Low back pain (LBP) is influenced by interrelated biological, psychological, and social factors, however current back pain management is largely dominated by one-size fits all unimodal treatments. Team based models with multiple provider types from complementary professional disciplines is one way of integrating therapies to address patients’ needs more comprehensively.This parallel group randomized clinical trial conducted from May 2007 to August 2010 aimed to evaluate the relative clinical effectiveness of 12 weeks of monodisciplinary chiropractic care (CC), versus multidisciplinary integrative care (IC), for adults with sub-acute and chronic LBP. The primary outcome was pain intensity and secondary outcomes were disability, improvement, medication use, quality of life, satisfaction, frequency of symptoms, missed work or reduced activities days, fear avoidance beliefs, self-efficacy, pain coping strategies and kinesiophobia measured at baseline and 4, 12, 26 and 52 weeks. Linear mixed models were used to analyze outcomes.P = 0.02). The long-term profile for pain intensity which included data from weeks 4 through 52, showed a significant advantage of 0.5 for IC over CC . The short-term profile (weeks 4 to 12) favored IC by 0.4, but was not statistically significant . There was also a significant advantage over the long term for IC in some secondary measures , but not for others . Importantly, no serious adverse events resulted from either of the interventions.201 participants were enrolled. The largest reductions in pain intensity occurred at the end of treatment and were 43% for CC and 47% for IC. The primary analysis found IC to be significantly superior to CC over the 1-year period (Participants in the IC group tended to have better outcomes than the CC group, however the magnitude of the group differences was relatively small. Given the resources required to successfully implement multidisciplinary integrative care teams, they may not be worthwhile, compared to monodisciplinary approaches like chiropractic care, for treating LBP.Trial registration NCT00567333. Low back pain (LBP) is one of the most prevalent and disabling chronic health conditions. An estimated 40–80% of adults experience low back pain (LBP) at some point in their lives , 2. FurtWhile the ‘biopsychosocial model’ for LBP has been promoted for decades, it is still incompletely and inadequately applied in both research and clinical practice –11. IndeIntegrating complementary modalities with conventional approaches has shown promise for LBP in previous studies . Team baThe purpose of this manuscript is to report the primary and secondary clinical outcomes of a randomized trial of monodisciplinary chiropractic care (CC), versus multidisciplinary integrative care (IC) for sub-acute and chronic LBP.This was a parallel group randomized clinical trial funded by the US Department of Health and Human Services. It was conducted from May 2007 to October 2009 with follow-up data collection through August 2010. Institutional Review Boards at participating institutions  approved the study protocol which has been described elsewhere . The stuThe study was conducted at Northwestern Health Sciences University . Participants were recruited from the surrounding Minneapolis/St. Paul metropolitan area primarily through targeted postcard mailings, brochures at community events, and advertisements in online local newspapers.Inclusion criteria were 18 years of age or older, LBP categories 1, 2, 3, or 4 according to the Quebec Task Force classification system  ; currentIndividuals were excluded if they had contraindications to study treatments  or who were pregnant or nursing, had current or pending spine-related litigation, a history of multiple lumbar surgeries, or progressive neurological deficits.The study statistician utilized a computer-generated randomization scheme and applied a 1:1 allocation ratio with randomly permuted block sizes that was concealed from the study team. As individuals became eligible, sequentially numbered sealed, opaque envelopes were drawn in consecutive order and opened in the presence of the study participant by the study team.All participants in the study received 12 weeks of either monodisciplinary chiropractic care (CC) or multidisciplinary team-based integrative care (IC). CC was delivered by a team of chiropractors allowed to utilize any non-proprietary treatment under their scope of practice not shown to be ineffective or harmful including manual spinal manipulation  and mobilization . Chiropractors also used hot and cold packs, soft tissue massage, teach and supervise exercise, and administer exercise and self-care education materials at their discretion. IC was delivered by a team of six different provider types: acupuncturists, chiropractors, psychologists, exercise therapists, massage therapists, and primary care physicians, with case managers coordinating care delivery. Interventions included acupuncture and Oriental medicine (AOM), spinal manipulation or mobilization (SMT), cognitive behavioral therapy (CBT), exercise therapy (ET), massage therapy (MT), medication (Med), and self-care education (SCE), provided either alone or in combination and delivered by their respective profession. Participants were asked not to seek any additional treatment for their back pain during the intervention period. Standardized forms were used to document the details of treatment, as well as adverse events. It was not possible to blind patients or providers to treatment due to the nature of the study interventions. Patients in both groups received individualized care developed by clinical care teams unique to each intervention arm. Care team training was conducted to develop and support group dynamics and shared clinical decision making. A clinical care pathway, designed to standardize the process of developing recommendations, guided team-based practitioner in both intervention arms. Evidence based treatment plans were based on patient biopsychosocial profiles derived from the history and clinical examination, as well as baseline patient rated outcomes. The pathway has been fully described elsewhere . Case maTo assess response to treatment during the intervention phase, patients completed a Patient Self-Assessment Form (PSAF) at each of their visits which was adapted from the Measure Yourself Medical Outcome Profile . PatientTable Participant demographic and clinical characteristics were collected during the baseline visits through self-report questionnaires and a health history and physical examination. Self-reported outcomes were collected at 2 baseline visits (7–14 days apart) and at 4, 8, 12, 26, and 52 weeks after enrollment using questionnaires administered independent of staff or clinician influence.The primary outcome measure was typical level of back pain over the previous week, using a numerical rating scale  –31.Back disability measured with the 23-item Roland Morris Disability Questionnaire  , 33Global improvement  , 35Days with medication use for back pain in the past week Quality of life measured with the EuroQol EQ5D-3L (− 0.109 to 1) Satisfaction with care  , 39Frequency of low back or leg symptoms  , 40Number of days in the past month with missed work or reduced activities due to back pain Work 0 to 42) and physical activity (0 to 24) subscales of the fear avoidance beliefs questionnaire [ and physPain self-efficacy (0 to 60) Pain coping strategies measured with the Vanderbilt Pain Management Inventory short form  , 45Kinesiophobia measured with the Tampa Scale for Kinesiophobia (17 to 68) .Secondary outcomes included:In a previous chronic LBP trial of exercise and spinal manipulation conducted by our team, group differences in pain of up to 8 percentage points after 3 months of treatment were observed. Informed by these results, the scientific literature at the time, and consensus of study investigators and clinicians regarding clinical importance, we were interested in detecting an 8-percentage point between group difference in pain after 12 and 52 weeks of treatment. Based on an α of 0.05 and 80% power, 85 participants per group were required. An allowance of 15% drop-out rate resulted in a total sample size of 200 participants.We used an intention-to-treat approach, analyzing all observed data from participants according to their allocated treatment assignment. Data were prepared for analysis by a data manager masked to group status; analyses were performed in SAS, version 9.1.The primary and most secondary outcomes were analyzed using linear mixed effect models including fixed effects for time, treatment, and a time-by-treatment interaction in addition to a random intercept to account for within-subject correlation. Hierarchical linear models are a robust method for analyzing ordinal outcomes using a Likert scale . Secondap-value ≤ 0.05) for group differences in the short term (weeks 4 through 12) to be determined. Clinical and demographic variables were included as covariates if they were at least moderately correlated (|r|≥ 0.5) with change in outcomes [The primary outcomes were short-term (4 to 12 week) and long-term 4 to 52 weeks) group differences in pain intensity derived from the linear mixed effect model. We used Fisher’s protected least significant difference approach to control for multiplicity . An areaoutcomes . Linear  weeks grSecondary analyses of the primary outcome measure included group differences at weeks 4, 12, 26, and 52. Additionally, responder analyses for no pain reduction, or pain reductions of 30% , 50% (moderate improvement), 75%, and 100%  were performed at weeks 12, 26, and 52 . DiffereAnalyses included group differences at the relevant individual time points for all measures, in addition to short-term (including 4 and 12 week outcomes) and long-term  profiles when possible.A summary of study participants is provided in Fig. Overall, 96% of study participants attended treatment visits as recommended; 93% for the CC group and 98% in the IC group. The mean number of visits in the CC group was 18.1 and for the IC group, was 23.8. Participants in the IC group received the following types of care: exercise therapy ; self-care education ; traditional Chinese medicine ; massage therapy ; chiropractic care ; cognitive behavioral therapy  and medication . The most frequent combinations were: TCM/SCE/ET (n = 22); ET, SCE, MT (n = 10); and ET, SCE, MT, CBT (n = 10). All participants received at least two types of treatment, and 27 received at least four. One participant received all of the treatments.During the 12-week intervention, 4 participants reported visits to other health care providers for their LBP: 3 from the CC group and 1 from the IC group. Between weeks 12 and 52, a total of 83 individuals sought additional health care: 46 in CC and 37 in IC.P = 0.02). The long-term profile for pain intensity (0–10) which included data from weeks 4 through 52, showed a significant advantage of 0.5 for IC over CC . The short-term profile (weeks 4 to 12) favored IC by 0.4, but was not statistically significant . Primary results are shown in Table The longitudinal omnibus test for pain showed IC to be significantly superior to CC over the 1-year period P = 0.0. The lonP = 0.04). On average, the difference in proportions for reduction of LBP intensity across all possible thresholds for improvement favored IC by approximately 6% at 12 weeks (95% CI − 3 to 13%), 7% at 26 weeks (95% CI − 2 to 15%), and 7% at 52 weeks (95% CI − 2 to 16%). Detailed results from the responder analyses are provided in Table Group differences for pain intensity at individual time points favored IC and ranged from 0.4 (weeks 4 and 12) to 0.6 week 52), with the only significant finding occurring at week 52  provided data on back pain intensity at every time point, and 166 (83%) provided the secondary outcomes at every time point. A total of 14 participants in the CC group and 5 in the IC group did not provide primary outcome data at all time points and the pattern of missingness seemed to be nonrandom. Participants with missing data at any time point reported higher pain intensity (when data was available) than participants with no missing data and this pattern was similar between groups. Sensitivity analyses for data missing not at random were performed using pattern mixture methods . MissingThere were five serious adverse events (SAEs) that occurred during the course of the trial ; all were classified as unrelated to study interventions. Four SAEs were reported by patients assigned to CC in which three were hospitalized , and one diagnosed with a brain tumor. One patient assigned to IC was hospitalized for nephrolithiasis evaluation and management.Minor self-limiting adverse events during the 12 weeks of intervention were reported with about equal frequency in both groups Table . The mosWhile there have long been calls to address LBP from a more comprehensive biopsychosocial perspective, there is still little research to date that has done so in a rigorous fashion. This study examines one approach for remedying this gap by comparing an integrative care (IC) intervention applying a multidisciplinary team-based approach to monodisciplinary chiropractic care. Overall, analyses demonstrated a consistent trend in favor of the IC group.Discussions of clinical importance for group differences requires consideration of the broader context regarding the condition being studied, what treatments are available, and the overall risk–benefit ratio of available options, which goes beyond criteria for establishing a clinically important change at the individual patient level . SeveralThe most recent Cochrane systematic review by Kamper et al.  examininThe Kamper et al. review reported on subgroup analyses assessing the impact of baseline symptom severity on the effectiveness of the multidisciplinary programs and found the results were inconclusive; however, given the modest benefits and relatively high resource and cost burden, the authors suggest these programs be reserved for the most severe cases where marked psychosocial distress is present. This approach is consistent with emerging risk-stratification and stepped care models aimed towards improving the efficiency and quality of care for musculoskeletal conditions .This study had several strengths, including the long-term follow up and high intervention adherence and follow up data collection rates. Expectations, a potentially important contextual factor that can influence treatment outcomes , 68 wereAs with any study there are also limitations that must be considered when interpreting the results. An important one which is common to many studies of complementary therapies, is the lack of representation of individuals from more diverse backgrounds, especially in terms of race and income. Also, despite careful attention to applying a clinical care pathway process , it was Because of the pragmatic nature of the study with intervention delivery designed to approximate how it could be implemented in practice, it is difficult to discern between the active elements of treatment and potentially important contextual and structural intervention qualities . Additionally, fidelity assessments of the interventions were limited to documentation by providers and patient-self report. Finally, another limitation of the study is the time it has taken from completion of the trial to publishing of the results, which came about from several members of the investigative team changing institutions. The findings however remain very relevant especially in light of continued and pervasive use of biophysically focused mono-disciplinary treatments  that still plague the LBP field, despite their limited effectiveness.Team based models of care with multiple provider types from complementary disciplines has been one way of integrating different therapies to more comprehensively address patients’ needs , 20. HowOne potential alternative to integrative multidisciplinary interventions is to train individual front-line providers to address patients’ biopsychosocial needs within the scope of their professional discipline, using multiple modalities to support guideline recommendations. Indeed, there have been shifts in both the chiropractic and physical therapy fields to take such an approach, integrating more psychosocial elements to these professional care models –75. To fAdditionally, future studies should consider ‘intervention mapping’ in the trial design phase, where the mechanisms of action of each element of multimodal care packages are more carefully aligned with patient needs and perhaps a greater range of more relevant psychosocial outcomes. This can facilitate results interpretations, as well as reporting and replication of interventions, and ultimately broader dissemination to those who could most benefit , 78. GreLow back pain patients who received integrative care by a multidisciplinary integrative care team tended to have better outcomes than those who received chiropractic care. However, given the relatively small magnitude of between group differences and the extensive resources required to successfully manage and implement, the team based integrative care might not be worthwhile. More efficient models for addressing biopyschosocial care for low back pain should be explored with greater emphasis on addressing the full spectrum of related psychosocial mechanisms and ensuring equitable access for all.


This study investigatesa fast dissolution and regeneration pretreatmentto produce regenerated cellulose nanofibers (RCNFs) via mechanicaldisintegration. Two cellulose pulps, namely, birch and dissolvingpulps, with degree of polymerizations of 1800 and 3600, respectively,were rapidly dissolved in dimethyl sulfoxide (DMSO) by using tetraethylammoniumhydroxide (TEAOH) as aqueous electrolyte at room temperature. WhenTEAOH (35 wt % in water) was added to the pulp–DMSO dispersion,95% of the dissolving pulp and 85% of the birch pulp fibers dissolvedalmost immediately. Addition of water caused the regeneration of cellulosewithout any chemical modification and only a minor decrease of DP,whereas the crystallinity structure of cellulose transformed fromcellulose I to cellulose II. The regenerated cellulose could thenbe mechanically disintegrated into nanosized fibers with only a fewpasses through a microfluidizer, and RCNF showed fibrous structure.The specific tensile strength of the film produced from both RCNFsexceeded 100 kN·m/kg, and overall mechanical properties of RCNFproduced from birch pulp were in line with reference CNF producedby using extensive mechanical disintegration. Although the thermalstability of RCNFs was slightly lower compared to their correspondingoriginal cellulose pulp, the onset temperature of degradation of RCNFswas over 270 °C. The sustainable use of naturally occurring components,such as cellulose,3 hemicelluloses,5 lignin,7 and starch,9 among others,12 in place ofplastic could overcome the above-mentioned shortcomings.Naturally derived materialsand chemicals are currently recognizedas sustainable alternative to the oil-based products used in everydaylife.13 Therefore, celluloseis among the most relevant raw materials in plastic replacements aswell as a starting point for a wide variety of novel products. Especiallythe nanosized cellulose, for example, cellulose nanofibers (CNFs)and crystals (CNCs), has been intensively studied to produce materialslike strong self-standing films.14 Theadvantages of nanocellulose films compared with plastics are theirvery high mechanical strength and oxygen barrier properties, especiallyat moderate humidity.15Celluloseis the most abundant natural polymer and is widely availablein nonedible plants.16 However, because of the strong hydrogen bondingability, microfibrils are aggregated into large-sized macrofibers,and a significant high amount of energy is requested to liberate nanosizedcellulose from these fibers. Nevertheless, we can lower energy consumptionby pretreating natural fibers using mechanical, enzymatic,17 chemical,19 or solvent-based methods.21In nature,cellulose is produced as cellulose microfibrils or elementalfibrils, which are nanosized constituents of molecular cellulose.23 Upon the dissolution, the natural cellulose structure, that is,the microfibrillar order, is lost, which could lower the energy consumptionduring the mechanical disintegration, especially if material is notdried in between treatments. So far, solvents such as ionic liquids,24 deep eutectic solvents,22 and N-methylmorpholine N-oxide25 have been used to dissolve celluloseto produce nanomaterials. In addition, aqueous LiBr systems have beenused to partially dissolve cellulose to liberate CNFs.26 Although systems like ionic liquids dissolvea substantial amount of cellulose (over 30 wt %),27 most of the above-mentioned solvents require long dissolutiontimes and/or high temperature. In addition, apart from aqueous LiBr,28 there is scarcity of information about productionof bulk materials, such as films, from regenerated cellulose nanomaterials.The dissolution and regeneration is also known to be an efficientmethod in producing nonconventional nanosized cellulose materials.29 where it helps decrease the viscosity of the solution.30 In addition, cellulose can be dissolved in DMSOin the presence of electrolytes, such as tetrabutylammoniumacetate31 and fluoride.32 DMSO is produced from dimethyl sulfide,33 a byproduct of the Kraft process used for cellulose pulping,thus being a sustainable solvent. DMSO itself has relatively low toxicity;however, it should be noted that it penetrates skin, thus allowingsolutes to pass into the living organism.34 Therefore, care should be taken when working with DMSO in the presenceof toxic solutes.In this study, we investigated the production of regenerated cellulosenanofibers (RCNFs) using novel solvent systems based on dimethyl sulfoxide(DMSO), which can dissolve cellulose rapidly in room temperature.DMSO is a common solvent used in cellulose dissolution and has beenstudied as a cosolvent with ionic liquids,35 or together with additional components,such as urea.36 Tetraalkylammoniumhydroxides are known to dissolve cellulose even at room temperature,and despite the apparent toxicity of alkyl cations, they are biodegradable.Moreover, it has been proposed that after use in dissolution and regenerationof biomass, they could be used as fertilizer.37 Previously, cellulose has been dissolved in DMSO by using aqueoustetrabutylammonium hydroxide (TBAOH). Here, dissolution of twocellulose pulps in DMSO using TEAOH at room temperature was investigated,and the production of RCNF was studied via mechanical disintegrationof regenerated cellulose. The films were produced from RCNFs, andthe mechanical and barrier properties of the films were compared tothe films produced from traditional CNFs obtained by using mechanicaldisintegration.In this study, DMSO was combined with aqueoustetraethylammoniumhydroxide (TEAOH). TEAOH belongs to the class of aqueous tetraalkylammoniumhydroxide ionic liquids that are studied for cellulose dissolutionas such39 DMSO and TEAOH (35 wt % in water)used for cellulose dissolution were obtained from VWR and Sigma-Aldrich,respectively. Deionized water was used for sample washing and dilutions.Birch pulp (BP) and dissolving pulp (DiP)were obtained as dry sheets. The properties of pulps are presentedin previous studies.Cellulose pulp (1 g) was dispersedin DMSO (90 g) by mixing with magnetic stirring for 15 min at roomtemperature. Approximately 9.8 mL (1 g) of TEAOH was then added inone portion, and the mixture was stirred for 5 min. Cellulose wasregenerated by adding 200 mL of water, after which the sample wasfiltered and washed with water until the suspension become neutral.The regenerated cellulose samples were stored at 4 °C.40 The amount of dissolved cellulose was studied in a similar way describedabove.To investigate the amount of dissolved cellulose, after the additionof TEAOH, the mixture was centrifuged at 10000 rpm for 10 min at 5°C with a Beckman Coulter Avanti J-26 XPI (USA) centrifuge. Approximately40 g of the upper layer was then removed from centrifuge tube, andcellulose was regenerated and washed as described above. Regeneratedcellulose was then dried in an oven at 60 °C. The concentrationof dissolved cellulose was calculated based on the dry mass of regeneratedcellulose. For the reference, dissolution of cellulose pulps in amixture of NaOH, urea, and water (7:12 :81 by weight) was investigatedin a similar way described in the literature.Optical microscope images of cellulose pulp dispersionin DMSObefore and after addition of TEAOH were acquired with a Leica MZFLIIImicroscope.The viscosity of the cellulosesolutionsin DMSO–TEAOH was investigated by using a TA Instruments DiscoveryHR-1 hybrid rheometer with cone–plate geometry (cone diameterof 40 mm and cone–plate angle of 1.999°). The measurementswere conducted at 20 °C.–1 by using a resolution of 2 cm–1. The number ofscans per sample was 40.Chemicalcharacterization of cellulose pulps before and after dissolution wasinvestigated by diffusion reflectance infrared Fourier transform spectroscopy(DRIFTS) using a Bruker Vertex 80v spectrometer (USA). The spectrawere collected in the range 4000–600 cmThe average degree of polymerization(DP) was determined by using the limiting viscosity method accordingto the ISO 5351 standard.41X-ray diffraction (XRD) of the cellulosepulps before and afterdissolution and regeneration and after fibrillation was conductedby a Rigaku SmartLab 9 kW rotating anode diffractometer (Japan) usingCo Kα radiation . The originaland regenerated cellulose were first pressed into pellets with a thicknessof 1 mm, and the nanofibrillated samples were measured from the self-standingfilms. Scans were taken over a 2θ (Bragg angle) range from 5to 50° at a scanning speed of 10°/s by using a step of 0.5°.The crystallinity index (CrI) was estimated from Gaussian fittingof the XRD diffractograms using an OriginPro 2020 by dividing thesum of the crystalline peak areas with the sum of all peak areas.The nondriedregenerated cellulose produced as described above was diluted to aconcentration of 0.5 wt % and disintegrated by passing the samplethree times at a pressure of 1000 bar through the 400 and 200 μmchambers of microfluidizer . Becauseof the minor dilution of the samples during the disintegration, thedry matter content of fibrillated samples was determined gravimetrically.Samples prepared from regenerated BP and DiP were named RCNF1 andRCNF2, respectively.42 At first, the sample(1.5% in water) was passed three times through −20 μm,twice through −40 μm, once through −60 μm,once through −80 μm, and twice through −90 μmwith a rotating speed of the grinding stones of ∼1500 rpm.The sample was then further diluted to 0.5% and passed through a microfluidizer:once through 400 and 200 μm chambers at a pressure of 1000 barand four times through 400 and 100 μm chambers at a pressureof 1500 bar.Reference CNFs were produced from DiP byusing combined disintegration with a friction grinder using a supermass colloider  and a microfluidizer .43 and thesamples were dried between two membranes by using a vacuum dryer  at 93 °C and at a negative pressureof 930 mbar for 10 min. Thus, the mass of the produced film was 60g/m2. The reference CNF film was produced with the samemethod.The RCNF films were produced via the filtration method.First, theRCNF dispersion with 0.33 g of dry matter was diluted into 100 g withwater. Air bubbles were removed by sonication in an ultrasound bath at room temperature with 37 kHzand power of 100% for 10 min by using the degas mode. The dispersionwas then filtered on a membrane  in a negative pressure of ∼800 mbar.Filtration was stopped after the time difference between two consequentdrops from the funnel was 30 s,The tensile propertiesof the films were measured at 23 °C and a relative humidity (RH)of 50%; these conditions were used on the sample for at least 48 hprior to the measurement. The films were cut to sample strips witha diameter of 5 × 70 mm, and the average thickness was measuredfrom the three random positions by using a thickness gage . The tensile test wasconducted by using a universal tensile machine with a 100 N load cell. During the test, the gage length was set to40 mm, and the strain rate was set to 4 mm/min. A prestrain of 0.1N was used.Themorphology of originaland regenerated cellulose was characterized by using a FESEM  with an acceleration voltage of 5.0 kV.Samples were first dispersed in water at a concentration of 0.5% overnightand then filtrated on the membrane. Samples were sputter coated withplatinum  beforeanalysis using a sputtering time of 30 s and a current of 40 mA.The morphology of RCNF films was observed with a SEM . The cross-section images were obtained from tensiletest samples after rupture.The transmittanceof the RCNF andCNF films was measured in the wavelength range 200–800 nm byusing a UV–vis spectrometer . To ensure thatthe films were perpendicularly aligned against the incoming beam andto avoid wrinkling, the films were put between two quartz glass slidesbefore they were set up in a cuvette stand.The thermal property ofthe original cellulose pulp and cellulose nanomaterials was characterizedby using a Netzsch STA 449F3 (Germany) thermogravimetric analyzer.Approximately 5 mg of dry sample was heated in aluminum oxide panfrom 30 to 950 °C at a rate of 10 °C/min under air flow(dynamic air) at a constant rate of 60 mL/min. The first-derivativecurves of the TGA (DTG) were calculated by using OriginPro 2019 software.p < 0.05).One-way analysis of variance (ANOVA)was conducted by using an OriginPro 2019 to determine the statisticalsignificance  and DP (3600) and DiP witha low hemicellulose content (4%) and a medium DP (1800). Prior tothe dissolution, cellulose pulp was dispersed in DMSO at room temperatureto obtain a turbid suspension. After the addition of TEAOH, the mixturewas significantly clearer and more viscose 1. HoweveBased on SEM images, most of the regenerated cellulose was uneven,flakelike material, indicating that the original fiber structure wasdisintegrated during the dissolution process 2. Few no–1) compared to the solution of BP (0.3 Pa·s at a shear rate of1 s–1). Despite the lower DP of DiP compared toBP, the higher solubility might result in higher viscosity. Nevertheless,viscosities of solution were relatively low, and the solutions ofDiP and BP in DMSO–TEAOH are poorly suitable for productionof regenerated cellulose films or filaments. Because of the low viscosity,the solution is quickly mixed with antisolvent used to regeneratecellulose, and therefore production of an intact film or a filamentis cumbersome. However, the low viscosity is advantageous for thepurification step of regenerated cellulose, as low viscosity allowsfast mass transport and thus limits the formation of gel-like material,where solutes (here DMSO and TEAOH) are trapped inside the regeneratedcellulose.The viscosity of the solution of DiP in DMSO–TEAOH was foundto be slightly higher  indicated the changes in the crystallinity of cellulose duringthe dissolution and regeneration.46During the dissolution, the crystallinity of originalcelluloseis lost, and the regeneration results in the rearrangement of orderedstructure of cellulose to form cellulose II crystalline structure.Hsystem 3.The in47 A weak signal was also observed around 40°(peaks around 10° and 34° are associated with sample holder48). Upon dissolution and regeneration, the peakof cellulose I disappeared, and new peaks were observed around 14°,24°, and 25°. The new peaks can be attributed to the celluloseII crystalline structure.47The changes in the crystallinestructure of cellulose during thedissolution and regeneration from DMSO-TEAOH were further indicatedin XRD measurements. Both BP and DiP exhibited typical cellulose Ipeaks around 17°, 19°, and 26° in XRD diffractograms4.47 A we49 Theincrease in DP was associated with radical formation from DMSO inthe presence of a strong base. In the case of TBAOH, the maximum radicalactivity was observed at a concentration of 20%, almost 2 times highercompared to the concentration of TEAOH in this study (11%). Therefore,it can be assumed that the presence of a radical in DMSO–TEAOHis small, and no radical-induced recombination of cellulose chainstook place. The small decrease in DP in the case of DMSO–TEAOHmight be due to the strong alkaline condition.50The slight decrease in the DP of both cellulosepulps was observedafter dissolution and regeneration using DMSO–TEAOH, and DPsof regenerated BP and DiP were 3100 and 1500, respectively. The decreasein DP contradicts the previous studies using DMSO with TBAOH, wherea minor increase in DP was reported.49 Although more studies regarding the dissolutionmechanism and dissolution limits  of cellulose in DMSO–TEAOH systems should be conducted,it is likely that dissolution is mostly due to the hydrogen bondingor deprotonation of cellulose with hydroxide ion resulting in thedissolution of cellulose in DMSO due to the electrostatic repulsionbetween cellulose chains. The cation in tetraalkylammonium hydroxidesystems is proposed to interact with carbon in position 1 in a cellulosering by either electrostatic or van der Waals forces.51 As stated previously, the radical formation in DMSO–TEAOHwas assumed to be minor and therefore does not take part in the dissolutionprocess to a large extent.The dissolution of cellulose in DMSO in the presenceof tetraalkylammoniumhydroxide is previously assumed to be the result of the combinationof the electron donor–acceptor complexation and radical reaction.Figure S1), similar to the CNFs produced from nonchemicallymodified cellulose. Based on the TEM imaging, RCNFs formed a weblikestructure, analogous to the more traditional CNFs . The weblike structureresults from the physical cross-linking and entanglement and is aresult of the long length and the lack of repulsion forces  between fibers. Because of the weblike structure, thelength of the RCNFs could not be measured. On the other hand, thewidths of nanofibers of RCNF1 and RCNF2 were 6.9 ± 4.9 and 5.9± 2.8 nm, respectively. The width values of the RCNFs were similarto those previously obtained from mechanical disintegration of chemicallymodified cellulose fibers,52 although CNFswith a diameter around half of the size of RCNFs can be produced byusing methods such as oxyl-mediatedoxidation.18 Previously, CNFs producedfrom mercerized cellulose fibers  were obtained with a width of 15–100 nm after mechanicaldisintegration.53 The reference CNFs producedfrom DiP by mechanical disintegration and microfluidization had anaverage nanofiber width 10.4 ± 5.4 nm .Both of theregenerated cellulose samples passed easily through a microfluidizerto form slightly gel-like and turbid suspensions S1, similFigure S5). Most of the RCNF2 fibers had a width around 5 nm. Minor amountsof large fiber bundles or aggregates and smaller few nanometer fiberswere observed. On the other hand, RCNF1 contained a large amount ofsmall (∼2 nm) fibers. In addition, there appeared to be morelarge (∼10–20 nm wide) fiber bundles in RCNF1 comparedwith RCNF2. It could be that the presence of hemicellulose, nondissolvedfibers, and cellulose DP has an impact on the fibrillation efficiencyof the regenerated cellulose. The hemicelluloses are known the helpin the nanofibrillation of natural cellulose fiber by creating stericand electrostatic repulsion between fibers, thus allowing the productionof few nanometer nanofibers.55 On the otherhand, nondissolved cellulose fibers can be more resistant to the mechanicaldisintegration compared to dissolved and regenerated cellulose particles,resulting in a higher amount of large aggregates in the case of RCNF1.Furthermore, cellulose with a longer molecular chain can form moretightly packed structure upon regeneration, thus requesting more energyto disintegrate into even-sized nanofibers.Although no statistical difference was observed between thewidthsof the two RCNF samples, it appears that regenerated cellulose fromDiP formed nanofibers with more narrow size distribution  fiberlike nanoparticlesafter mechanical disintegration of regenerated cellulose.23 or very short fiberlike particles,22 and it can be assumed that they do not form an entangled network,thus resulting in the formation of brittle film structure, similarto the CNCs.61 On the contrary, RCNFs appearedas long fiberlike materials forming weblike structure, and by useof the filtration method, both RCNFs formed self-standing films thatwere slightly opaque and easy to handle .One of the mainadvantages of cellulose-based nanofibers is that they can form strongfilmlike structure by using the water-based filtration method, similarto that used in papermaking, and could thus enable the continuouslarge-scale production of nanomaterial films. Previously, the regeneratedcellulose nanomaterials are obtained as sphericalo handle 6  (43 kN·m/kg),64 a well-known bioplastic. The mechanical propertiesof BP-derived RCNF1 film were higher compared to the RCNF2 film, likelydue to the higher DP of BP compared to DiP. The notable exceptionwas in the specific yield strength, as there was no static differencebetween two samples. A similar specific yield strength indicates thatboth RCNF films tolerate similar force before enduring permeated deformation;that is, the original form of the samples is not regained when forceis removed. After the yield point, the RCNF1 showed longer elongationand thus was subjected to more extended strain hardening, typicalto fiberlike cellulose nanomaterials.65 Therefore, the specific tensile strength of RCNF1 was higher thanthat of RCNF2.Good mechanical properties, such as tensile strength, are fundamentalfor cellulose nanomaterials films since they are expected to be exposedto various mechanical stress during their use . As stated above, both RCNF films were easy to handle,and they could be bent, similar to more common types of CNFs 6c. The s67 The specific tensile strength, modulus, yield strength,and strain of film of CNF produced for DiP by intensive mechanicaldisintegration  were significantly lowercompared to the CNF film (1.25 g/m3). Therefore, the tensilestrength and modulus of CNF film were higher compared to RCNF filmsand similar to those reported in the literature.Inthe literature, a wide range of mechanical properties of CNFfilms have been reported with specific tensile strength ranging fromaround 100 to over 200 kN·m/kg.68 Furthermore, cellulose films regenerated from N,N-dimethylacetamide–LiCl and aqueousLiOH–urea systems with tensile strengths of 170 MPa69 and 263 MPa,70 respectively,have been reported, being notably higher compared to RCNF films. Therefore,there is still room for improvement of the mechanical properties ofRCNF films. Nevertheless, fast room temperature dissolution, relativelyeasy washing steps, and simple film preparation using vacuum filtrationof RCNFs are advantageous compared to the many regeneration processesreported in the literature.Different regeneratedcellulose-based films with high mechanicalstrength have been reported. Commercially available cellophane hasbeen reported to have tensile strengths in the range 50–120MPa, being similar to those of RCNF films.36 and DMSO–ionic liquid systems have been used as a recyclablesolvent for cellulose.71 The simple evaporationof water from the washing system could facilitate the recycling ofthe DMSO–TEAOH system. However, as cellulose was first dispersedin pure DMSO, removal of TEAOH from washing liquor by using ion exchangecould be a suitable method for separation of two solvent components,which could then be used for further dissolution. The recycling aswell as the dissolution mechanism will be investigated in future studies.Although the mechanical propertiesof RCNF2 film were slightlylower compared to the CNF film, having same raw materials ,the production of CNF requests two types of mechanical treatment,that is, grinding and microfluidization. The grinding step was necessaryduring the production of CNF, as DiP could not be passed through themicrofluidizator. On the other hand, regenerated DiP was easily processedwith microfluidization after the fast room temperature dissolutionand regeneration process. Therefore, DMSO–TEAOH pretreatmentis a potential pretreatment method for cellulosic nanomaterial production.However, it should noted that more research is needed to optimizethe dissolution process as well as to calculate the energy consumptionof whole process  for proper comparison between different cellulosicnanomaterials. Especially the recycling of the solvent system is crucialas the large consumption of solvent chemicals is both economicallyand ecologically nonsustainable. TEAOH together with urea is demonstratedto the recyclable solvent for cellulose,72 The DiP exhibited a strong mass loss rate peakwith a maximum at 320 °C, whereas the maximum mass loss rateof BP was at 310 °C.Purecellulose has relatively good thermal properties, making itsuitable material for application requesting elevated temperature.However, chemical and physical alteration of the cellulose structurecan have a notable effect on the thermal behavior of cellulose. Therefore,the thermal properties of original cellulose and RCNFs were investigatedby TGA 8.In the73Thermal properties of RCNFs were found to be onlyslightly lowercompared to the original cellulose pulp. The onset temperature ofRCNF1 and RCNF2 was 271 and 280 °C, respectively, being around10–20 °C lower compared to their corresponding startingmaterials. Similarly, the maximum mass loss rate for both RCNFs wasslightly lower compared to their corresponding raw materials. Theonset temperature of CNF was slightly higher compared to RCNF2, sharingthe same raw materials. Overall, because of the good thermal stabilitytogether with high specific tensile properties, it can be assumedthat RCNFs could be used as a reinforcement agent for polymeric composites,which many times request a temperature around 200 °C during theprocessing.53 and CNCs74 obtained from cellulose II is more stable comparedto the corresponding nanomaterials from cellulose I. The higher thermalstability of cellulose II nanomaterials has been ascribed to the strongerhydrogen bonding. However, other factors, such as hemicellulose content,play a significant role in thermal stability as the alkaline mercerizationcan remove some of the more thermally labile components of cellulosepulp.53 Other factors such as DP and theamount of crystalline fractions can have an important role in thermaldegradation of cellulose. Previous studies reported that celluloseII from microcrystalline cellulose has a poorer thermal stabilitycompared to the original microcrystalline cellulose.75 Here, both RCNFs exhibited slightly lower thermal stabilitycompared to their corresponding raw materials. Both regenerated cellulosesamples exhibited slightly lower crystallinity compared to the originalpulp . Nevertheless, the CrIof CNFs was still somewhat higher compared to RCNFs, which might explainits slightly higher thermal stability. Despite the decreased thermalstability of RCNFs compared to original pulps, their thermal stabilitiesare higher compared to CNFs produced by using chemical pretreatments.77 For example, oxyl-mediatedoxidation, postesterification,78 and heat-inducedconversion of ionic bonds to amide bonds79 to produce CNFs have been used to improve thermal stability.The cellulose II crystallinestructure is described as the thermodynamicallymost stable cellulose polymorph, and there has been a report thatthe thermal stability of CNFsnalpulp 4b, andtIt was shown that cellulose pulps can be rapidly dissolved in DMSOby adding aqueous TEAOH. Only a minor decrease in DP of cellulosewas observed, and the regenerated cellulose was found to be suitablefor the production of RCNFs by using mild mechanical disintegration.Thus, the width (around a few nanometers) of the nanofibers producedwas in line with more traditional CNFs produced by mechanical disintegrationof chemically modified natural cellulose fibers. The RCNFs could beused to produce self-standing films with comparable mechanical propertiesand higher transparency than that of CNFs produced via intensive mechanicaldisintegration. Therefore, it was shown that mild, room temperaturedissolution and regeneration of cellulose fibers is an efficient pretreatmentmethod to produce a novel type of cellulosic nanomaterial with goodproperties.


The broad application of high-resolution chromosome detection technology in prenatal diagnosis has identified copy number loss (CNL) involving autosomal dominant (AD) genes in certain fetuses. Exon sequencing of fetuses exhibiting structural anomalies yields diagnostic information in up to 20% of cases. However, there is currently no relevant literature about the genetic origin and pregnancy outcome of CNL involving AD genes in fetuses without structural abnormalities.This was a prospective study involving pregnant women who underwent amniocentesis for fetal copy number variation sequencing (CNVseq). Detection of parent-of-origin was suggested in cases of samples with CNL involving AD genes and the pregnancy outcome was monitored. Amniotic fluid samples from 24,844 fetuses without structural abnormalities were successfully tested via CNVseq. The results showed that 134 fetuses (0.5%) had small CNL (< 10 Mb) containing AD genes, after excluding microdeletion and microduplication syndrome and polymorphisms. By monitoring the pregnancy outcomes of the 134 fetuses, we found that 104 (77.6%) were good, 13 (9.7%) were adverse, and 17 (12.7%) pregnant women voluntarily chose to terminate pregnancy. Of the 13 fetuses with adverse pregnancy outcomes, only 2 fetuses had phenotypes consistent with those of diseases caused by AD genes involved in CNL.The overall prognosis for fetuses without family history or structural abnormalities but with small CNL containing AD genes detected during pregnancy is good. The genetic origin, overlap status of established haploinsufficient gene and/or region, size of the CNL, and genetic mode may affect the pathogenicity of the CNL.The online version contains supplementary material available at 10.1186/s12864-022-08340-y. Prenatal ultrasound is an established screening tool in obstetrics that provides increasingly high resolution to identify fetal structural abnormalities in approximately 5% of pregnancies . The ideIn some prenatal diagnostic centers, up to 74.8 – 96.8% of the fetuses undergoing invasive prenatal diagnosis did not exhibit structural abnormalities before amniocentesis . CompareFor fetuses without ultrasound structural abnormalities, if high-resolution chromosome detection techniques indicate that there are CNL including AD genes in fetuses during pregnancy, what are their genetic origins and pregnancy outcomes? At present, such literature is lacking. This study sought to analyze the above contents to provide useful information for prenatal genetic counseling of mothers carrying fetuses with CNL.NIPA1 was the most common gene (25 samples), followed by CTNNA3 (10 samples). According to the genomic location of genes  had CNL involving AD genes (< 10 Mb) after excluding microdeletion and microduplication syndrome (MMS) and polymorphisms; the average size of the CNL was 1.11 Mb, CNL involved a total of 202 AD genes, covering 128 different AD genes. Among the 134 samples, 37 carried two or more AD genes and one contained seven different AD genes. Among the 128 different genes, 23 (18.0%) were detected in multiple samples; P = 0.000); the proportion of abnormal pregnancy outcomes was lower, but the difference was not statistically significant . In the three groups, the rate of voluntary TOP in the de novo group was much higher than that in the other two groups . These details are presented in Table Our follow-up on the pregnancy outcomes of the 134 fetuses found that 104 (77.6%) had good outcomes, 13 (9.7%) had adverse outcomes, and 17 12.7%) pregnant women voluntarily chose termination of pregnancy (TOP). Details of the fetuses with adverse pregnancy outcomes are summarized in Table .7% pregnhttps://omim.org/), we analyzed information regarding gene pathogenicity. Among the 128 different genes, 65 (50.8%) exhibited multiple genetic modes. A total of 202 AD genes were detected in 134 samples, which were divided into inherited, de novo, and unknown origin groups. Our results showed that, in the de novo group, the proportion of genes with multiple genetic modes was higher than that in the inherited group. The specific information is listed in Table Based on content in the Online Mendelian Inheritance in Man® database . The smaller the fragment, higher the proportion of normal pregnancy outcomes and lower the proportion of abnormal pregnancy outcomes, but there was no significant difference . The relevant information is shown in Table In the nearly 60 years since prenatal diagnosis of genetic diseases was first proposed, the field has made great progress. While invasive fetal sampling technology has improved, the technological progress of cytogenetics and molecular biology has expanded the scope of genetic disease diagnosis even further. Presently, chorionic villus sampling and amniocentesis can be used to diagnose the majority of diseases with known genetic causes; moreover, the genomes and exomes of abnormal fetuses can be sequenced to help identify potential genetic vulnerabilities –19.Theoretically, full gene, single-exon, or multi-exon deletion is often assumed to disrupt gene function by causing the complete absence of the gene product due to lack of transcription or nonsense-mediated decay of an altered transcript as identified by very strong evidence of pathogenicity in the ACMG/AMP guidelines . HoweverThe results of this study showed that in the de novo group, the proportion of samples that overlap of HI was the highest, average size of the CNL was the biggest, and proportion of fetal adverse pregnancy outcome was the highest. At the same time, larger the CNL fragment, the higher the proportion of adverse pregnancy outcomes. Whether CNL overlaps the HI gene or region, the number of protein coding genes involved in genomic variation and genetic origin are important components of the pathogenicity classification of CNV , 27. GenAt present, neither CNVseq nor CMA technology can determine the accurate breakpoint location of CNVs . Among 1The overall prognosis for fetuses without family history or structural abnormalities but with small CNL containing AD genes (< 10 Mb) detected during pregnancy, after excluding MMS and polymorphism, is good. Protocols that combine the CNL data of parents with the results of AD gene function tests are critical to inform medical consultation and decision rules.This study involved pregnant women who underwent amniocentesis for fetal CNVseq during their second or third trimester due to reasons, such as advanced maternal age, increased risk of a screening test, ultrasound soft markers, or maternal request at West China Second University Hospital of Sichuan University from February 2017 to June 2020. Fetuses with structural abnormalities, based on the ultrasonogram before amniocentesis and family history of a genetic condition, were excluded.Based on routine collection procedures, 20 mL amniotic fluid was extracted and separated into 4 sterile centrifuge tubes containing 4 mL, 4 mL, 6 mL, and 6 mL, respectively. CNVseq was performed using the 6-mL amniotic fluid samples and quantitative fluorescence PCR (QF-PCR) was performed using one of the 4-mL amniotic fluid samples. The other two tubes of amniotic fluid were stored in a 2–8 ℃ refrigerator. According to the manufacturer's instructions, DNA was extracted from amniotic fluid samples using a DNeasy Blood and Tissue Kit . QF-PCR was performed using 21 trisomy/sex chromosome/polyploidy and 18 trisomy/13 trisomy/polyploidy detection kits . When QF-PCR results indicated the presence of maternal cells in the samples, CNVseq and QF-PCR were repeated on the spare samples after cell culture.DNA libraries were prepared using a Chromosome CNV Detection kit  and subsequently sequenced on the Illumina NextSeq500 sequencing platform using a NextSeq500 High Output kit  according to the manufacturer's instructions. We compared the reads obtained by next generation sequencing with the GRCh37 reference genome and performed bioinformatics analysis to obtain the genomic copy number information of the samples as described previously . In thisWhen CNL involving AD genes was identified in the amniotic fluid sample, we recommended that the biological parents of the fetus undergo CNVseq to determine the origin of the fetal CNL; 2 mL of peripheral blood was collected from each parent and anticoagulated with EDTA. DNA extraction and CNVseq methods were performed as described for the amniotic fluid samples.One year after amniocentesis, the researchers contacted the mother or father of the fetus for follow-up. The information discussed during the inquiry included fetal ultrasound results; pregnancy complications; pregnancy loss; TOP and the causes; date and mode of delivery; weight and length of the newborn; the Apgar score; physical appearance of the newborn ; feeding conditions after birth; and examination results of pediatric outpatient services. The flowchart of the study is shown in Fig. Additional file 1. Supplementary Table: The genomic positions, occurrences, genetic origins, and genetic modes of all 128genes.


It was determined that six out of eleven traits (dry leaf weight (DLW), canopy temperature (CT), relative water content (RWC), flag leaf area (FLA), green leaves area (GLA) and leaf area index (LAI)) loaded the highest onto PC1 and PC2 (with scores of >0.27), and five of them had a positive trend with GY, while the CT trait had a negative correlation determined by principal component analysis (PCA). Genetic parameters and multidimensional analyses  showed that CT, RWC, GLA, and LAI were the most important interpretive traits for GY. Selection based on these four interpretive traits might improve genetic gain for GY in environments that are vulnerable to water shortages. The membership index and clustering analysis based on these four traits were significantly correlated, with some deviation, and classified genotypes into five groups. Highly tolerant, tolerant, intermediate, sensitive and highly sensitive clusters represented six, eight, two, three and six genotypes, respectively. The conclusions drawn from the membership index and clustering analysis, signifying that there were clear separations between the water shortage tolerance groups, were confirmed through discriminant analysis. MANOVA indicated that there were considerable variations between the five water shortage tolerance groups. The tolerated genotypes  can be recommended as interesting new genetic sources for water shortage-tolerant wheat breeding programs.Water shortages have direct adverse effects on wheat productivity and growth worldwide, vertically and horizontally. Productivity may be promoted using water shortage-tolerant wheat genotypes. High-throughput tools have supported plant breeders in increasing the rate of stability of the genetic gain of interpretive traits for wheat productivity through multidimensional technical methods. We used 27 agrophysiological interpretive traits for grain yield (GY) of 25 bread wheat genotypes under water shortage stress conditions for two seasons. Genetic parameters and multidimensional analyses were used to identify genetic and phenotypic variations of the wheat genotypes used, combining these strategies effectively to achieve a balance. Considerable high genotypic variations were observed for 27 traits. Eleven interpretive traits related to GY had combined high heritability ( Water shortages and changes in the world′s climate will lead to an increase in the occurrence of drought phenomena in arid and semiarid areas ,2,3, andPlant breeding has proven successful in sustained improvement of drought tolerance in rainfed crops and dry-land crops throughout the years. There are three approaches that might be taken to improve yields with water shortage. The first approach is breeding segregation for yield under water shortage , assuming that this will provide a yield advantage under water shortage conditions. The positive correlation between yield under water shortage and optimal conditions ,8,9, andAchieving complementarity between physiological and genetic information can provide a more comprehensive genotype model of environment interaction . The int2-spread pathway via the stomata [2 may considerably affect genotype performance, and its responses differ from one plant to another, largely because of the differences of genotypic influences on stomatal  and nonstomatal  limits of net CO2 assimilation rate [Water shortage increases in plants due to potential losses by evapotranspiration (the aridity index) by preventing the flow of water from soil. Water phase-out from a week to months occurs, depending on the soil characteristics in which plants are cultivated under natural conditions . Water s stomata , or an i stomata  and rais stomata . It was ion rate .It was found that water status measurements are useful indicators of tolerance to water shortage stress in plants ,20,30, aThe inaccurate depiction of interpretive  traits related to water shortage stress, and low genetic variation of available wheat varieties, is currently one of the leading causes for limited success when breeding drought-tolerant wheat varieties ,36. BreeThe principal aim of this study was to describe the characteristics of double haploid wheat lines compared to numerous cultivars in response to water shortage stress, and to identify interpretive traits associated with water shortage tolerance based on their heritability, genetic gain and multidimensional evaluation. Another aim was to identify genotypes with higher water shortage tolerance that can be utilized to breed water shortage-tolerant varieties.−2, and the fertilizing rates used were 1.2 kg m−2 N and 0.8 kg m−2 P2O5. During the growing season, the temperature and rain were 12.9–32.2 °C, and 8.0–25.0 mm, respectively. The soil texture was classified by electrical conductivity as sandy loam (2.89 dS m−1). Two irrigation regimes were used two weeks after sowing. The first regime (full irrigation) was watered to 100% field capacity upon reaching a cumulative evaporation of 50 mm, and the second regime (limited irrigation) was watered to 33% field capacity upon reaching a cumulative evaporation of 150 mm. The main plots were allocated to two irrigation regimes, while the subplots were allocated to the genotypes.A set of 25 genotypes (seven varieties and 18 lines) of bread wheat were used in this study . The varLeaf water status was measured by canopy temperature (CT), leaf water content (LWC), relative water content (RWC) and leaf equivalent water thickness (LEWT) at the flowering stage on the flag leaf. CT was measured under a cloudless sky before noon (11:30 to 12:00 h) using an infrared thermometer . The images were captured at a height of approximately 1 m over the top of the ear and converted into CT using the FLIR Quick Report 1.2 software package. LWC and RWC were measured for five leaves collected randomly from each genotype, and then weighed directly to capture data related to fresh weight (FW). The same leaves were flooded with water at 25 °C until they were fully turgid, then dehydrated with tissue paper to remove any excess water, and weighed to capture data on turgid weight (TW). Finally, the same leaves were oven-dried for 48 h at 70 °C and weighed to capture data on dry weight (DW). Data taken from FW, TW and DW were used to calculate LWC, RW, and LEWT, according to the following equations ,41,42:(12 concentration (Ci), and atmospheric CO2 concentration (Ca) were measured at a saturating photosynthetic photon flux density of 700 μmol photons m−2 s−1. Temperature, CO2 concentration, and relative humidity were measured at 26 ± 2 °C, 485 ± 23 μmol/L and 65 ± 7%, respectively. Instantaneous water use efficiency (WUE), intrinsic water use efficiency (WUEi), and stomatal limitation value (Ls) were calculated using data taken from Pn, E, Gs, Ci and Ca, according to the following equations [Photosynthetic parameters were measured in the grain-filling stage using a Li-6400 gas exchange system , starting at 10:00 AM until 12 noon. The upper third of the flag leaves was used. Photosynthesis rate (Pn), transpiration rate (E), stomatal conductance (Gs), intracellular COquations :(4)WUE =Five plants from the middle rows of each pilot module were randomly selected to calculate green leaf number (GLN), flag leaf area (FLA), and green leaf area (GLA) at the mid anthesis stage. All green leaves were removed from the five plants and run through an area meter  to calculate surface GLA. Thereafter, all parts of the five plants  were oven-dried at 70 °C for 72 h to a constant weight and then weighed to obtain dry stem weight (DSW), dry leaf weight (DLW), and total dry weight (TDW). Data taken from GLA (A), number of plants in 1 m (B), number of plants used , and distance between rows  were used to calculate leaf area index (LAI), according to this equation [−1) and spike length  were measured in 10 plants chosen randomly from each plot after excluding awns and upon reaching maturity. The number of spikes , and number of spikelets  were counted at the same time. The plants were threshed to measure the number of kernels , thousand-kernel weight , and grain yield . Three rows (2 m long) were used to estimate GY.The middle rows were used to lower the environmental impact in each plot for measuring agronomic and yield traits. Days to heading  were recorded when 50% of plants had headed and flowered, and days to maturity  were recorded when the tops of the peduncles of 50% of plants were yellow. Grain filling duration  was calculated during the period from DM and DH. Plant height  for each individual season with full and limited irrigation. A combined analysis was performed across the two seasons to test the homogeneity of error variance assuming season is a random effect, and genotypes and irrigation regimes are both fixed effects. The results indicated homogenous variance across two seasons for all measured traits, and based on this test the data of the two seasons were combined and analyzed according to Gomez and Gomez . Subsequith genotype, jth trait, and i > 0.8), tolerant , moderately tolerant , sensitive , and highly sensitive .The multicollinearity test was used to uncover multicollinearity sources in a correlation matrix of interpretive traits to their exclusion. Multidimensional modeling was used to better understand correlations between a large number of measured traits, their Path coefficient analysis to partition variation into multiple direct and indirect effects, depending on SMLR output and path coefficient analysis and a membership index, was used to characterize the drought tolerance index (DTI) values of all tested genotype contributions and their encapsulation into major components. Principal component analysis (PCA) was performed, based on data provided by the correlation matrix, to reduce the dimensions of the data space and reliance on the first two components of PCA. Stepwise multiple linear regression analysis (SMLRA) was used to determine the most powerful interpretive traits that contribute to the most variability in the intrigued variable (GY), depending on the output of PCA. The DTI values of the genotypes were calculated using four influential interpretive traits , and the membership index was used in the computation . The memThe membership index value (score) used in the calculation of the genetic similarity matrix  between genotypes from three interpretive traits was based on the Euclidean distance dissimilarity coefficient using UPGMA (Unweighted Pair Group Method with Arithmetic Mean). The matrix of Euclidean distance dissimilarity coefficient was used in the calculation of the principal coordinate analysis (PCoA) to reduce the dimensions of data space. Discriminant function analysis (DFA) and MANOVA used the same data as the DTI score per genotype  in order to confirm the categorization of genotypes. All genotypes were then given an equal prior probability to be classified into the five classes of drought tolerance. Statistical analysis  was carried out using the XLSTAT statistical software package .p < 0.01) for treatments (irrigation levels and genotypes) and their interaction in each season, based on ANOVA. The only exception was interaction of DH in S1, irrigation levels of NKS in S2, and levels of irrigation of Pn in S1 and S2, being nonsignificant , genotypic (GCV), and phenotypic coefficient of variability (PCV), genetic advance (GA), and genetic gain (GG)) varied greatly for all measured traits. The h2 demonstrated a wide range between measured traits, which varied from 29.99% for WUEi to 97.82% for DH. The ratio of PCV to GCV was proximate for some traits and the GCV was smaller than the PCV, except for ten traits , which showed a greater percentage of PCV to GCV. Genetic advance (GA) and genetic gain (GG) showed high diversity for all measured traits  and variance inflation factors (VIF) ranged from 0.00 to 0.465 and from 0.00 to 10.00, respectively, except for three traits , which showed VIF values >10.0, before excluding traits . After e2 of SMLR model was 0.886. The five components of GY variation partitioned into multiple direct and indirect effects using path coefficient analysis. Each direct and indirect effect contributed to 0.565  and 0.321, respectively. The R2 value was the same as that in the SMLR model (0.886), with a noise value of 0.338. It was concluded that these traits  may be relevant and important selection criteria and can be relied on in defining the levels of drought-tolerant and drought-sensitive genotypes of wheat, coupled with high correlation with yield.The relationships between all traits suggested to be very important by PC1 and PC2 were analyzed using SMLR and path coefficient analysis in order to understand the best-measured and yield-related traits and their contribution to yield performance . The resi > 0.8), and included eight genotypes . The second group was classified as tolerant , and included six genotypes . The third group was classified as moderately tolerant , and contained four genotypes . The fourth group was classified as sensitive , and contained seven genotypes . In each growing season (S1 and S2) separately, 20 out of 25 genotypes were similarly categorized from their combined scores. Some genotypes in the fourth group were classified as highly sensitive  with some traits and/or season combinations such as DHL21 with LAI in S1, DHL14 with LAI in S2 and DHL06 with CT in S2 (fifth group).The four traits  were used in classification of tested genotypes after the Gs trait was removed owing its low-level heritability and genetic gain. We used these four traits in finding a membership index for the phenotypic profiling of 25 wheat genotypes to assess the extent of their drought-tolerance. The membership index was obtained by scores calculated from these four traits into five main clusters . RegardlWe decided to use the membership index scores of the four traits  to create a cluster analysis for the phenotypic profiling for drought tolerance in the wheat genotypes. The clustering was the genetics dissimilarity matrix based on Euclidean distance using Ward’s method of agglomeration. The genotypes produced five major clusters, clearly separated with a dissimilarity coefficient of 0.599 and reflected the distance between the five clusters based on the tolerance or sensitivity of wheat genotypes for drought. The highly tolerant, tolerant, intermediate, sensitive and highly sensitive clusters represented six, eight, two, three and six genotypes, respectively .p < 0.0001) according to the Mantel test. The first three PCoAs  had eigenvalues greater than 1, which explained 88.46% of the total variation . PCoA resulted in a clear separation between genotypes groups based on tolerance or sensitivity for drought. S and HS groups were distributed into quadrants 1 and 2, respectively, but the I group was distributed in both quadrants , so we were prompted to use quadratic discriminant analysis (QDA). Its results had a 0.00% error rate, confirming that the classification of our genotypes using clustering based on membership index was an influential analysis. Discriminant analysis was used to better understand the grouping and evaluate the extent of the differences between drought groups. The four selected measures  were high and significant with each statistical multivariate analysis used, thus confirming the odds of prediction by clustering based on membership. The discriminant functions  of five groups and four selected measures were closely associated for the prediction of membership into drought groupings for the 25 genotypes used  operates in a manner similar to MANOVA, which initially computes the Mahalanobis distance of each genotype to a group and then uses this distance to categorize a genotype into the group with the smallest generalized squared distance . The hompes used . The firpes used .Loading the four variables in S1 and S2 to canonical discriminant functions showed that GLA, LAI, RWC and CT were positive and highly correlated to Can1 . In addiMultivariate analysis of variance (MANOVA) indicated that the five groups were significantly different for four traits in S1 and S2 . This alPlant breeders rely on several interpretive traits as screening criteria to evaluate the water shortage tolerance of genotypes and increasing access to sustainable genotypes appropriate for water shortage ,49. Intep < 0.01) between treatments and their interactions  and genetic gain (>20%), coupled with their approximate values for GCV and PCV (h2 (<60.0%), genetic gain (>20.0%), and GCV for the trait, evidenced that the variation between genotypes was largely due to the additive genetic part. The selection process had a large confidence interval with these traits [There were eleven  traits with high heritability  of 0.886. Many investigators have used multivariate analyses [Multivariate analyses (SMLR and path coefficient) are effective instruments for understanding the relationship between interpretive traits for yield ,61. It hanalyses ,64. Baseanalyses . The sepCT and RWC are useful indicators of water status in plants ,53, and 2 fixation [RWC is a robust mechanism to preserve cellular hydration by osmotic adjustment as a barometer of plant water status. Trials can be rapidly performed to identify genotypes that preserve high leaf RWC values during water deficit stress. Tolerant genotypes have the potential to minimize stress by keeping leaves turgid under water deficit stress through their possession of certain physiological advantages, which protect and preserve the photosystem complex, plant growth and stomatal activity . The lowfixation .p < 0.0001, alpha = 0.05).The clustering analysis results, based on a dissimilarity matrix and Euclidean distance using Ward’s method of agglomeration, showed that all genotypes were assigned to five groups (clusters) based on phenotypic profiling for drought tolerance . The fivFurthermore, PCoA revealed compatible relationships with cluster analysis of these genotypes, which showed that the genotypes’ stability in their classification groups . This ish2 > 60%) and genetic gain (>20%), compared to GY, which had moderate values of both heritability (57.60%) and genetic gain (16.89%). Selection of interpretive traits could be a substitute for tools for indirect selection of GY under water shortage stress conditions for these reasons. Multidimensional analyses determined four effective interpretive traits  proposed as comprehensive criteria for selecting water shortage-tolerant genotypes, and selection based on them might improve genetic gain for GY in environments that are vulnerable to water shortages. Discriminant analysis confirmed the results obtained. There were clear separations between water shortage tolerance groups. MANOVA indicated that there was considerable variation between the five water shortage tolerance groups. The six genotypes  can be recommended as interesting new genetic sources for water shortage-tolerant wheat breeding programs.Eleven interpretive traits to yield combined high heritability (


Most of the historical and old building stock in Europe are constructed from masonry, when brick, stones, or their combination are bound with traditional mortars. Rising damp, due to accompanying effects, is the main factor influencing the quality of indoor climate as well as having an important impact on the durability of masonry structures. In this study, new types of lightweight concrete with waste aggregate content as a suitable material for remediation of damp damaged masonries were designed and tested. Alternative aggregate served as silica sand substitution in the range of 0–100 vol.%. Basic structural properties, mechanical resistance, water, and water vapor transport properties were measured after 28 days of water curing and were compared with dense reference concrete and with traditional masonry materials as well. Moreover, the porous structure of produced concretes and changes caused by usage of alternative aggregate usage were evaluated with the mercury intrusion porosimetry (MIP) technique. Obtained experimental data showed the suitability of modified concretes with 25–50 vol.% of waste aggregate content to ensure acceptable strength and hydric properties, and these properties were found to be comparable with masonry structures and materials used in the past. Most of the building stock in Europe was being built during the 20th century as a consequence of recovery efforts after the Second World War . NowadayThis study focuses on the rising damp that constitutes a still-discussed phenomenon causing the deterioration of indoor and outdoor appearances of historical as well as modern buildings built using traditional porous materials. This effect is closely connected with the presence and condition of horizontal water-proof insulations that can be absent or damaged . Thus, dSo, the initial damages have to be repaired and rehabilitated as soon as possible to prevent other decay of structures caused by moisture . In caseOn the other hand, the long-term MPC resistance against water and moisture effect is questionable or not easily accessible ,18. TorrA considerable amount of old masonry-based buildings or historical monuments suffer from moisture problems, which can lead to further serious deterioration of the masonry’s state and durability. This paper is focused on the design and testing of lightweight concretes with alternative waste aggregate content and disposing of advanced liquid water and water vapor transport properties to be used for remediation of damp induced damaged buildings structures where masonry elements or even masonry parts are absent. Experimentally obtained data indicated a high rate of lightening of produced composites accompanied by growing porosity caused by the increasing amount of incorporated silica sand alternative. However, high porosity results in mechanical properties reduction. It was concluded that the optimized amount of LA ranged in the interval of 25–50 vol.%, when sufficient mechanical strength and improved hygric properties, comparable with traditional masonry materials , were ensured.Ordinary Portland cement (OPC), meeting the strength class of 42.5 according to the standard EN 197-1 , manufacFive mixes, indicated in Input raw materials were homogenized in the laboratory mixing device E095  according to the procedure specified in the standard EN 196-1 . Fresh mcap) was calculated. Finally, the apparent moisture diffusivity (Χapp) was obtained as the relationship between the absorption coefficient and capillary saturated water content, as was reported by Luikov [−1·s−1·Pa−1), water vapor diffusion coefficient D (m2·s−1) and water vapor resistance factor µ (-) were determined with the use of the cup method formulated in the standard EN ISO 12572 [3 that provided a high relative humidity of 93% [Comprehensive material specification of both natural and waste aggregate was performed at the first stage of experimental investigation. The bulk density of aggregates in both loose and compacted states was determined using the method described in the standard EN 1097-3 . The comy Luikov . Tests wSO 12572 . Disk-shy of 93% . Both arThe material characteristics of natural aggregate, as well as lightweight replacing alternative, are summarized in 50) and 0.13 mm (d90). Only 10% of particles by weight are lightly finer in the case of LA. Differences in particle size distributions of particular materials are prescribed to the blended composition of waste aggregate with the predominance occurrence of plastic and paper-based components. The grinding of plastics can be a relatively complicated process in obtaining fine graininess because of their low melting point in dependence of plastic type [The particle size distribution of both aggregate types is presented in tic type .−3 (C-R), through 1720 kg·m−3 (C-LA 25), 1420 kg·m−3 (C-LA 50), and 1160 kg·m−3 (C-LA 75), up to 760 kg·m−3 (C-LA 100) were mitigated. From the indicated trend, one can note that all concretes with silica sand substitutions with LA can be included in the lightweight composites class. Similar observations were concluded in the study performed by Allahverdi et al. [−3 up to approx. 1250 kg·m−3 attributed to materials with 45% of EPS. The graphical illustration of open porosity development for different composites mixes is presented in 2~0.99). In this context, open porosity of 19.1% was measured for the control material. For other samples with partial or full substitution of NA, porosity values were detected to increase with respect to C-R by: 10.6% (C-LA 25), 20.2% (C-LA 50), 28.7% (C-LA 75), and 42.5% (C-LA 100). It was reported that the porosity of historical bricks from the 19th century had open porosity of about 35% [Hardened concretes were, after 28 days of the curing period, subjected to structural properties determination. The dry bulk density development in dependence on lightweight aggregate content measured on produced composites is depictured in i et al. . Expandebout 35% , and bribout 35% ,48. In tbout 35% ,50. Commbout 35% . In this−3 (C-R) up to almost 1400 kg·m−3 (C-LA 50). Overall, the huge reduction of dynamic moduli from approx. 35 GPa recorded for C-R to 2 GPa recorded for C-LA 100 was observed and such behavior clearly corresponds to the obtained compressive strength data mentioned above.Strength properties obtained for reference samples and concretes lightened with silica sand replacing aggregate are summarized in cr) in dependence on waste aggregate content in mixtures is depictured in cr is determined as the first peak of local maximum in incremental pore volume curves . As is clearly visible in cr show a gradually increasing exponential trend (regression coefficient of ~0.99), with values in the range of approx. 0.1 µm up to 11.4 µm. Thus, compared to the reference sample, concretes with high content of applied waste aggregate represent much more permeable material for water vapor. In addition, these composites allow more efficient transport of liquid water.The mercury intrusion porosimetry (MIP) data measured on control samples and concretes enriched with waste aggregate are presented in −2·s−1/2 [One-dimensional liquid water transport properties with dependence on LA content in hardened concretes are summarized in −2·s−1/2 ,63,64, wapp) was recorded. In comparison with control material, determined values are higher by 16% (C-LA 25), 37% (C-LA 50), 68% (C-LA 75), and 80% (C-LA 100). Compared with absorption coefficient values, percentage increments of apparent moisture diffusivities showed a growing trend as well. However, it is not so sharp. The reason for this may be found in the larger moving rate of the wet front, and thus capillary saturated water content (wcap) was achieved faster with increasing LA content in concretes. Such behavior could be ascribed to the higher abundance of macropores, and larger capillaries in the structure of waste aggregate modified samples [cr) analysis as described in the previous text. LA adjusted concretes with increased liquid water transport ability could be successfully applied in view of, e.g., damp suffering historical/modern masonry structures as a filling or supplementing material that allows effective moisture wicking.Likewise, the increase of apparent moisture diffusivity  can be considered.This study investigated the potential application of lightweight concrete with varying waste aggregate content as a suitable material for remediation of damp-induced and damaged masonries. At the first stage, the physical properties of raw materials intended for concretes preparation were determined and assessed. Further, structural properties, mechanical resistance, the transport of liquid water, and water vapor transmission properties were measured on hardened water cured concretes, and obtained results were compared with traditional dense concrete basically applied in remediation interventions. Moreover, the changes in the porous structure of composites caused by waste aggregate usage with the help of MIP were observed. The comprehensive investigation pointed out that the effect of alternative silica sand replacing aggregate on aforementioned properties of manufactured concretes, and several conclusions can be deduced, such as:Tested lightweight concretes are proven to be a possible alternative to traditional and historical masonry materials. Thus, composites with the waste aggregate content of 25–50 vol.% are able to ensure sufficient strength properties and comparable hydrothermal behavior like building materials used in the past. Furthermore, composites with natural aggregate substitutions above 50 vol.% with applied waste aggregate showed importantly reduced weight, and this is beneficial for their possible applications for non-bearing structures. Moreover, very effective transports of liquid water and water vapor were revealed, and thus these composites may find usage in remediation interventions of damp suffering old and/or historical buildings.