory mutations,M tuberculosis and its transmission is often analysed independently from patient-related factors, some of which can be linked to tuberculosis transmission.The role of compensatory mutations in alleviating the fitness cost of resistance has also been experimentally established in mycobacteria and other bacteria. In particular, compensatory mutations in M tuberculosis genotypes as reported from previous in-vitro experiments, with the corresponding transmission fitness in a defined epidemiological setting.Here, we explored the possible links between the fitness of different drug-resistant We performed whole-genome sequencing (WGS) of the available isolates from a retrospective cohort study of individuals routinely diagnosed with multidrug resistant (MDR) or rifampicin-resistant tuberculosis in primary care and hospitals Khayelitsha, Cape Town, South Africa, over a period of 10 years.Khayelitsha is a poor, peri-urban subdistrict in Cape Town, South Africa. Among the population of approximately 400 000 people, antenatal HIV prevalence is 34% and tuberculosis case notification rate is 1600 per 100 000 people per year.The storage of MDR or rifampicin-resistant isolates in the biobank was approved by the Stellenbosch University ethics committee (N09/11/296), and linkage of WGS data to patient clinical data was approved by the University of Cape Town ethics committee (HREC 416/2014). Permission to access Western Cape Provincial Health Data Centre data was granted by the Western Cape Department of Health. There was no written informed consent from patients, because biobanked specimens were linked with routinely collected data with the data subsequently de-identified for analysis.M tuberculosis complex lineage, genotypic resistance profiles, and infer the patient-to-patient transmission of M tuberculosis complex strains, we performed WGS of the available isolates from the study cohort. To detect drug resistance, we compared the mutations detected in each strain with an in-house catalogue of known drug-resistance-conferring mutations.To identify the M tuberculosis complex lineages,r=1 and off-p=0·5). To test the robustness of our results to changes in the model priors, we performed sensitivity analyses by using a range of wide generation and sampling time γ distributions . Contingency tests were performed using Fisher's exact test. The difference in the number of drug-resistant-conferring mutations between groups was tested using two-sided Wilcoxon sum rank tests, given the non-normality of the data as tested with the Shapiro-Wilk test. In multivariable regression models, factors were included on the basis of univariate significance and presumed relevance on the basis of the literature. Individuals with missing data for the response variable were excluded when applicable. All models were corrected by individual age (as a continuous variable), sex, and HIV status. The significance threshold used was α=0·05 in all cases. Factors associated with the number of drug-resistance-conferring mutations were analysed in an adjusted multivariable Poisson regression. The phylogenetic multivariable logistic regression analyses of factors associated with MDR or rifampicin-resistant tuberculosis phenotypes and the transmission of these resistant diseases were performed using the package phylolmThe funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.M tuberculosis isolates  unique individual isolates . Appendiisolates .M tuberculosis isolates with WGS data, the majority belonged to lineage 2 (785 [67·2%]), followed by lineage 4 (349 [29·9%]), lineage 3 (28 [2·4%]), and lineage 1 (six [0·5%]). Given the small numbers of lineages 1 and 3 isolates, we focused our other analyses on lineages 2 and 4. Based on our WGS data, 866 (74·1%) of strains were multidrug resistant, 243 (20·8%) were RMR, and 59 (5·1%) were extensively drug resistant. rpoB S450L was the most common rifampicin-resistance-conferring mutation (730 [62·5%]), followed by D435V (83 [7·1%]), H445Y (64 [5·5%]), L452P (49 [4·2%]), L430P (45 [3·9%]), H445D (44 [3·8%]), and D435Y (37 [3·2%]), as well as other less frequent mutations (rpoB S450L (Fisher's exact test p<0·0001), with most compensated strains carrying this mutation (318 [97·2%]). rpoB mutations, the drug-resistant profile, number of drug-resistant-conferring mutations, and whether the individual was inferred to have transmitted rifampicin-resistant tuberculosis.Of the 1168 utations . 327 strrpoB S450L mutation , and additionally carrying a compensatory mutation  were positively associated with multidrug resistance. Extrapulmonary tuberculosis  and previous tuberculosis treatment episodes  were negatively associated with multidrug resistance. Of note, the high odds ratios and wide CIs observed for compensated rpoB S450L were because of the fact that virtually all compensated strains were multidrug resistant (323 [98·8%]), and only four strains harbouring compensatory mutations were RMR.We first looked for individual and bacterial factors associated with multidrug resistance as opposed to RMR. In a phylogenetic multivariable logistic regression , we founrpoB S450L mutation and additional compensatory mutations contribute to the amplification of drug resistance in M tuberculosis. In support of this notion, we observed that compensated M tuberculosis genotypes carried more drug-resistant-conferring mutations on average , and additionally carrying a compensatory mutation . When repeating the multivariable regression analysis including only the first sampled isolate of each cluster, we found the same results  were positively associated with smear positivity, whereas culture-negative conversion was negatively associated , and being infected with an sociated .Figure 3rpoB S450L mutation, and even higher if the strain carried an additional compensatory mutation , an earlier diagnosis year during the study , and individual age  were positively associated  of MDR or rifampicin-resistant M tuberculosis in this setting.We studied the in vivo fitness of MDR or rifampicin-resistant M tuberculosis to evolve drug-resistant-conferring mutations under antibiotic treatment pressure directly depends on the replicative capacity within a host. This notion implies that, hypothetically, the lower the fitness cost associated with a particular drug-resistant-conferring mutation, the higher the probability of acquiring novel mutations conferring drug resistance to additional drugs. Our results support this idea. We observed that rifampicin-resistant strains carrying the low fitness cost mutation rpoB S450L were associated with a higher number of drug-resistant-conferring mutations, and that the number of drug-resistant-conferring mutations further increased when the strain also carried compensatory mutations. Compensated strains have been previously found to carry more drug-resistant-conferring mutations in other settings.rpoB mutations (S450L or non-S450L) are associated with a higher number of drug-resistant-conferring mutations. Additionally, we found high-fitness drug-resistant genotypes to be associated with sputum smear-positivity, which further supports the notion that genotypes with high-fitness mutations (as measured in vitro), might also replicate better within the host. Another question is whether lineage 2 strains are also more prone to acquire drug-resistant-conferring mutations. One study showed that the risk of drug resistance acquisition is higher for lineage 2 compared with lineage 4 strains.The potential of rpoB S450L and compensatory mutations showed an increased likelihood of MDR or rifampicin-resistant tuberculosis transmission. At the same time, these genotypes showed an increased number of drug-resistant-conferring mutations and were more likely to be multidrug resistant (as opposed to RMR) and to present smear-positive microscopy. In turn, individuals carrying MDR strains and presenting smear-positive microscopy were also more likely to transmit the disease than rifampicin monoresistant strains and smear-negative microscopy. Overall, other factors associated with the transmission of rifampicin-resistant tuberculosis in this study were in line with the literature. Several studies have shown the MDR tuberculosis epidemic to be driven mainly by transmission.We also found that bacterial factors play a direct role in the transmission of MDR or rifampicin-resistant tuberculosis in Khayelitsha. Remarkably, these factors were interconnected, and seem to affect the transmission of rifampicin-resistant tuberculosis in a synergistic manner. Genotypes carrying both the low fitness cost mutation In contrast with classic molecular epidemiological studies based on genetic clustering, we used TransPhylo to answer the question of who transmitted to whom. Although previous evaluations have shown that TransPhylo has a good performance identifying tuberculosis transmitters, this method still presents some limitations.M tuberculosis strains.Whether our results can be generalised to different settings warrants future research. However, taken together, the available evidence suggests that the effect of compensatory evolution in the emergence and spread of drug-resistant tuberculosis is not limited to Khayelitsha. Our results are relevant for the control of drug-resistant tuberculosis worldwide, given the new treatment regimens based on novel drugs that are increasingly being rolled out to treat MDR or rifampicin-resistant tuberculosis. We need to design appropriate surveillance and intervention strategies to deliver the best possible treatment for individuals and prevent the emergence and spread of highly drug-resistant and transmissible All sequencing data are available online in the European Nucleotide Archive repository under accession numbers PRJEB45389 and PRJNA670836. Accession numbers for the samples analysed in this study and associated metadata can be found in RMW acknowledges baseline support from the South African Medical Research Council. All other authors declare no competing interests.


Lumbar disc degeneration (LDD) is associated with low back pain (LBP). Although both insomnia and mental distress appear to influence the pain experience, their role in the association between LDD and LBP is uncertain. Our objective was to investigate the role of co-occurring insomnia and mental distress in the association between LDD and LBP-related disability.A total of 1080 individuals who had experienced LBP during the previous year underwent 1.5-T lumbar magnetic resonance imaging, responded to questionnaires, and participated in a clinical examination at the age of 47. Full data was available for 843 individuals. The presence of LBP and LBP-related disability  were assessed using a questionnaire. LDD was assessed by a Pfirrmann-based sum score . The role of insomnia  and mental distress (according to the Hopkins Symptom Check List-25) in the association between the LDD sum score and LBP-related disability was analyzed using linear regression with adjustments for sex, smoking, body mass index, education, leisure-time physical activity, occupational physical exposure, Modic changes, and disc herniations.A positive association between LDD and LBP-related disability was observed among those with absence of both mental distress and insomnia , and among those with either isolated mental distress  or isolated insomnia . However, among individuals with co-occurring insomnia and mental distress, the association was not significant .LDD does not associate with LBP-related disability when insomnia and mental distress co-occur. This finding may be useful when planning treatment and rehabilitation that aim to reduce disability among individuals with LDD and LBP. Future prospective research is warranted.The online version contains supplementary material available at 10.1186/s12891-023-06365-2. Low back pain (LBP) causes a significant burden to both individuals and societies worldwide . It is eMental distress often co-occurs with insomnia  and it hMental distress has been found to modify the association between LDD and LBP; if mental distress was not present, there was an association between LDD and LBP, but if mental distress was present, the association between LDD and LBP was lost . HoweverThe aim of this study was to investigate the potential role of co-occurring mental distress and insomnia  in the association between LDD and LBP-related disability using a large sample of Northern Finns reporting LBP. Based on our previous findings , we hypoCross-sectional study.The Northern Finland Birth Cohort 1966 (NFBC1966) is a prospective longitudinal population-based cohort study (n = 12 058 live births) that initially comprised inhabitants of the two northernmost provinces of Finland (Oulu and Lapland). Pregnant women whose expected date of delivery was between January 1st and December 31st, 1966, were invited to the cohort. Cohort members have been followed since 1966 via, for example, regular postal questionnaires and clinical examinations (which comprised objective measurements of weight and height) .In this cross-sectional study, we used data  scans, and clinical examination) from the most recent data collection point at the age of 47 in 2012–2014. A total of 7146 of the NFBC1966 cohort participants answered the questionnaires, 5832 participated in the clinical examinations, and 1540 underwent MRI of the lumbar spine. Of these 1540 participants, 1080 individuals had experienced LBP during the previous year prior to MRI, and were therefore included in the study. Those who had not had any LBP within this time period were excluded. The study sample used in the main analyses was based on 843 individuals who had the full data available.A total of 1540 NFBC1966 participants underwent MRI of the lumbar spine in 2012–2014. For the lumbar MRI we used 1.5-T equipment  and T2-weighted fast-recovery fast spin-echo (frFSE) images in the sagittal and transverse planes, and T1-weighed fluid-attenuated inversion recovery sequence images in the sagittal plane. A detailed description of the MRI protocol has been presented in a previous publication [A Pfirrmann-based LDD consensus reading was pursued as previously described . In the Based on the final LDD consensus, the overall burden of LDD was quantified by means of a Pfirrmann-based sum score variable by categorizing Grades I and II as 0, and Grades III, IV and V as 1, 2 and 3, respectively. The LDD sum score for five lumbar discs could thus theoretically range from 0 to 15, with higher values indicating higher LDD burden , 21.Data on LBP were collected using a questionnaire issued to the participants at the time of the MRI . LBP wasAs part of the questionnaires targeted to the cohort members, participants filled out the Hopkins Symptom Check List-25 (HSCL-25) , 23, and21. In this study, the participants were divided into two categories depending on the presence of clinically relevant mental distress. The cut-off for clinically relevant mental distress in the HSCL-25 was set at 1.55 , as a cut-off of 1.55 is suggested by the previous literature [The HSCL-25 is a widely used instrument for screening symptoms of anxiety and depression , 25, 26.terature , 25–27. The AIS-5 elicits the participants’ sleep problems over the past month , 28. It To study the influence of co-occurring insomnia and mental distress on the association between LDD and LBP-related disability, we formed a variable with the following four categories: (1) absence of both mental distress and insomnia, (2) isolated mental distress, (3) isolated insomnia, and (4) co-occurring mental distress and insomnia.Based on previous studies, sex, smoking, body mass index (BMI), education, leisure-time physical activity, occupational physical exposure, and Modic changes and disc herniations presenting in lumbar MRI were considered potential confounders in the association between LDD and LBP –41. The 2) and categorized according to the World Health Organization definition  [The participants’ weight and height were measured in the clinical examination by a trained nurse. BMI was calculated using the participant’s weight and height as kilograms per meter squared (kg/mMI > 30) .Education level was elicited using the number of school years: < 9 school years, 9–12 school years, > 12 school years. This classification has also been used in a previous study  and is bSmoking was determined by two questions: (1) “Have you ever smoked cigarettes (yes/no)?” and (2) “Do you currently smoke (yes/no)?” Based on the answers, the participants were classified into three groups: (1) never-smokers, (2) former smokers and (3) current smokers .To determine physical activity during leisure time, the participants were asked to estimate how often they take part in physical activity that causes at least some sweating and breathlessness (corresponding to moderate-to-vigorous intensity). The response alternatives were (1) daily, (2) 4–6 times a week, (3) 2–3 times a week, (4) once a week, (5) 2–3 times a month, and (6) once a month or less often. The participants were divided into three categories on the basis of their responses: “active” (at least four times a week), “moderately active” (1–3 times a week), and “inactive” (less than once a week) .Occupational physical exposure was assessed as described previously . IndividThe presence of lumbar disc herniations and Modic changes were also used as covariates. The protocols have been published previously , 43. An Statistical analysis was performed using SPSS Statistics, version 27, 64-bit edition . The threshold of statistical significance was set at P = 0.05. Descriptive statistics were used to present the distributions of LDD, LBP-related disability, and background variables in the pooled study sample and stratified by the co-occurring mental distress and insomnia categories; frequencies (n) and percentages (%) were used for categorical variables; and means with standard deviations (SD) or medians with interquartile ranges (IQR) were used for continuous variables. Differences between the co-occurring mental distress and insomnia categories were tested by Kruskal-Wallis and Chi-square tests; Bonferroni correction was applied in pairwise comparisons. Cronbach’s α was calculated as a measure of internal consistency for HSCL-25 and AIS-5.The association between the LDD sum score (continuous predictor) and LBP-related disability (continuous outcome) was modeled using linear regression, with the beta coefficient (B), 95% confidence interval (CI) and P value. Both unadjusted and adjusted models were constructed. The final, adjusted, analyses were made on a complete-case basis.To study the roles of mental distress and insomnia in the association between the LDD sum score and LBP-related disability, we stratified the regression models by the presence of mental distress and insomnia (the four-category variable). This approach was justified, as we observed a significant association between insomnia and LBP-related disability, and as we have also previously shown that mental distress modifies the association between LDD and LBP-related disability .The study followed the principles of the Declaration of Helsinki and was approved by the Northern Ostrobothnia Hospital District Ethical Committee 94/2011 (12.12.2011). The NFBC1966 members took part voluntarily and signed their informed consent. All personal identity information was encrypted and pseudonymized before being granted to the researchers.The sample comprised 1080 individuals with LBP. Table Table The aim of this study was to explore the role of co-occurring mental distress and insomnia in the association between LDD and LBP-related disability among a large sample of Northern Finns with LBP. A positive association between LDD and LBP-related disability was observed among those with absence of both mental distress and insomnia, and among those with either isolated mental distress or isolated insomnia. However, among individuals with co-occurring insomnia and mental distress, the association was not significant.The prevalence of insomnia has been estimated to range between ∼5% and ∼15% –46 in thEven though research activity around the predictors of LBP has been extensive, many questions remain unanswered . AssociaHealthcare resources are limited and should be properly targeted. Although 85% of individuals with LBP are on sick leave for only a few days at most, the remaining 15% have longer sick leaves and account for half of the total number of sick leaves . Data frThe strengths of this study were manifold. We used a general population sample of Northern Finns reporting LBP, and the sample size was relatively large. The assessments of insomnia and mental distress were based on reliable and validated questionnaires. LBP-related disability was chosen as the primary pain dimension, because it was perceived as a wide concept that captures pain-related disability at work, during leisure time, and during sleep. Adjustments were made for several potential confounders.Our study also had limitations. As it had a cross-sectional design, neither causality nor the direction of associations could be addressed. The interrelationships between insomnia, pain and mental distress are potentially complex. Mental distress and insomnia were treated as dichotomous variables with two categories, which can be seen as a rough simplification of the underlying spectrum of symptoms. However, clinical use in Finland and previous studies , 23–28 hLDD does not seem to associate with the LBP-related disability when both insomnia and mental distress are present. A positive association between LDD and LBP-related disability was observed among those with absence of both mental distress and insomnia, and among those with either isolated mental distress or isolated insomnia. This finding may be useful when planning treatment and rehabilitation that aim to reduce LBP-related disability among individuals with LDD and LBP. In order to reduce the disability related to LDD, it may be beneficial to take co-occurring insomnia and mental distress into account in the treatment and rehabilitation of LBP. Further research with longitudinal data and prospective designs are urgently warranted.Below is the link to the electronic supplementary material.Supplementary Material 1


Smoking is a risk factor for gastric cancer. Studies have shown that the risk of gastric cancer can vary by smoking status and smoking amount at a single point in time. However, few data have been reported about the effect of changes in smoking status over time on the risk of gastric cancer.This study collected data from the National Health Insurance Corporation in Korea on 97,700 Korean men without gastric cancer who underwent health check-ups from 2002 to 2013. The smoking status  of study participants was assessed in 2003-2004 and 2009, and the results were categorized into 7 groups: never-never, never-quit, never-current, quit-quit, quit-current, current-quit, and current-current. Participants were followed until 2013 to identify incident gastric cancer. A multivariate Cox proportional hazard model was used to calculate adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) for incident gastric cancer according to changes in smoking status and smoking amount (pack-years).Compared with group 1 (never-never), participants currently smoking in 2009  had higher HRs for gastric cancer . The risk for gastric cancer was highest in heavy smokers, followed by moderate smokers.In all categories of smoking status, current smoking was associated with the highest risk of gastric cancer. Heavy smoking was associated with an increased risk of gastric cancer, even in former smokers. Although the incidence and mortality of gastric cancer have gradually decreased worldwide, gastric cancer is still the fifth most common malignancy and third leading cause of cancer death globally . East AsSmoking is a major concern in global public health. Despite a global decline in the proportion of smokers, the total number of smokers has increased due to population growth . SmokingMost studies have investigated the association between smoking and gastric cancer based on smoking status or smoking amount at a single point in time. However, smoking status can change over time. Social policies and anti-smoking campaigns recognize the common cycles of smoking status  that result in smoking status changes. However, no studies have yet assessed the risk of gastric cancer according to these changes in smoking status.The purpose of our study was to identify the effect of changes in smoking status on the risk of gastric cancer. Therefore, we investigated the risk of incident gastric cancer according to changes in 3 categories of smoking status  over time.In Korea, the National Health Insurance Corporation (NHIC) provides the National Health Insurance Service (NHIS), which covers 97% of the Korean population. Medical information from the NHIS, which represents the medical service usage of the entire Korean population, is stored in the National Health Information Database (NHID) , includiMost Korean medical institutions have a contract with the NHIC to provide the medical information of their healthcare users and patients. Therefore, the NHID contains the medical information and socio-demographic variables of the Korean population collected from health care utilization and health check-ups. In recent years, the NHIS in Korea has provided access to the sampled database for research purposes after deleting personal identification information. The sampled data includes information from health check-ups and is linked with Statistics Korea, which contains data on the development of gastric cancer in Korea.Based on the NHID information, 194,444 subjects who underwent health check-ups between 2003-2004 and in 2009 and had valid cigarette smoking records were included in our study. Of these, 10,742 participants whose smoking status was not valid (quit-never and current-never) were excluded. In addition, we excluded 2,347 participants who were diagnosed with gastric cancer between 2002 and the date before their medical health examination in 2009, as well as 83,655 women participants. The final analysis included 97,700 participants who were monitored for incident gastric cancer. The total follow-up period was 423,288.7 person-years and the average follow-up period was 4.33  person-years.Changes in smoking status were assessed by answers to a selfadministered questionnaire during the biennial health check-up. According to responses about smoking status at the time of their health check-up in 2003-2004 and again in 2009, study participants were categorized into 3 groups: never (participants who never smoked), quit (participants who had quit smoking) and current (participants currently smoking). Study participants were then further categorized into 7 groups based on their change in smoking status between the first examination (2003-2004) and the second examination (2009) as follows: group 1 (never-never), group 2 (never-quit), group 3 (never-current), group 4 (quit-quit), group 5 (quit-current), group 6 (current-quit), and group 7 (current-current). Smoking amount was determined in 2009 and was defined in pack-years (1 pack year= 1 year× 1 pack per day). Participants were divided by smoking amount into 4 groups: never smokers, light smokers (0-10 pack-years), moderate smokers (10-20 pack-years), and heavy smokers (> 20 pack-years).The NHIC general health examination is conducted in 2 stages. The first stage is a comprehensive screening test to determine the presence or absence of disease among the general population without symptoms. The second stage includes consultation screening tests and a more detailed examination to confirm the presence of disease. These health examinations also include a questionnaire covering lifestyle and past medical history. Our study data included information provided by these questionnaires, as well as anthropometric measurements and laboratory measurements. Alcohol intake was defined as consuming alcohol > 3 times per week. Physical activity was defined as moderate-intensity physical activity (at least 30 minutes per day > 4 days each week) or vigorousintensity physical activity (at least 20 minutes per day > 4 days each week). Body mass index (BMI) was calculated as weight (kg) divided by height squared (meters).Systolic blood pressure (BP) and diastolic BP were measured by trained examiners. The following laboratory data were measured when these participants underwent health examinations: fasting blood glucose, total cholesterol, triglyceride, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, aspartate aminotransferase, alanine aminotransferase and gamma-glutamyl transferase (GGT) levels.The NHID was linked to data on diagnosed diseases from Statistics Korea. In this study, the entry date was the time of the first health check-up after 2009, and the last follow-up date for diagnosis of gastric cancer was December 31, 2013. The primary endpoint of this study was the identification of gastric cancer based on the International Classification of Diseases, 10th revision, Clinical Modification (ICD-10-CM) codes registered in the NHID. Gastric cancer was diagnosed based on biopsy results after endoscopy with an ICD-10 code assigned at the time (C16).Data were expressed as mean±SD or median (interquartile range) for continuous variables and percentages for categorical variables. One-way analysis of variance and the chi-square test were used to analyze statistical differences among the characteristics of the study participants at the time of enrollment in relation to changes in smoking status. Person years were calculated as the sum of the follow-up years for all participants from the baseline entry date until the date of gastric cancer diagnosis or until December 31, 2013, whichever came first.To evaluate associations among the 7 groups according to changes in smoking status and incident gastric cancer, we used Cox proportional hazards models to estimate adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) for incident gastric cancer. The Cox proportional hazards models were adjusted for multiple confounding factors. In the multivariate-adjusted models, we included variables that might confound the relationship between smoking status change and smoking amount and incident gastric cancer, such as age, BMI, systolic BP, fasting blood glucose level, total cholesterol level, alcohol intake, and physical activity. To test the validity of the Cox proportional hazard models, we checked the proportional hazard assumption. The proportional hazard assumption was assessed using a log-minus-log survival function and was found to be graphically unviolated. A p-value < 0.05 indicated statistical significance. All statistical analyses were performed using SAS version 9.4 .Ethics approval for the study protocol and analysis of the data was obtained from the Institutional Review Board (IRB) of Kyung Hee University Hospital. The informed consent requirement was exempted by the IRB because researchers retrospectively accessed an anonymized database for analysis purposes.During 423,288.7 person-years of follow-up, 1,119 (1.15%) incident cases of gastric cancer developed between 2009 and 2013. The baseline characteristics of the study participants in relation to the 7 groups of smoking status change are presented in The aim of this study was to quantify the risk of gastric cancer in Korean men according to changes in smoking status over time. We found that the risk of gastric cancer was relatively higher in men currently smoking in 2009 regardless of previous smoking status, compared with the never-never group. Although group 3 (never-current) had a widely dispersed 95% CI , the 3 currently smoking groups in 2009  had higher HRs for gastric cancer than the never-never group. This result suggested that a currently smoking status was the strongest risk factor for the future development of gastric cancer among all smoking status groups.Our results are in line with previous studies that investigated the relationship between smoking and gastric cancer. Two meta-analyses of 46 case-control studies and 32 cohort studies demonstrated that “currently smoking” was associated with an increase in the risk of gastric cancer by 57% and 62%, respectively ,13. In aThe mechanisms by which smoking is involved in gastric carcinogenesis may help explain our results. Cigarettes contain multiple components  that can be toxic and carcinogenic ,15. StudWe assessed the risk of gastric cancer by smoking amount. Compared with never smokers, moderate smokers and heavy smokers showed an increased risk of gastric cancer in a dose-dependent pattern. This finding indicates that greater smoking amounts lead to a higher risk of gastric cancer. Interestingly, the former smokers in group 6 (current-quit) showed a significant association with an increased risk of gastric cancer. A plausible explanation for this finding may be the smoking amount in group 6. The mean smoking amount in group 6 was 23.2± 16.7 pack-years (heavy smokers). This finding suggests that high smoking amounts are linked to an increased risk of gastric cancer even in former smokers. Although never smoking is most effective at preventing gastric cancer, it is necessary to encourage smokers who are not ready to quit smoking to reduce their smoking amount.The merits of the present study include reliable nationwide data, including serial evaluations for smoking status and diagnosis of gastric cancer based on ICD-10 codes. These advantages enabled us to quantify the risk of gastric cancer according to changes in smoking status and smoking amount.Helicobacter pylori infection in our study participants because our data source did not contain information about H. pylori infection, which is a strong risk factor for gastric cancer. It has been reported that the adverse effect of H. pylori infection on gastric cancer can be exacerbated by smoking [H. pylori. Moreover, the prevalence of H. pylori infection is > 50% in Korean adults [H. pylori should be recognized as a major limitation in our study.Nonetheless, our study had several limitations. First, we could not identify  smoking ,24. Butt smoking  demonstrn adults . TherefoSecond, the assessment of smoking status and amount was dependent on a self-reported questionnaire. Although the validity and reliability of the NHID have been confirmed in studies, recall bias is possible.Third, there were no data for the location or histology of the gastric cancers in our study. Studies have shown heterogeneous results about whether smoking is primarily associated with cardia gastric cancer or with non-cardia gastric cancer -10. HoweIn conclusion, current smoking was associated with an increased risk of gastric cancer, regardless of previous smoking status. Although former smoking was not associated with an increased risk of gastric cancer, former smokers with a history of heavy smoking had an increased risk of gastric cancer. These results suggest that smoking cessation and reducing smoking amounts are both important factors in reducing the risk of gastric cancer.


In the inspiratory phase, TrA-IO activity was significantly affected by breathing pattern and EO activity with posture (p < 0.05). TrA-IO activity significantly increased during Forced-Expi in the supine posture (47.6% of the maximum voluntary contraction) and Exertion-Inspi in the elbow-toe posture (35.7%), whereas no differences were found during Deep-Bre or Q-Bre (< 20%). EO activity increased in the elbow-toe posture (22.5–30.6%) compared with that in the supine posture (< 5%) during all breathing tasks. IAP values were low during all tasks (< 15%) except for Forced-Expi (24.9%). Abdominal muscle activation and IAP interacted with the breathing pattern and posture.We aimed to assess the effects of interaction between several breathing patterns and postures on abdominal muscle activation and intra-abdominal pressure (IAP). This comparative cross-sectional study enrolled fourteen healthy university students majoring in sports science and/or physical education. They performed four active breathing tasks: quiet nasal breathing (Q-Bre), nasal deep breathing (Deep-Bre), completely forced expiration (Forced-Expi), and exertional nasal inhalation with abdominal muscles with isometric contraction (Exertion-Inspi) in the elbow-toe plank and supine postures. Breathing volume; IAP; and transverse abdominis-internal oblique muscle (TrA-IO) and external oblique muscle (EO) activities were recorded. Abdominal muscle activity and IAP significantly interacted with breathing pattern and postures during the expiratory phase ( These exercises may be effective in preventing low back pain in athletes7 or as therapeutic exercises in patients with low back pain with a positive lumbar instability test2. In addition, inspiratory muscle training has been found to improve dynamic balance in healthy older adults, resulting in improved walking speed and inspiratory muscle function5. Thus, breathing exercises could improve physical function, human behavior, and quality of life, which are not merely for improving breathing function3.Breathing exercises, such as maximum expiration training10. There is a trade-off between the task demand such as posture or limb movements and respiratory demand of the respiratory muscles , which are called the local muscles15. For instance, during heavy weight-lifting, spinal stability is the priority, at the expense of a steady breathing pattern13. However, as respiratory demand is increased, such as in certain exercises or in cases of respiratory disease, the contribution of the transverse abdominal muscle and diaphragm to spinal stability could be compromised12. These basic studies have led to the principle of performing exercise in conjunction with the correct posture and breathing for injury prevention and enhancing physical function.Breathing exercises are often combined with the motion of the limbs or trunk, such as with the plank, elbow-toe, and postural stability exercises. Combining exercises could lead to a synergistic effect because the respiratory muscles are used for both respiration and postural control2. Another clinical practice study has shown that the activity of the abdominal muscles increased significantly when combined with maximum expiration compared with resting expiration during side-bridge exercises16. In addition, inspiratory muscle training combined with the Pilates methods was found to provide an enhancement in pulmonary function and physical conditioning in older patients3. However, basic and clinical practice studies have not bridged between several breathing patterns and postures on abdominal muscle activation and intra-abdominal pressure (IAP) caused by these muscles co-contraction. Clinicians should elucidate how much IAP would increase by exercises18 and consider whether exercises allow individuals to sustain exercise without cardiorespiratory burden13. Thus, understanding the effects of breathing pattern and posture from a physiologic standpoint is necessary for effective exercise instruction. This study aimed to examine the effects of interaction between several breathing patterns and postures on abdominal muscle activation and IAP.Similarly, some clinical practice studies have demonstrated the effect of exercises combined with breathing. Abdominal draw-in lumbar stabilization exercises with respiratory resistance resulted in decreased low back pain and dysfunctions and increased muscle thickness in contraction, contraction rate, and pulmonary functionFourteen healthy men , 5.0 years], mean height, 172.1 cm [4.2 cm], and mean weight, 73.3 kg [13.4 kg]) volunteered to participate in this comparative cross-sectional study. The participants were university students majoring in physical education. The study sample comprised four football players, two rugby players, two long-distance runners, a sprinter, a tennis player, a baseball player, a volleyball player, a handball player, and a soccer player. The participants had no neuromuscular, orthopedic, or respiratory abnormalities.All procedures involving human participants were performed in accordance with the ethical standards of the Institutional and/or National Research Committee and the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Written informed consent was obtained from all participants. The protocol was approved by the Ethics Review Board of Tokai Gakuen University (# 24-19).The participants performed four active breathing tasks: quiet breathing (Q-Bre), deep breathing (Deep-Bre), pursed-lips forced expiration (Forced-Expi), and exertional nasal inhalation with expanding the thorax not to abdominal wall expansion (Exertion-Inspi). Specifically, Q-Bre was normal nasal tidal breathing (as a control). Deep-Bre was a complete nasal inhalation with submaximal expansion of the abdominal wall and relaxed expiration through the mouth. Forced-Expi was a completely forced exhale with pursed lips until the abdominal wall was hollowed. Exertion-Inspi was a complete nasal inhalation, sufficient to expand the thorax while maintaining the abdominal muscles in isometric contraction, not to abdominal wall expansion. The respiratory rhythm was applied by examiner cueing. The participants practiced repeatedly during a lesson from breathing exercise instructors (T.K. and J.U.). The four breathing tasks were performed in a random order in the supine (as a control posture) and elbow-toe postures for 30 s. Regarding the elbow-toe posture, the participants performed a prone plank posture on the floor, maintaining a straight trunk and lower extremities, with only their toes and forearms touching the floor Fig. . They re20. Briefly, IAP was measured using a pressure transducer  placed intra-rectally21. Before the actual measurements were taken, the maximum IAP was obtained based on the maximum voluntary pressurizations produced during Valsalva maneuvers performed in a standing posture (maxIAP). The highest value among three trials was used as the maximum. The IAP during the task was normalized using maxIAP (%IAP). The breathing volume of all the tasks was measured with a pneumotachograph  using a face mask covering the nose and mouth . Respiratory volume was calibrated using a syringe calibrated at 3 L .Respiratory and IAP measurements were performed as previously described22. Preparing for the EMG, the surface of the skin was cleaned with alcohol and rubbed with sandpaper. Surface bipolar electrodes  were placed on each of the muscles using Kinesio tape. Before measurements and throughout the testing period, all EMG signals were monitored and checked using a real-time oscilloscope display. EMG signals from the right transverse abdominis-internal oblique muscle (TrA-IO) and right external oblique muscle (EO) were recorded during the tasks. Electrodes for the TrA-IO were placed 2 cm inferomedially to the anterior superior iliac spine23 at an angle following the inguinal ligament. The activity of the TrA-IO could be best assessed from this surface location25. Electrodes for the EO were placed at the junction of a line drawn from the umbilicus to the anterior axillary line at an oblique angle following the muscle fibers. A reference electrode was placed on the right iliac crest. The EMG signals were amplified differentially using an AC amplifier . Moreover, band-pass filtering was set at both low (time constant of 0.03 s) and high (2 kHz) cut-off filters . Before performing the actual tasks, all participants completed three consecutive trials of maximum voluntary contraction (MVC) of the isometric muscles21. MVC was performed to sustain the posture against the external force applied in the supine (for TrA-IO) and side-lying (for EO). The participants kept the area beyond their iliac crest hanging over the examination table in the supine or side-lying posture, with their pelvis and lower leg held in place by two examiners. Thereafter, another examiner applied the maximal resistance to the participant’s upper trunk with the hands using the whole body weight. The participants horizontally kept their trunk position against resistance, trying to hold the maximum force for approximately 5 s. The highest root mean square of EMG amplitude of each muscle during the three MVC trials  was adopted as the MVC value used for subsequent statistical analysis (100% MVC). The root mean square of EMG data of the MVC trial was calculated for 100 ms . Moreover, Valsalva maneuver test was performed in the standing position to develop the maximal IAP (100% IAP)20. Valsalva maneuver test comprised a complete nasal inhalation followed by holding breath with maximal voluntary pressurization to the abdominal compartment as forcefully as possible.Surface electromyogram (EMG) measurements were conducted as previously describedIAP and airflow and EMG data were simultaneously recorded on a computer  using an analog-to-digital converter  at a sampling rate of 2 kHz. Representative data are shown in Fig. p-value < 0.05 was considered statistically significant. In addition, the magnitude of changes in the values from the reference value (Q-Bre in the supine posture) was expressed as the effect size. Each outcome was classified as a small (0.2), moderate (0.5), or large (≥ 0.8) effect26. Finally, the muscle activity ratio between the local and global muscles was expressed as a relative value (TrA-IO/EO).All data are expressed as mean ± standard error of mean. A two-factor (breathing and posture) repeated-measures analysis of variance was performed to assess statistical significance of the effects of interaction. Based on the model of Mauchly’s test, if the assumption of sphericity was not met, the Greenhouse–Geisser correction was applied. Then, Bonferroni post hoc testing was used for pairwise comparisons. All statistical analyses were performed using SPSS software, version 22 . A Respiratory volume was remarkably different between breathing patterns and posture during both the inspiratory and expiratory phases Table . Forced-TrA-IO and EO activity including IAP only had a remarkable effect on the breathing pattern and posture during the expiratory phase Table . In the The greatest TrA-IO activity was observed in Forced-Expi at the expiratory phase Fig. . The nexEO activity in the elbow-toe posture induced 22.5–30.6% of the MVC regardless of the type of breathing pattern  were > 1.0 in all breathing tasks in the supine posture Table . In the The maximum IAP during the Valsalva maneuvers was 196.9 mmHg . The relative IAP value was substantially lower in all tasks, at < 15% of the IAP Fig. , Table 3In this study, we found novel findings showing an effect of interaction between several combinations of breathing patterns and postures on abdominal muscle activities based on EMG and IAP, while observing respiratory volume. We found that TrA-IO and EO activity including IAP only interacted with breathing pattern and posture during the expiratory phase. Additionally, in the inspiratory phase, TrA-IO activity and IAP were remarkably affected by breathing pattern, while EO activity and IAP were affected by postural tasks. Thus, our results may fill the gap between basic and clinical practice regarding the importance of combining posture exercises with breathing tasks.28. TrA-IO activity was primarily affected by Forced-Expi, as evidenced by the greatest IAP development, which induced the greatest respiratory volume approximately 4–5 times, compared with that in Q-Bre  was moderate and comparable with the result of 45% maximal lifting effort during isometric weight-lifting found in a previous study19. If a few breathing trials did not show any issues, but were repeated several times, it was still necessary to consider the cardiorespiratory burden.The greatest TrA-IO activity was observed in the Forced-Expi breathing pattern at the expiratory phase in the supine posture (47.6% of MVC). Even in the supine posture, the TrA-IO activity level in Forced-Expi breathing was still relatively higher than that in some of the plank exercises without breathing tasks reported in previous studiesre Table . In the 30, which was a complete nasal inhalation while maintaining the abdominal muscles in isometric contraction, not to abdominal wall expansion. Although the respiratory volume was not highly increased, to maintain the isometric contraction, the TrA-IO activity required to be balanced against the increasing pressure produced by the descent of the diaphragm in inhalation32. Thus, endurance-related training is recommended for the sustenance of TrA/IO activity without being affected by the breathing event, as TrA/IO activity plays a role in respiration and the postural control effect10. Moreover, it would improve the risk–benefit ratio, because the IAP value in Exertion-Inspi was smaller (12.4% of IAP) than that in Forced-Expi. This low value allows for sustaining exercise without cardiorespiratory burden19. Regarding exercise instruction, due to the small respiratory volume in Exertion-Inspi, the instructor emphasized the need for isometric contraction of the TrA-IO.The second greatest TrA-IO activity value was observed in the Exertion-Inspi pattern throughout the inspiratory and expiratory phases in the elbow-toe posture (approximately 30–40% of MVC). Despite this finding, no statistical differences in respiratory volume were found between Exertion-Inspi and Q-Bre Table . The TrA28. Even if we focus on the large inspiration pattern, which induced a 2.0–2.4-fold respiratory volume in Deep-Bre compared with that in Q-Bre, the relaxed-deep breathing (Deep-Bre) resulted in insufficient TrA-IO activity. In this breathing pattern, the abdominal muscles only expanded passively, contrary to what was observed in Exertion-Inspi. Although the IAP value in Deep-Bre was low, the elasticity of the passive expansion of abdominal muscles nullified the IAP produced by the descent of the diaphragm during inhalation32. Thus, the Deep-Bre pattern did not affect TrA-IO activity and IAP dynamics and only promoted the passive expansion of the abdominal muscles, even in the elbow-toe posture.Compared with the Q-Bre task, there was no increase in the activity of the TrA-IO in the Deep-Bre. TrA-IO activity in Deep-Bre in the elbow-toe posture was only approximately 20% of the MVC, which was the same as that of some of the plank exercises without breathing in previous studies28, it is impossible to compare them directly due to differences in MVC trials and lack of supine posture data in the previous studies28. Imai et al.27 and Okubo et al.28 found that EO activity increased in several additional postural tasks in addition to the elbow-toe posture, rather than in the local muscles. These results in combination with ours suggest that EO activity contributes more to posture tasks than to breathing-related tasks.EO activity was affected by the posture tasks. The activity was 3–6 times larger in the elbow-toe posture than that in the supine posture, regardless of the type of breathing pattern. Although this result is in accordance with the results of previous studiesWe found a unique result regarding the relative values of TrA-IO and EO activity. The TrA-IO/EO ratio of < 1.0 in the elbow-toe posture indicated that the contribution of EO activity was higher than that of TrA-IO activity. Conversely, a ratio > 1.0 in Exertion-Inspi in the inspiratory-expiratory phase and in Forced-Expi in the expiratory phase indicated that the TrA-IO was also a contributor, together with the EO. Performing the dual task of posture and breathing may be useful for activating all the abdominal muscles. The dual task is also likely to have an effect on muscle strength and physical performance. Additional research is required for determining the training effect on physical performance and synergistic effects of muscle function.2. On the other hand, we could not evaluate the prospective study about the trainability or sustained effects of training according to each sport’s characteristics in this study. Second, we used only surface EMG rather than fine-wire EMG. Fine-wire EMG could have been used to clarify each muscle activity with less cross-talk effect28. However the muscle activity of the TrA-IO and EO significantly differed among several breathing patterns and postural tasks in this study. Thus, our goal might have been achieved. Lastly, we could not directly assess the activity of the diaphragm due to technical reasons, such as using fine-wire EMG as an invasive method for the diaphragm12. Instead, we considered that measuring IAP value played a role in estimating the activity of the diaphragm including the abdominal muscles.There are several limitations to this study. First, the participants were university students majoring in physical education who were not accustomed to routine practice of the breathing methods used, such as the Pilates method. Abdominal draw-in lumbar stabilization exercises in the resistance respiratory group were received three times per week for 4 weeks, resulting in reduced pain and abdominal dysfunction compared with the control middle-aged groupThe TrA-IO, EO, and IAP were remarkably affected by breathing pattern and posture in the expiratory phase. Meanwhile, in the inspiratory phase, TrA-IO activity and IAP affect the breathing pattern, whereas EO activity had a relationship with the posture. For appropriate exercise load on the abdominal muscles, it is recommended to combine postural tasks with forced breathing, not relaxed-deep breathing.