Short read sequencing has been used extensively to decipher the genome diversity of human cytomegalovirus (HCMV) strains, but falls short to reveal individual genomes in mixed HCMV strain populations. Novel third-generation sequencing platforms offer an extended read length and promise to resolve how distant polymorphic sites along individual genomes are linked. In the present study, we established a long amplicon PacBio sequencing workflow to identify the absolute and relative quantities of unique HCMV haplotypes spanning over multiple hypervariable sites in mixtures. Initial validation of this approach was performed with defined HCMV DNA templates derived from cell-culture enriched viruses and was further tested for its suitability on patient samples carrying mixed HCMV infections.4 copies/ml. PCR products of up to 7.7 kb and a GC content < 55% were efficiently generated when DNA was directly isolated from patient samples. In a single sample, up to three distinct haplotypes were identified showing varying relative frequencies. Alignments of distinct haplotype sequences within patient samples showed uneven distribution of sequence diversity, interspersed by long identical stretches. Moreover, diversity estimation at single polymorphic regions as assessed by short amplicon sequencing may markedly underestimate the overall diversity of mixed haplotype populations.Total substitution and indel error rate of mapped reads ranged from 0.17 to 0.43% depending on the stringency of quality trimming. Artificial HCMV DNA mixtures were correctly determined down to 1% abundance of the minor DNA source when the total HCMV DNA input was 4 × 10Quantitative haplotype determination by long amplicon sequencing provides a novel approach for HCMV strain characterisation in mixed infected samples which can be scaled up to cover the majority of the genome by multi-amplicon panels. This will substantially improve our understanding of intra-host HCMV strain diversity and its dynamic behaviour.The online version contains supplementary material available at 10.1186/s12864-021-08272-z. It is well-known that human cytomegalovirus (HCMV) seropositive persons can be infected with more than one HCMV strain, either synchronously or sequentially –6. SolidThe genome sequence of an individual HCMV strain is characterised by a unique combination of linked sequence patterns of all polymorphic regions along the whole genome . IndividDue to short read lengths of second generation sequencing techniques, determination of individual genomes within mixtures can be challenging. It requires model-based analysis to reconstruct unique haplotypes from short sequencing reads , 25, 26 In contrast to short read sequencing platforms, single molecule real-time (SMRT) sequencing from Pacific Biosciences (PacBio) and MinION Nanopore sequencing from Oxford Nanopore Technologies exceed with their ability to perform novel genome analyses, due to their extended read length , 29. UnlStill, there is limited data on the number of unique HCMV DNA molecules within mixed HCMV populations, which in the following will be termed HCMV haplotypes. It is still unknown how specific combinations of polymorphic regions present on an individual HCMV DNA molecule affect pathogenicity or replication efficiency.In the present study we assessed how enrichment of HCMV DNA by long range PCR and long read sequencing enables the identification of linkages between non-adjacent polymorphic regions present on single HCMV DNA molecules. The newly developed protocol was successfully applied to a selection of bronchoalveolar lavage samples (BALs) displaying mixed HCMV infections. This approach provides novel insights into the composition of individual haplotypes within a mixed HCMV strain population.First, a long range PCR approach was established by targeting two genomic regions, spanning from UL55 to UL76 (30 kb), and from UL139 to UL146 (6.7 kb), respectively Fig. .Fig. 1ScE.coli. As shown in Table To find the best conditions for sensitive and specific long range amplification, three different commercially available PCR enzymes were compared . AmplifiFor further optimization 10-fold dilution series of human foreskin fibroblast (HFF)-derived HCMV DNA were used, as this source of HCMV DNA in the background of an excess of genomic DNA is comparable in composition and integrity to DNA derived from clinical samples. The highest sensitivity was obtained for the shortest amplicons, F3 and F4  were sequenced by PacBio and error rates introduced by PCR and sequencing were estimated and compared to Illumina sequencing. Total DNA was directly isolated from HFFs infected with HCMV strain TB40-BAC4-luc or Merlin and used either as PCR template (amplicons) or directly without any further enrichment (non-enriched) before sequencing. After demultiplexing, circular consensus sequencing reads (ccs) for each forward and reverse strand were generated to prevent incorrect analysis due to heteroduplexes. Resulting ccs reads per sample showed an average length of 3583 to 6162 bases Table  and PacBThe error rate was estimated from mapped reads either quality trimmed with pError of 0.01 or 0.001. A comparison between non-enriched and amplicon-enriched DNA allowed us to specifically determine the error rates introduced by long range PCR. Substitutions, deletions, and insertions relative to the reference sequence were counted and the percentage of mismatches out of the total number of matched bases was calculated. As shown in Table Illumina sequencing shows a very low error rate for insertions (0.001%) and deletions (0.004%) whereas substitutions were 2.0-fold higher compared to PacBio sequencing Table  which coFinally, a detailed analysis of substitution errors revealed that A and T substitutions are 2.0-fold more frequently found than G and C substitutions Table . This wa6 to 1 × 107 copies per ml. Amplicons were subjected to Illumina sequencing. Quality trimmed and filtered reads were mapped against a total of 18 unique genotype reference sequences which comprise highly polymorphic regions within gN, gO, and gH  and F4 region (UL146) were generated, pooled and sequenced on an Illumina MiSeq instrument  was used as template. Irrespective of the average GC content target regions which comprise a GC content of 72% over a 1 kb segment  were especially difficult to amplify. Hence, for GC rich regions alternative target enrichment techniques such as solution-based capture , 32, 33 In addition to genome specific limitations, successful amplification of long range amplicons depends on the integrity of input DNA. The extent of DNA fragmentation when directly isolated from patient samples may vary between different sample types and different storage conditions . In thisTaking these findings together it appears that fragment lengths up to 7 kb and an average GC content of less than 55% are best suited to generate long range amplicons from clinical samples.Both, PCR target enrichment and sequencing are prone to false introduction of substitutions and indels . MoreoveWhen we applied the long read sequencing protocol to determine haplotypes in patient samples we chose a PHRED score of ≥20 for quality trimming. This approach ensures that a higher number of long reads is retained at the expense of a higher indel error rate. Further removal of short reads guarantees that only long reads spanning over multiple non-adjacent polymorphic sites are used for haplotype determination. Depending on the distribution of diversity along the target sites it will be necessary to adapt these analyses parameters accordingly. Alternatively, filtering of ccs reads using a cutoff for the number of full passes and a predicted accuracy will avoid read length reduction, but this filtering strategy requires a sufficiently high number of full passes.Other artifacts known to be generated by PCR of highly homologous sequences are chimeric and heteroduplex sequences. In samples with multiple HCMV strains, we cannot fully rule out the possibility of chimera formation which might artificially increase the observed diversity in a mixed sample. We used manual inspection of final haplotypes to rule out that chimeric sequences are mistaken as additional haplotypes Fig. . ChimeraThe formation of heteroduplexes is another concern in amplicon sequencing. DNA variants derived from closely related templates especially have a higher chance to form heteroduplexes during PCR amplification, which makes our application prone to such error. To prevent this false interpretation in our data, we decided to split our double stranded subreads into individual forward and reverse reads.It is critical that each haplotype sequence within a mixture is equally amplified by PCR in order to accurately determine and quantify all haplotypes. For validation, a set of artificial mixtures of two related HCMV DNAs were sequenced. Our data show that the mixed ratios can precisely be determined. Even low abundance haplotypes of 1% were correctly detected down to about 20 copies of the minor haplotype sequence. This finding correlates well with the before mentioned sensitivity limit of about 30 copies per reaction and underlines that both, the total HCMV DNA load and the sensitivity limit of the PCR are critical for detection of minor haplotypes in mixtures, given that the subsequent sequencing step was performed to an appropriate read depth. Since our protocol was validated solely for two haplotypes in mixtures it cannot be excluded that distinct haplotype mixtures or mixtures with more than two haplotypes may influence the PCR performance. All primer pairs, however, were designed to bind to highly conserved regions of the HCMV genome, thus it can be assumed that distinct haplotype sequences are similarly well amplified.Interestingly, BAL samples which have the same number of haplotypes for regions F3 and F4, displayed almost identical frequencies of occurrence of individual haplotypes for both target sites. It is tempting to speculate that these individual haplotypes are found on the same HCMV DNA molecule within the HCMV population. Such observations may be verified in future studies through a long read based genome sequencing approach with overlapping long amplicons. HCMV displays several diversity hot spots spread across the genome with 30/170 genes having a dN (nonsynonymous substitutions per nonsynonymous site) value of > 0.025 . Our two4 copies/ml) necessary for long range amplification. We have tested BAL samples for this study, however our lab has successfully used plasma samples as input material as well (data not shown). In clinical samples with a minimum viral load of 104 copies/ml, minor haplotypes of 20% can be detected with the determined sensitivity limit of 20 copies. Since the initial PacBio requirement of a minimum of 700 ng DNA input for library preparation has been reduced to 100 ng, the use of clinical samples with restricted material availability has become even more feasible. Additionally, using multiplex PCRs for long range amplicon generation, patient samples can be used more efficiently. This approach was already tested within our lab and promising results multiplexing F3 and F4 have been achieved.Mixed strain infections in transplant recipients are common and have been associated with higher viral loads, delayed viral clearance and poorer clinical outcomes . PotentiIn the present study, the newly developed long read PacBio sequencing approach was applied to four BAL samples that carry multiple genotypes as determined by short amplicon sequencing of gN, gO (located in the F3 target region) and UL146 (located in the F4 target site). Actually, also multiple haplotype sequences were identified by long amplicon sequencing. Comparison of sequencing data from both approaches clearly demonstrates limitations as well as main advantages of long amplicon sequencing. First, our findings indicate a potentially higher detection rate of low abundance populations by short amplicon compared to long amplicon sequencing as observed in two samples. Since short range PCRs usually show a higher sensitivity and are less prone to fragmented DNA found in patient samples, low abundance templates are easier to amplify. This makes this genotyping approach particularly suitable for initial screening of mixed infections. Second, short amplicon genotyping may strongly underestimate the intra-host diversity.Our data show that individual haplotypes can carry the same genotype sequences across the respective target region but are otherwise substantially diverse. Moreover, SNVs of the same genotype which can be detected by short amplicon sequencing will not represent the overall haplotype diversity. Third, intra-host haplotype sequences show stretches of identity interspersed by diverse sections. This observation underlines the usefulness of long sequencing reads to identify co-linearity of multiple polymorphic regions of individual haplotype sequences which could not be assessed by short reads. Third generation long read sequencing platforms, such as SMRT sequencing from PacBio and Nanopore sequencing from Oxford Nanopore Technologies open this possibility due to its extended read length , 29.In this study, we established and successfully applied a long read sequencing technique to long amplicons and identified co-linear genome stretches (haplotypes) in patient samples with mixed HCMV populations. This strategy for haplotype determination allows linkage analysis of multiple non-adjacent polymorphic sites along up to 7.7 kb. This allows a better approximation to the true strain diversity in mixed samples, which short read sequencing approaches failed to do. Taken together, this study provides the basis of a novel HCMV genome characterization strategy which will lead to an improved understanding of intra-host diversity and the dynamics of mixed HCMV strain populations. This could have important implications for diagnostics, treatment, and vaccine development.Primers are depicted in Supplementary Table E. coli overnight culture using the Nucleobond BAC 100 kit (Machery-Nagel) for low-copy plasmid purification. All steps were done according to the manufacturer. Purified BAC-DNA was eluted in 50 μl nuclease-free deionized water, quantified using Nanodrop and stored at 4 °C and under no conditions frozen to avoid DNA fragmentation. Purified BAC-DNA was used to establish long range PCR assays.TB40-BAC4-DNA was purified from 400 ml of Cryopreserved virus-stock vials of strains Merlin (AY446894) and TB40-BAC4-luc  derivedSix BAL samples stored at − 20 °C, all from patients who received lung transplants at the Medical University of Vienna between 2014 and 2016, were investigated. DNA was isolated using the QIAamp Viral RNA Mini kit (Qiagen). 250 μl of BAL solution was lysed and further purified as described in the manufacturer’s protocol. DNA was eluted from columns with 70 μl elution buffer. HCMV DNA was quantified by HCMV-specific qPCR (see below) and stored at 4 °C before being subjected to short and long range PCR.6 to 1 × 107 copies per ml) was used as template DNA for amplification by long range PCR.HCMV DNA either purified from Merlin or TB40-BAC4-luc virus stocks were diluted to the appropriate concentrations and mixed to achieve the ratios as listed in Table Purity and content of BAC-DNA and cell-culture-derived DNA was quantified using the NanoDrop 1000 tool (Peqlab). Amount of PCR amplicons was determined using the Qubit™ double-stranded DNA High-Sensitivity Assay according to the manufacturer’s instructions (Thermo Fisher Scientific) on the Qubit 2.0 fluorometer. HCMV-specific DNA of cell-culture-derived DNA and BAL samples was quantified by an in-house qPCR as previously described  and cellPCR enzymes initially used for evaluation of sensitivity and specificity of long range PCR are listed in Table HCMV DNA positive BAL samples were screened for mixed genotype infections by short range PCR amplification of polymorphic target regions within envelope glycoprotein N (gN), envelope glycoprotein O (gO), and UL146 (nested PCR). Detailed description of PCR amplicons, associated primers and respective annealing temperatures are listed in Supplementary Table SMRT bell library preparation and sequencing were performed by the Next Generation Sequencing Facility at Vienna BioCenter Core Facilities. Long range PCR amplicons and non-enriched DNA were further purified using the QIAEX II Gel extraction Kit (Qiagen). Due to indispensable loss of HCMV DNA (> 80%) during gel extraction, both non-enriched DNA and PCR products were subjected to the protocol: Desalting and Concentrating DNA Solutions. All steps were performed as advertised in the protocol and the HCMV DNA was eluted in 20 μl of Tris buffer. For library preparation a minimum DNA input of 700 ng and 2.5 μg for amplicon and whole genome sequencing was required, respectively. Initial quantification and purity were measured with the NanoDrop 1000 tool (Peqlab) and a Qubit 2.0 fluorometer (Thermo Fisher). Large fragment analysis was performed with a Femto Pulse system and the genomic DNA 165 kb Kit (Agilent). Non-enriched DNA was sheared to an expected fragment length of < 10 kb. Samples were indexed and multiplexed according to the Sequencing facility. Subsequent to library preparation a blue pippin size selection (Sage Science Inc.) was used to isolate target fragment lengths of long range PCR amplicons. Sequencing was performed on a PacBio-Sequel system for 10 and 20 h for cell-culture derived and BAL samples, respectively. Details on PacBio sequencing run information is listed in Two ng input DNA per sample was used for library preparation using the Nextera XT library preparation kit, and samples were indexed using the Nextera XT index kit (Illumina). After index PCR, samples were purified with 45 μl of Agencourt AMPure XP magnetic beads with a sample to beads ratio of 3:2 (Beckman Coulter), normalized by Qubit quantification and pooled to generate a 4 nM library. To compensate for low diversity libraries, a 12 pM PhiX control spike-in of 2.5% was added. Single and paired-end sequencing  was done on an Illumina MiSeq instrument with automatic adapter trimming (Illumina). Raw reads in fastq format that passed filters were used for analysis using CLC Genomics Workbench 12.0 (Qiagen). Detailed information on MiSeq sequencing runs is listed in https://github.com/pacificbiosciences/barcoding/) with default parameters and symmetric options. In order to generate Highly Accurate Single-Molecule Consensus Reads, demultiplexed subreads were aligned with the PacBio ccs tool of Bioconda (https://github.com/PacificBiosciences/ccs). During this process, multiple reads of the same SMRTbell sequence are combined to produce high quality consensus sequences. To prevent incorrect analysis due to heteroduplexes, consensus sequences for each strand of a molecule were separated by strand. For all following steps, each forward and reverse strand was treated as individual single-strand reads. No full-length subreads were required for ccs generation, a minimum predicted accuracy of 0 and a minimal number of passes for ccs of 0 was chosen  (position in CDS of Merlin strain: 1 to 180), gO (1 to 680), and gN (1 to 220), were considered. In total, 2 gH, 8 gN, and 8 gO genotype sequences were used as reference sequences for mapping . MappingIllumina fastq reads were quality trimmed with a base-calling error probability of 0.001, a minimum number of nucleotides in reads of 100, and human genomic DNA reads were removed. Then, quality trimmed and filtered reads were mapped to highly polymorphic regions within gO (1 to 680), gN (1 to 220), and UL146 (position in CDS of Merlin strain: 1 to 360) with default setting parameters for match/mismatch scores and insertion/deletion costs, a length fraction of 0.3 and a similarity fraction of 0.8. A total of 30 genotypic reference sequences were used as previously described  and listA positive genotype mapping was scored if the number of reads was > 10 and the consensus length corresponded to at least 80% of the reference length. Consensus sequences were derived from the mappings by using a minimum read depth of 5 reads per base with low coverage regions coded as ambiguities (Ns). Ambiguity codes were inserted using a noise threshold of 0.1 and a minimum nucleotide count of 5. Alignments of derived consensus sequences were screened visually and unique consensus sequences were counted as independent genotypes.To retain PacBio ccs reads that span the complete amplicon length, all ccs were initially length trimmed. For this, F3 amplicon-derived reads below 7500 and above 8000 and F4 amplicon-derived reads below 6500 and above 7000 number of nucleotides in reads were excluded. Remaining reads were processed with following parameters: quality trimming with a base-calling error probability of 0.01, ambiguous limit of 2 in a read and exclusion of low quality reads. Only reads that span more than half of the amplicon length (4000 nucleotides for F3- and 3500 nucleotides for F4-derived amplicons) were retained and human genomic DNA reads removed. Remaining reads were mapped to 236 HCMV full genomes , with deAdditional file 1 Supplementary Tables 1- 12b. Supplementary tables_final.Additional file 2: Supplementary Figs. 1–3. Supplementary_Figures


A comparative study focusing on the visco–elastic properties of two series of carbon black filled composites with natural rubber (NR) and its blends with butadiene rubber (NR-BR) as matrices is reported. Strain sweeps at different temperatures are performed. Filler network-related contributions to reinforcement ( Nano-sized filler particles incorporated into a rubber matrix result in materials offering an array of synergistic mechanical properties. Filler particles modify in particular the visco–elastic properties of rubber composites ,2,3. AltThe nature of the filler network is debated already for more than 50 years ,5. VarioVarious findings suggest that immobilized rubber bridges connecting neighbored nanoparticles are of extraordinary importance for the properties of the filler network in highly filled rubber composites. A conclusion of relevant studies is that the filler network contains not only solid-like filler particles, but also glassy rubber bridges, which will soften in the application relevant frequency–temperature range. Accordingly, the filler network shows visco-elastic properties ,26. An aus model  has beennetwork, ,28. It iIn a previous communication , we haveThe objective of this work was to get deeper insights towards the molecular origin of reinforcement and dissipation in rubber composites. Temperature-dependent trends in dissipation and reinforcement are compared for natural rubber and natural rubber–butadiene rubber blends filled with an identical grade of carbon black. The importance of glassy rubber bridges being part of the viscoelastic filler network for both—reinforcement and dissipation—is demonstrated.N-cyclohexyl-2-benzothiazylsulfenamide  as accelerators and diphenylguanidine , together with 99% pure zinc oxide and 98% pure stearic acid  as activators. Both NR and NR-BR blend (ratio 70:30) are filled with varied volume fractions of N234  being Standard Vietnamese Rubber 10 (SVR 10) and a high cis 1,4 butadiene rubber (BR) from Trinseo Deutschland GmbH Schkopau with the trade name BUNARubber mixing is carried out in two-steps. The first step involves mechanical mixing of rubber and incorporation of carbon black particles into the rubber matrix along with other additives . The mixing machine used was a HAAKE PolyLabDynamic shear measurements with variable strain amplitude were done using an Anton Paar MCR501 Twin-Drive rheometer. Rectangular specimen of dimension 2 × 8 × 30 mmTransmission electron microscopy (TEM) studies on rubber composites were performed by means of a FEI Tecnai G2 TEM operated at 200 kV. Ultra thin sections of a thickness of 60 nm were prepared by cryo-ultramicrotomy using a RMC Power Tome with CRX cryo chamber equipped with a Diatome diamond knife. The software cellStorage modulus m (fixed here to 0.6) describes the shape of the sigmoidal decrease in In order to quantify the filler network contributions to reinforcement the Equation (1)Gγ′=GKraus fits with ite well . The pard to G∞′ . The dirediction  probablyr matrix b.Already Payne  and Krauby Ulmer  without the formGγ″=G0″−GThe fit lines in  network ,27 is inAn arising question is why there are significant differences between the mechanical properties of rubber composites with NR and NR-BR matrix. In order to contribute to a better understanding of these differences TEM images have been prepared  for reprposition .Temperature-dependent strain sweep measurements on NR and NR-BR composites with variable filler contents  the topology of the filler network and (ii) the chemical composition of the glassy rubber bridges could be of major importance. Both features are influenced by various factors, in particular by the chosen recipe and the processing conditions. The topology of the filler network be can changed not only by the chosen mixing procedure ,35, but behavior , making Since it is found that the filler network topology is only weakly influenced in case of the carbon black filled NR and NR-BR composites investigated in this work , the chemposites ,38. Evensipation . To valiA comparative study focusing on the visco-elastic properties of two series of identically filled NR and NR-BR model composites has been performed. The major importance of the filler network in rubber composites containing high filler fractions for both—reinforcement and dissipation—is highlighted. It is demonstrated based on strain sweeps conducted on rubber composites with various filler fractions at different temperatures that (1) the filler network has visco-elastic properties and that (2) a modified Kraus equation promoted recently  is able Future studies should focus on additional insights regarding the influence of glassy bridge composition and filler network topology on the mechanical behavior of rubber composites. This may include approaches where composition and processing conditions are varied, improvised methods providing specific information as well as numerical simulations. The common molecular origin of filler network related contributions to reinforcement and dissipation demonstrated in this work is an important insight needed to fulfill the functional requirements of rubber composites in tire treads application where dissipation and mechanical strength have to be optimized in parallel.


Gossypium hirsutum L.) is crucial for efficient utilization and conservation of finite groundwater resources of the Ogallala Aquifer in the Texas High Plains (THP) region. The goal of this study was to suggest optimum irrigation termination periods for different Evapotranspiration (ET) replacement-based irrigation strategies to optimize cotton yield and irrigation water use efficiency (IWUE) using the CROPGRO-Cotton model. We re-evaluated a previously evaluated CROPGRO-Cotton model using updated yield and in-season physiological data from 2017 to 2019 growing seasons from an IWUE experiment at Halfway, TX. The re-evaluated model was then used to study the effects of combinations of irrigation termination periods (between August 15 and September 30) and deficit/excess irrigation strategies (55%-115% ET-replacement) under dry, normal and wet years using weather data from 1978 to 2019. The 85% ET-replacement strategy was found ideal for optimizing irrigation water use and cotton yield, and the optimum irrigation termination period for this strategy was found to be the first week of September during dry and normal years, and the last week of August during wet years. Irrigation termination periods suggested in this study are useful for optimizing cotton production and IWUE under different levels of irrigation water availability.Determining optimum irrigation termination periods for cotton ( About 95% of the water pumped from this aquifer is used for irrigated agriculture2 and more than 90% of the region’s total water needs are met from this aquifer5. Producers in this region are finding it difficult to provide full crop water needs due to the rapid decline in groundwater levels and increased pumping costs8. The average decline in groundwater level between pre-development9  and 2015 was maximum in the Texas portion of the Ogallala Aquifer among eight states and it was estimated as 12.5 m10. The average decline across the entire Ogallala Aquifer region during the same period was estimated as 4.6 m10.Irrigated agriculture in many semi-arid and arid regions across the globe has been threatened due to diminishing water resources. The semi-arid Texas High Plains (THP) is one of the most productive agricultural regions in the United States (US), mainly due to the availability of irrigation water from the underlying vast Ogallala Aquifer that is spread over eight statesGossypium hirsutum L.) is a major irrigated crop grown in the THP region, contributing to about 25% of the US cotton production and 64% of the Texas cotton production12. Like other crops, cotton production in this region relies upon irrigation with water mined from the Ogallala Aquifer. Projected increase in temperature and more erratic precipitation patterns in the future could further intensify existing water shortages and adversely affect crop production in this region15. To prolong the Ogallala Aquifer’s life and sustain an agriculture-based economy, water districts in the THP regions have been restricting groundwater withdrawals from the aquifer. For example, the High Plains Water District (HPWD)16 and the North Plains Underground Water Conservation District17 have set an annual groundwater extraction limit at 460 mm (18 inches).Cotton  and yield under different irrigation conditions (full/deficit) over multiple decades could result in more appropriate calendar-based recommendations for the THP region. Implementing appropriate irrigation termination strategies based on water availability could provide water savings with negligible/no yield losses.Rapidly declining groundwater levels and restrictions on the use of groundwater for irrigation necessitate the adoption of irrigation management strategies focused on water savings and economic enhancement. Even though cotton is highly adaptable to limited/deficit water conditions31. No gain in yield or fiber quality was observed with irrigation after bolls begin to open. Spray irrigation following boll opening provided moisture in the crop canopy which enhanced the environment for the plant pathogens that cause boll rot or hardlock31. As compared to pivot irrigated fields, irrigation termination in furrow irrigated fields was recommended a few days before31. Some researchers suggested irrigation termination periods based on growing degree days (GDD) after physiological cutout and their recommendations varied substantially33. They have considered the appearance of five nodes above white flower on the main stem as the physiological cutout stage. Lascano et al.24 evaluated irrigation termination thermal times based on accumulated heat units from crop emergence  Cropping System Model (CSM) can serve as an effective tool for simulating cotton growth and yield under varied soil, crop management and weather conditions43. The DSSAT CSM CROPGRO-Cotton model has been used extensively by researchers worldwide for suggesting efficient irrigation management strategies for cotton production49.Evaluating efficient irrigation strategies through field experiments alone is rather difficult as late-season rainfall events can lead to the loss of an experimental site-year and results need to be accumulated over different weather scenarios . Crop growth models, on the other hand, are very useful to rapidly simulate different crop growing conditions and assess their long-term impacts on crop production and enable the development of efficient irrigation strategies for different agro-climatic conditions20. In our previous study20, we suggested ideal irrigation termination periods for the THP using the DSSAT CSM CROPGRO-Cotton model, which was calibrated by Adhikari et al.50 using measured data from 2010 to 2013 growing seasons from an IWUE experiment conducted by Bordovsky et al.51 at the Texas A&M AgriLife Research Center at Halfway in the geographic center of the THP. However, those irrigation termination recommendations were given for a full and three deficit irrigation treatments implemented by Bordovsky et al.51 and such recommendations for commonly adopted ET-replacement-based deficit irrigation strategies are lacking. The IWUE experiments were resumed at Halfway in 2016 with additional late-season irrigation treatments, and detailed in-season crop physiological data was collected from this experiment during the 2017—2019 growing seasons. Re-evaluating the CROPGRO-Cotton model based on this updated yield dataset, which includes late-season irrigation termination treatments, should enhance confidence in the use of the model for determining optimum irrigation termination dates for commonly adopted ET-replacement based irrigation strategies. The objectives of this study were therefore to: (1) re-evaluate the Adhikari et al.50 calibrated CROPGRO-Cotton model using additional in-season crop physiological and yield data available from 2017 to 2019 field experiments conducted at Halfway, TX, (2) assess the long-term (1978–2019) effects of irrigation termination periods on seed cotton yield and IWUE under commonly adopted deficit/excess ET-replacement practices, and (3) suggest the optimum irrigation termination periods for cotton production under strategic deficit/excess irrigation regimes during dry, normal, and wet years.Limited studies have used crop growth models for determining optimum irrigation termination periods for cotton3 cm−3, respectively. Results from calibration of the model for simulation of cotton phenological stages, canopy height and seed cotton yield are presented in the following sections.Parameters adjusted during the DSSAT-CSM-CROPGRO-Cotton model calibration are presented in Supplementary Table Simulated dates of onset of different cotton phenological stages were within the range of measured dates during both calibration and evaluation of the model , TRIFL  and RHGHT (relative height of the ecotype in comparison to the standard height per node) were found to affect canopy height simulation and hence they were calibrated to values of 1.0, 0.18 and 0.80, respectively  than the evaluation (water limiting treatments)  when compared to full/excess irrigation strategies (100% and 115% ET-replacement) Fig. . Based o55, and wet weather conditions for prolonged duration could lead to yield loss58. In general, simulated seed cotton yield under deficit irrigation strategies  was found to be higher under normal/wet years as compared to dry years. However, in the case of full/excess irrigation strategies (100% and 115% ET-replacement), no significant differences in simulated seed cotton yield were found between dry and normal/wet years, especially when irrigation was terminated at later dates.The simulated seed cotton yield was found to be the lowest under 55% ET-replacement strategy followed by the 70% ET-replacement strategy, as expected Fig. a,b. The 20 also reported that cotton irrigation water use under full irrigation may exceed the HPWD’s pumping limit if irrigation is terminated after 5 September in dry years and after 12 September in normal and wet years. Overall, these results indicate that adoption of appropriate deficit irrigation strategies along with suggested termination dates could enable producers achieve higher IWUE with minimum/no reduction in seed cotton yield while being compliant with the HPWD regulations.The simulated median irrigation water use increased continuously as the irrigation termination date moved towards the end of the growing season Fig. . As expeIn general, as the irrigation termination date moved towards the end of the growing season, the median simulated IWUE increased until a certain termination date and then decreased for the later termination dates. Ideal irrigation termination dates were identified based on our selected IWUE criteria (date on which simulated median IWUE was the highest or the earliest irrigation termination date in the case of same maximum IWUE on multiple dates). Based on the simulated median IWUE, the ideal irrigation termination dates in normal years were identified as September 19, September 5, September 5, September 5 and August 29 under 55%, 70%, 85%, 100% and 115% ET-replacement strategies, respectively Fig. . In the 58.As expected, simulated median IWUEs were lower in wet years under all irrigation strategies, except for the 55% ET-replacement strategy in which simulated IWUE was higher in wet years as compared to dry years Fig. . The sim59. Interestingly, simulated IWUE in the case of 115% ET-replacement was lower than that of 100% and 85% ET-replacement strategies in all weather conditions. A similar trend was also reported in other studies60. The THP region receives an average annual rainfall of 470 mm, out of which a major portion (about 330 mm) occurs during the cotton growing season51, and this rainfall is generally adequate to achieve a good yield of a drought-tolerant crop such as cotton under deficit irrigation strategies. Modala et al.50 also reported a marginal reduction in yield in the adjacent Texas Rolling Plains region when irrigation was decreased from 100 to 66% ET-replacement under normal rainfall conditions; however, the reduction was substantial under dry conditions. On the other hand, due to semi-arid and windy climate along with uncertain rainfall patterns, maintaining high IWUE is a challenge in the THP region51, and irrigation as per lower ET-replacement  strategies could lead to substantial yield loss and reduction in IWUE.Simulated IWUE was the highest for 85% ET replacement strategy and the lowest for 55% ET replacement strategy among the most termination dates and categories of years Fig. . Other sP-value = 0.39), and from September 12 to September 19 in the case of 55% ET-replacement strategy   varied from the last week of August to the first week of September while those for the deficit irrigation strategies  varied from the first week of September to the third week of September. The optimum irrigation termination periods suggested in this study are either the same or one week earlier than those suggested by Ale et al.64. Simulated seed cotton yield and IWUE were found to be the lowest in the case of 55% ET-replacement strategy in all categories of years 66, which considers the contribution from rainfall and soil water content (in addition to irrigation) to crop yield, could serve as a better indicator for determining optimum irrigation periods than IWUE and our future efforts will focus on this improvement.In comparison to 100% ET-replacement strategy, simulation of 115% ET-replacement strategy  resulted in only a small improvement in yield , but caused a substantial decline in IWUE  , 3.2 mm d−1–Medium (M), and 6.4 mm d−1–High (H)) implemented in combination with three cotton growth stages 49. These three growth stages were defined based on accumulated growing degree days at a threshold temperature of 15.6 °C51. Treatments were labeled by the irrigation water level during each growth stage . A low energy precision application (LEPA) center pivot irrigation system was used for irrigation applications. More details about the field experiment and previous model evaluation can be found in Bordovsky et al.51 and Adhikari et al.50, respectively. Following a two-year period (2014 and 2015) used to equalize soil water and residual nutrient content across plots, IWUE experiments were resumed at the same location as the 2010–2013 experiments in 2016 by modifying treatments to include late, as well as more typical irrigation termination treatments. We collected in-season crop physiological data  and end-of-the-season yield data from this new experiment during the 2017–2019 growing seasons and we re-evaluated the Adhikari et al.50 CROPGRO-Cotton model based on this new data as a part of this study. Irrigation treatments considered for model re-evaluation included LMM, LMH, HHH, LMM + , LMH + , and HHH + (+ symbol indicates late irrigation termination). The soil at the study site is Pullman clay loam . The average (1978–2019) growing season (April–October) and annual rainfall at the study site were 376 mm and 463 mm, respectively. The recorded minimum and maximum temperatures were − 21.3 °C and 44.4 °C, respectively.A cotton IWUE experiment was conducted at the Texas A&M AgriLife Research Center at Halfway   as well as different tools to facilitate the effective use of the models67. The model calculates and reports model state variables over daily time steps69. Long-term (1978–2019) daily weather data for this study were obtained from an on-site weather station. Daily weather data input to the model included precipitation, maximum and minimum air temperature, solar radiation, dew point temperature, relative humidity and wind speed. The missing weather data were filled with the data retrieved from nearby Olton 6S and Plainview 1S weather stations, which are part of the West Texas Mesonet70. A summary of monthly average weather parameters during the model re-evaluation period of 2017–2019 cotton growing seasons (April through October) is presented in Supplementary Table 50.The DSSAT CSM CROPGRO-Cotton model (version 4.7) was used in this study. The model uses weather data, genetic parameters, and soil and crop management data as inputs to simulate crop growth and development over time in response to different crop and soil management practices71 and the calibrated values reported for the THP region in previous studies52. The testing ranges used in Adhikari et al.50 were slightly modified in this study to account for the differences in cultivars. Some of the sensitive cultivar and ecotype parameters were then adjusted until a good match between the measured and simulated values was achieved. The parameters related to crop growth were initially adjusted to achieve a good match between the simulated dates of onset of emergence, anthesis and physiological maturity stages, and the measured dates from the field experiment. The parameters related to crop development and yield were then adjusted until a good agreement between the simulated and measured seed cotton yield was achieved. A similar approach of calibrating phenological stages before calibrating parameters related to yield was adopted by several researchers72.The CROPGRO-Cotton model was re-evaluated using the most recent in-season crop phenological data and cotton yield data collected during 2017–2019. The model was calibrated based on measured data from two high irrigation treatments (HHH and HHH +) and evaluated against measured data from four deficit irrigation treatments . A new cultivar FiberMax 2011GT was added to the DSSAT cultivar database. The cultivar and ecotype parameters for this variety were populated based on the literature values40. DSSAT partitions potential ET into the potential crop transpiration (Ep) and potential soil evaporation (Es)67. While actual soil evaporation depends on the potential Es and soil water content, actual crop transpiration is the minimum of potential Ep or root water uptake73. The DSSAT model provides two methods for estimating daily potential ET, the FAO-56 Penman–Monteith method74 and the Priestley–Taylor75 method. The Priestley–Taylor method was used in this study due to unavailability of continuous weather data on wind speed and relative humidity, which are required by the FAO-56 method. Adhikari et al. (2016) used a modified ET method suggested by Thorp et al.40, the Priestley–Taylor method gave a good match between the simulated and measured seed cotton yield in this study. The Priestley-Taylor evaporation method was recommended under soil water limited conditions76 and it was used in the majority of other DSSAT cotton modeling studies also78.Accurate calculation of daily potential evapotranspiration (ET) is very important for good model performance2), percent root mean square error (%RMSE) and percent error (PE) were employed for evaluating the model performance. The d-index is a standard measure of the degree of model simulation error79 and it varies between 0 (no agreement) and 1 (perfect agreement) , coefficient of determination r, percentto ∞ Eq. . The PE . 83 Eq. . A value84. The ‘Automatic when required’ irrigation scheduling method was used in this study by keeping the threshold to trigger and stop irrigation at 50% and 100% of available water content, respectively, in the top 30 cm management depth. The estimated amounts of daily irrigation water were considered as the irrigation water requirement for a 100% ET-replacement strategy. Irrigation water requirements for other ET replacement strategies were then estimated by multiplying 100% ET replacement irrigation prescriptions with an appropriate multiplier . The date-wise estimated irrigation amounts for different ET-replacement strategies were finally input to the model manually. Estimated irrigation applications on all dates after the simulated irrigation termination date were eliminated. Two pre-plant irrigations of 25.4 mm each were applied on 25th and 29th of April in all years for all scenarios to represent a typical practice followed in the study region and adopted in the Halfway experiments61.Using the evaluated CROPGRO-Cotton model, long-term (1978–2019) simulations were run for five deficit/excess irrigation strategies (55% to 115% Evapotranspiration (ET)-replacement in 15% increments) with eight irrigation termination dates with a one-week interval between 15th August and 30th September. The ET-replacement refers to the ET demand which is the accumulation of daily potential plant transpiration plus actual soil evaporation minus infiltration from rainfall (rain—runoff)85. Years 1985, 1992 and 2015 (extreme wet years) and year 2011 (extreme dry year) were found to be outliers and hence they were also excluded from the analysis. After sorting the remaining 37 years by growing season precipitation, the bottom 12 low-precipitation years were classified as ‘dry’ years and the top 12 high-precipitation years were classified as ‘wet’ years. The remaining 13 years were classified as ‘normal’ years.The impacts of environmental conditions during the growing season on cotton IWUE and seed cotton yield under different irrigation termination dates and deficit/excess irrigation strategies were then studied by dividing the simulation period into (i) dry, (ii) normal and (iii) wet years according to the total precipitation received from April 1st to the simulated termination date during the growing season  simulations were run with the evaluated model for a hypothetical dryland scenario, and the simulated average yield was considered as the dryland yield for estimating IWUE. Based on the simulated yield, the earliest termination date at which the median seed cotton yield reached a peak value or when an increase in median seed cotton yield from that termination date to the next termination date was < 3%, was considered ideal. With reference to IWUE, a termination date corresponding to the maximum simulated IWUE was considered ideal. When the same maximum IWUE was simulated on multiple irrigation termination dates, the earliest irrigation termination date among them was considered ideal. The ideal irrigation termination periods identified based on the simulated median seed cotton yield and simulated median IWUE were then compared, and if they were found to be different, a statistical analysis was carried out using a student’s t-test86 to decide an optimum termination period that provides higher seed cotton yield and IWUE. A paired two-tailed t-test was performed (considering the same population twice for two different irrigation termination periods identified based on yield and IWUE) at a significance level of 95%  using the T.TEST command in Microsoft Excel.Recommendations on ideal irrigation termination periods for cotton production under different ET-replacement strategies were made based on simulated IWUE and yield while keeping in view the HPWD’s annual groundwater extraction limit for irrigation of 460 mm. The IWUE was estimated as the ratio of the difference between irrigated and dryland cotton yield, to the amount of seasonal irrigation water applied50 was re-evaluated using in-season crop phenology data and seed cotton yield data collected from a cotton IWUE experiment at Halfway, TX during 2017–2019. The evaluated model performed well in simulating the dates of onset of different cotton phenological stages, canopy height and seed cotton yield. The optimum irrigation termination dates for 55%, 70%, 85%, 100% and 115% ET-replacement strategies were identified as September 12, September 5, September 5, August 29 and August 29, respectively under normal weather conditions. The optimum irrigation termination periods were found to be similar or a week earlier/later than those in normal years in wet/dry years. The 85% ET-replacement strategy along with suggested optimum irrigation termination dates of September 5 in dry and normal years and August 29 in wet years was found to be the most efficient strategy for saving 17 to 41 mm of irrigation water annually. Simulated median irrigation water use was less than the annual groundwater extraction limit (460 mm) specified by the HPWD under all simulated strategies (except for late September irrigation termination dates in case of 115% ET-replacement strategy during dry years) indicating that adoption of appropriate deficit irrigation strategies based on available irrigation capacities could enable producers achieve higher seed cotton yield and IWUE while complying with the HPWD regulations. However, irrigation termination decisions should also consider the differences in water holding capacity of the soil, crop management practices, cultivar characteristics, crop health, and the amount and distribution of late-season rainfall. In addition, extending these simulations to different sites across THP could strengthen the recommendations on irrigation termination decisions. Furthermore, irrigation termination decisions based on modeled soil water content towards the end of the growing season (the time when a producer starts thinking about irrigation termination) could be practically relevant. Our future research efforts will focus on these limitations.The DSSAT CSM CROPGRO-Cotton model was used in this study to assess the effects of irrigation termination dates on IWUE and seed cotton yield under strategic deficit irrigation strategies. Previously evaluated DSSAT CROPGRO-Cotton modelSupplementary Information.


Vascular smooth muscle cells (VSMCs) remodel vascular walls actively owing to mechanical cues and dedifferentiate to the synthetic phenotype from contractile phenotype in pathological conditions. It is crucial to clarify the mechanisms behind the VSMC phenotypic transition for elucidating their role in the vascular adaptation and repair and for designing engineered tissues. We recently developed novel micro-grooved collagen substrates with “wavy wrinkle” grooves to induce cell–substrate adhesion, morphological polarization, and a tissue-like cell arrangement with cytoskeletal rearrangements similar to those in vascular tissue in vivo. We found that cultivation with this micro-grooved collagen significantly induced VSMC contractile differentiation. Nonetheless, the detailed mechanism underlying the promotion of such VSMC differentiation by micro-grooved collagen has not been clarified yet. Here, we investigated the detailed mechanism of the cell arrangement into a tissue and contractile-differentiation improvement by our micro-grooved collagen substrates in terms of nuclear–cytoskeletal interactions that possibly affect the nuclear mechanotransduction involved in the activation of transcription factors. We found that VSMCs on micro-grooved collagen manifested significant cell arrangement into a tissue and nucleus slimming with a volume reduction in response to the remodeling of the actin cytoskeleton, with consequent inhibition of nuclear shuttling of a transcriptional coactivator, Yes-associated protein (YAP), and improved contractile differentiation. Furthermore, VSMC nuclei rarely deformed during macroscopic cell stretching and featured a loss of nesprin-1–mediated nuclear–cytoskeletal interactions. These results indicate that our micro-grooved collagen induces a cell alignment mimicking in vivo VSMC tissue and promotes contractile differentiation. In such processes of contractile differentiation, mechanical interaction between the nucleus and actin cytoskeleton may diminish to prevent a nuclear disturbance from the excess mechanical stress that might be essential for maintaining vascular functions. Vascular smooth muscle cells (VSMCs) constitute the main components of the arterial wall, and these regulate contraction and dilation of the walls as well as vigorously remodel the arterial wall in which they reside via biochemical cues from endothelial cells and mechanical signals that are self-applied ,2,3, andFrom a biomechanical viewpoint, influences of mechanical characteristics of cell culture substrates have been in the spotlight of recent studies. Previous studies have addressed cellular responses to substrate stiffness ,7,8 and To overcome these issues, we recently devised a cell culture micro-grooved substrate consisting of only a type I collagen matrix that offers a substantial basal level of adhesivity by means of RGD peptide sequences, to bring about substrate–cell adhesion, elongation of cells, and a tissue-like cell arrangement with cytoskeletal rearrangements similar to those in vascular tissue in vivo . We founCells on a substrate generate internal forces through contraction of the actin–myosin cytoskeletons. These forces are crucial for cellular mechanotransduction and are potentially affected by substrate geometry. The forces are transmitted from the cytoskeleton to the nucleus via nuclear-membrane proteins that are constituents of a LINC (linker of nucleoskeleton and cytoskeleton) complex , which pThus, in this study, we used primary porcine VSMCs to investigate the detailed mechanism of the cell arrangement into a tissue and contractile-differentiation improvement by our originally designed micro-grooved collagen substrate in terms of nuclear mechanics and nuclear–cytoskeletal interactions. Incidentally, a Yes-associated protein (YAP), Hippo effector, and transcriptional coactivator with PDZ-binding motif (TAZ) serve as a mechanosensing on–off switch that regulates multiple cellular processes. YAP and TAZ localize into the nucleus and become transcriptionally active in the cell in response to mechanical cues such as substrate stiffening  and mechThe micro-grooved collagen substrates were fabricated as described previously . BrieflyE is the elastic modulus, F is the applied force, α is an opening angle of the cone of the cantilever tip, ν is the Poisson ratio , and δ is indentation depth of the sample registered in the force–displacement curves. From the results of the Hertzian model approximation, we identified elastic modulus and Z contact points (specimen surface) of the substrates in each pixel and generated a surface topography map and an elastic modulus map of the substrates.To determine the surface topography and elastic modulus of the collagen substrates, AFM analyses were carried out by means of a NanoWizard IV AFM instrument  mounted on top of an inverted optical microscope  equipped with a digital CMOS  camera . The substrates were immersed at room temperature in phosphate-buffered saline [PBS(-)]. AFM quantitative imaging (QI) mode was then applied to construct a force–displacement curve for each pixel of a measured area of 128 × 128 pixels (100 × 100 µm) obtained by a precisely controlled high-speed indentation test. We used rectangular silicon nitride cantilevers equipped with a cone probe tip  with a nominal tip radius of 10 nm and a spring constant of 0.06–0.08 N/m. These high-speed indentations were carried out until a force was reached at a preset force of 1.5 nN. Substrate elasticity was computed from the obtained force–displacement curves via application of the Hertzian model , which a2. The cells were subcultured repeatedly in a 1:4 split ratio when they reached approximately 80% confluence. Primary VSMCs at passages 6–8 were employed in all the experiments. VSMCs were seeded onto either the flat collagen or micro-grooved collagen substrate. Initial cell density was maintained at a relatively high value of ~200 cells/mm2 since low initial cell density  decreased cell–cell contact guidance, which also inhibited tissue formation from aligned cells. The VSMCs cultured on either substrate for 3 days that gave rise to confluent tissues were subjected to subsequent experiments.Porcine aortic VSMCs served as the test model. Primary VSMCs were prepared by the explant method described elsewhere . They weThe VSMCs cultured on either the micro-grooved or flat collagen substrate were fixed for 10 min with PBS(-)  supplemented with 3.7% of formaldehyde, were permeabilized for 5 min with PBS supplemented with 0.5% of Triton X-100 , and were rinsed with PBS supplemented with 1% of bovine serum albumin (BSA) to block nonspecific binding of proteins. The fixed cells were incubated for 30 min with this blocking solution before treatment with staining reagents. The major protein markers of smooth-muscle contractile differentiation—α-SMA, calponin, and transgelin—or nesprin 1 (a component of the LINC complex) were stained fluorescently as follows: The samples were probed with a mouse antibody against α-SMA , mouse antibody against calponin , mouse antibody against transgelin , or rabbit antibody against nesprin 1  overnight at 4 °C. This was followed by 1 h incubation at room temperature with a secondary antibody  for α-SMA, calponin, and transgelin or with a goat anti-rabbit IgG antibody conjugated with Alexa Fluor 488  for nesprin 1. All the antibodies were diluted with PBS(-) containing 1% of BSA. Additionally, actin filaments inside cells were fluorescently stained by incubation for 60 min with Alexa Fluor 546–conjugated phalloidin . For fluorescent visualization of nuclei, we stained intranuclear DNA for 30 min with Hoechst 33342 .Stacked fluorescent cross-sectional images of the stained VSMCs were captured by means of a confocal fluorescence microscope in the range of cell thickness (~15 µm) with 0.5 µm intervals by means of a 60× oil immersion objective lens  or a 40× objective lens , a confocal system  with a multicolor fluorescence system , and a CMOS digital camera . A nucleus outline was traced automatically, and the projected area, length, width, and volume of the nucleus were determined in the ImageJ software . The obtained stacked cross-sectional images of the cells were also transformed into 2D images of superimposed projection to evaluate fluorescence intensity of the intranuclear DNA and contractile proteins .EdU is a nucleoside analogue of thymidine and is incorporated into DNA during only DNA synthesis, thus enabling the visualization of newly synthesized DNA . This teFor a comparison of mechanical properties of nuclei, force–indentation responses of a nucleus were determined by AFM as described in 2 incubator [Either the micro-grooved or flat collagen substrate on the PDMS membrane described in ncubator . Before First, we examined the effects of surface topography of the micro-grooved collagen substrate on cell alignment and intracellular structures, especially, the actin cytoskeleton and nucleus. Confocal fluorescent imaging revealed that VSMCs cultured on the conventional flat collagen substrate possessed irregular shapes with actin fibers of random orientation, and nucleus orientation differed among the cells A,C. SomeAfter examination of the mechanical environment around the nucleus, thick bundles of mature stress fibers in the VSMCs on the flat collagen substrate were found to be distributed on the apical and basal surfaces of the nucleus C. In conBy fluorescent image analysis, we next assessed the impact of the tissue-like cell alignment (caused by micro-grooved collagen) on VSMC differentiation . FluoresWe also demonstrated that both the fluorescence intensity of Hoechst-stained DNA and the proportion of S phase cells detected by the 5-ethynyl-2′-deoxyuridine (EdU) assay were significantly lower among the VSMCs cultured on micro-grooved collagen than among those cultured on flat collagen A–D. FurtTo assess changes in the nucleus–cytoskeleton interactions, we investigated stretch-induced deformation of the nucleus in the VSMCs whose contractile differentiation was facilitated by the micro-grooved collagen substrate, which enabled the modeling of the in vivo arrangement of cells in a tissue. VSMCs cultured on either flat collagen or micro-grooved collagen were subjected to simple uniaxial stretching on the stage of an inverted microscope under physiological cell culture conditions, and morphological changes of the cell body and nucleus were monitored continuously A,B. The The elongation of the nucleus during cell stretching was significantly depended on the intracellular F-action structures: the nuclei in VSMCs whose actin filament were completely disrupted by treatment with cytochalasin D, rarely elongated during stretching in both groups .To clarify this difference in nucleus deformability during cell stretching, we quantified nuclear stiffness by AFM indentation analysis. We depolymerized actin filaments inside cells by treatment with cytochalasin D  just before AFM indentation analyses to preclude mechanical effects of actin filaments A. AroundHence, we finally examined the expression of nesprin 1, which tethers the actin cytoskeleton to the outer nuclear membrane (by serving as a component of the LINC complex) as another factor affecting nuclear–cytoskeletal interactions . The synThese findings meant that the actin–nucleus connection via the nucleus–cytoskeleton linker protein was weaker in the aligned VSMC tissue whose contractile differentiation was induced by the cultivation on the micro-grooved collagen substrate, thereby causing significant inhibition of the nucleus elongation and deformation during macroscopic cell/tissue stretching.We fabricated a collagen micro-grooved substrate to bring about tissue-like cell alignment related to the mechanical environment of an in vivo single vascular elastic lamina. The fabrication was facile because the type I collagen matrix sheet attached to the prestretched PDMS membrane was buckled by the release of the sheet strain, thus promoting the formation of wavy-wrinkle–like micro-grooves on collagen surfaces . The obtThe tissue-like alignment of VSMCs on micro-grooved collagen also induced F-actin remodeling: the mature stress fibers distributed in the apical part of the cells almost disappeared and became concentrated in the cells’ both side regions consisting of cell–cell junctions D. ConseqFurthermore, YAP nuclear localization turned out to be significantly inhibited in the aligned VSMC tissue on the micro-grooved collagen substrate J,K. TranLevels of contractile proteins’ expression were significantly greater in the aligned VSMC tissue on the collagen micro-grooved substrate, implying that VSMC differentiation was more active on the micro-grooved collagen . The mosThe finding of cell deformation under macroscopic stretching revealed that the synthetic VSMCs featuring mature stress fibers around the nucleus on the conventional flat collagen closely follow the stretch deformation imposed by the substrate, and their nucleus also deforms significantly under the applied substrate stretch, although the deformation value of the nucleus was ~40% of that of the cell body A,C. This


Membrane proteins work within asymmetric bilayers of lipid molecules that are critical for their biological structures, dynamics and interactions. These properties are lost when detergents dislodge lipids, ligands and subunits, but are maintained in native nanodiscs formed using styrene maleic acid (SMA) and diisobutylene maleic acid (DIBMA) copolymers. These amphipathic polymers allow extraction of multicomponent complexes of post-translationally modified membrane-bound proteins directly from organ homogenates or membranes from diverse types of cells and organelles. Here, we review the structures and mechanisms of transmembrane targets and their interactions with lipids including phosphoinositides (PIs), as resolved using nanodisc systems and methods including cryo-electron microscopy (cryo-EM) and X-ray diffraction (XRD). We focus on therapeutic targets including several G protein-coupled receptors (GPCRs), as well as ion channels and transporters that are driving the development of next-generation native nanodiscs. The design of new synthetic polymers and complementary biophysical tools bodes well for the future of drug discovery and structural biology of native membrane:protein assemblies (memteins). Obtaining accurate kinetic and structural information about molecular interactions of biologically relevant states of therapeutic targets is the foundation of modern drug discovery. However, this endeavour is complicated by the fact that most targets are embedded in asymmetric lipid bilayers with signaling lipids, post-translational modifications, cofactors and heteromeric subunits. The native assemblies are essentially impossible to reconstitute using recombinant methods and detergents, which strip off the lipids that are bound in vivo. Overcoming this challenge represents a key goal of an open innovation community known as the styrene maleic acid lipid particle (SMALP) network. Our challenge is to progress from in vitro to ex vivo structural biology, allowing currently inaccessible molecular machines to be excised from endogenous sources and purified intact for structure–function analysis. Targeting native states while keeping the original lipids, cofactors, modifications and partner proteins bound is clearly critical for discovery of drug molecules with higher selectivity and fewer side effects.This effort has led to the development of a growing set of synthetic SMA-derived copolymers including SMA and its derivatives  that conSeminal studies highlighting the potential to characterize native-state complexes within SMA nanodiscs are detailed in Understanding native GPCR function is fundamentally important to the pharmaceutical industry. These receptors bind more therapeutic agents than any other family, and are targeted by approximately one-third of all marketed drugs . Genomic2AR), as expressed both in Pichia (P.) pastoris and human embryonic kidney (HEK) cells [w/v) and purified using N-terminal His10 tags, it displayed ligand binding activity similar to that of the protein in yeast membranes, and exhibited a stable helical structure that withstood repeated freeze–thaw and storage cycles. The change of positions of Trp residues from accessible to buried positions as ligands bind to wild-type and mutant A2AR can be monitored by fluorescence spectroscopy [The first GPCR to be solubilized without detergents using the SMALP system is the human adenosine A2A receptor (AK) cells . After ttroscopy . Attachmtroscopy .Pichia pastoris membranes using SMA(2:1) or SMA(3:1). With the yeast preparations, a polymer concentration of 2.5% w/v, low salt and low temperature are optimal for retaining MT1R stability and for binding G protein and arrestin [Dopamine receptors are GPCRs that contain a cysteine-rich extracellular and a long intracellular C-terminus, and can form functional heterodimers with adenosine receptors . The doparrestin . Anotherarrestin . The pro2AR bind most tightly to PtdInsP2, which serves to increase G protein coupling and GTPase activity. An exogenous PtdInsP2 molecule is seen to bridge the complexed structure of NTSR1 and β-arrestin, stabilizing their active conformation [q and Gi1 heterotrimers than detergent micelle preparations [The stability, activity and selectivity of GPCRs depends on their interactions with lipids. The array of lipids complexed with the β1 adrenergic receptor (β1AR), β2AR and neurotensin receptor 1 (NTSR1) include cholesterol, phosphatidylserine, phosphoinositide and phosphatidic acid molecules based on tandem mass spectrometry analysis of their collisional dissociation ,44,45,46ormation  and suggormation  or classormation , illustrarations . PMA off2AR and human V1a vasopressin receptor (V1aR), as expressed in transfected HEK cells [2+ and Mg2+ tolerance as well as milder solubilization activity, and yields slightly smaller particles with diameters from 6–12 nm. While the most widely used SMA(2:1) copolymer generally offers the highest membrane solubilization efficiency, its activity is compromised at low pH or high polycation levels where the polymer can precipitate. The related DIBMA copolymer has been used to solubilize the β2-adrenoceptor (β2AR), which is sensitive to phospholipid composition [A variety of additional copolymers are being developed to purify such transmembrane targets and investigate biological lipid and ligand interactions. This has been demonstrated with the AEK cells . They weposition . Compariposition . Given scis–trans isomerization, inducing a conformational change in the protein. In particular, light converts the inverse agonist-bound off position to a full agonist state. The latter state catalyzes the replacement of GDP by GTP on transducin, which is an associated heterotrimeric G protein that triggers a response in rod cells of the retina. These 7TM bacterial proteins are topologically similar to mammalian class A GPCRs, which bind diffusible agonists and exhibit analogous lipid-dependent dynamics and conformational changes that also transduce allosteric information through G proteins.Bacteriorhodopsins form a large superfamily of seven transmembrane helix (7TM) proteins that all contain a covalently bound retinal molecule. When this chromophore absorbs light, it undergoes Haloquadratum walsbyi was resolved to 2.0 Å following its purification with a C-terminal double His6 tag in the presence of 2.5% (w/v) SMA(3:1) copolymer. The protein had been transferred from Escherichia (E.) coli membranes which were spiked with 1.5% (w/v) DMPC to increase the yield relative to levels achievable with DDM detergent. This allowed gentle release of the memteins into a lipidic cubic phase (LCP) system for in meso crystallization. The resulting structure reveals the orientation of a set of interacting monoolein molecules as well as the bound trans-retinal, with SMALPs offering an improvement in resolution [The first atomic resolution structure of a lipid-bound protein determined using SMALPs appeared in 2017. The crystal structure of a bacteriorhodopsin trimer from solution   and Halobacterium salinarum sensory rhodopsin I (HsSRI). Both were produced in E. coli with C-terminal His6 tags and then solubilized in SMA(2:1) copolymer [w/v) was used to increase protein yield without needing to add exogenous DMPC. Four sets of trimers of RxR are evident by dynamic light scattering analysis of the 54 nm diameter nanodiscs, which are five times larger than typically seen for most SMALPs. Average particle diameters of 30 nm were observed by AFM, with nanodiscs stacked in sets of one, two or three. Approximately 700 lipid molecules were present per nanodisc along with the four trimers. This corresponds to an average ratio of 57 lipids for every protein molecule, which is higher than earlier estimates of lipids associated with the E. coli PagP β barrel in SMALPs, potentially indicating greater membrane retention or a more ordered lipid microenvironment. The time-course of rhodopsin photocycles in SMALPs was very similar to that measured in egg PC liposomes but differs from that in DDM detergent preparations, which hinder proton pumping activity. The more unstable HsSRI protein is deactivated by DDM but retains its color and activity when solubilized and purified in SMALPs. The SMA–HsSRI complex binds NaCl with a dissociation of 34 mM, and this interaction influences its absorption of light, indicating that SMALPs provide a more native-like membrane environment.A recent study compared thermally stable and unstable rhodopsins from opolymer . A high The endogenous lipid interactions of archeorhodopsin and bacteriorhodopsin, which are lost with detergent, are maintained in SMALPs. Native mass spectrometry revealed that these receptors complexed with diphytanyl glycerol lipid ligands (S-DGD and 2DP) in native membranes isolated using SMA(3:1) copolymer and DMPC . This stNatronomonas pharaonis (NpSRII), which mediates photorepellent responses to light. This receptor and its transducer protein were purified from E. coli membranes using single C-terminal His6 tags and 2.5% w/v SMA(2:1) copolymer [E. coli membranes are similar to those from DDM and liposome preparations. However, the decay of the M photointermediate is accelerated in SMALP preparations. This can be attributed to high local proton concentrations as a result of the carboxylic groups on the SMA molecules. In contrast, this decay was decelerated in membranes which have altered lipid compositions and lower charge densities, as this reduces reprotonation rates for key intermediates in the reaction pathway. Hence, both lipid and polymer charge can affect signaling, providing an impetus for designing uncharged or zwitterionic copolymers to retain endogenous activity. This protein has also been solubilized into larger nanodiscs (up to 35 nm) using DIBMA copolymer [Another study focused on the sensory rhodopsin II of opolymer . The phoopolymer . The ratFlavobacterium johnsoniae that was solubilized using 1% SMA3000 as well as XIRAN 25010 copolymer [The confluence of native nanodisc and cryo-EM technologies has allowed determination of several memtein structures, revealing the positions of asymmetric bilayer leaflets and endogenous post-translational modifications in multisubunit complexes. The first memtein to be resolved was Alternative Complex III (ACIII), a 464 kDa complex from opolymer , which iw/v), and were more pure and stable than with detergent preparations [E. coli revealed the associated asymmetric bilayer at high resolution [E. coli membrane. A set of 31 lipid molecules form visible hexagonal patterns in the inner leaflet and ordered disarray in the outer leaflet. The first structure of the Salmonella (S.) typhimurium AcrB has been resolved at 4.6 Å following solubilization in SMA, allowing the conformational effects of disease-resistant mutation and dynamic changes to be modelled [The superfamily of ATP-binding cassette (ABC) transporters contains targets involved in cancer drug resistance and genetic diseases including adrenoleukodystrophy and cystic fibrosis. Five mammalian transporters were solubilized using SMA2000 (2.5% arations . The humsolution . Followimodelled . This prmodelled  within tw/v), and purified by metal affinity and size exclusion chromatography for cryo-EM analysis. Multiple structures of agonist-bound open, closed, and desensitized states were only achievable in SMALPs, with resolutions down to 2.9 Å. This allowed the positions of the bound glycine agonist, inhibitory γ-aminobutyric acid (GABA) neurotransmitter and taurine to be elucidated, with binding activities quantified with scintillation-proximity assay (SPA) [Xenopus oocytes and HEK cells also forms a pentamer that can both be solubilized with SMA(3:1) copolymer (2% w/v). Comparison of the agonist binding responses indicates significant differences between the two preparations, suggesting that HEK cell lipid compositions are better suited for pore opening [Ion channels form selective pores for ions to cross the membrane in response to changes in chemical stimulation, mechanical forces or temperature, and play roles in a wide range of physiological processes, including neuronal disorders. These multisubunit proteins respond to signaling lipids, which in some cases can regulate channel opening . The zebay (SPA) . The α1  opening .Bacillus subtilis KimA protein is a K+/H+ symporter that has been solubilized with SMALP 30010 [2 and water vapor in plants is mediated by the slow anion channel 1 (SLAC1). The structure of this channel from a brome grass has been resolved at 2.97 Å after expression in Schizosaccharomyces (S.) pombe and solubilization with SMA copolymer [The acid-sensing ion channel ASIC1 is found in neurons and has been solubilized and resolved with bound lipids using SMALP 30010 copolymer. The structure of the trimeric channel was determined at pH 7 and 8 to resolutions of ~2.8 and 3.7 Å, respectively. These illuminate the long-lived desensitized and resting state conformations, which are distinguished by the cavities and re-entrant loop positions within the channel. Several lipid densities are also uniquely evident in the SMALP preparations, indicating the positions of biological-source lipids along the transmembrane helices . The BacLP 30010 . This alopolymer . The pos2 lipids indicate effects on pore conformations and specific activation [2 and calmodulin with the Ca2+ selective channel TRPV5 in its open state has also been resolved by cryo-EM, revealing structural rearrangements around the gate [2 ligand reveals that this lipid occupies a distinct interfacial cavity, indicating variable mechanisms for lipid-activation in this family [+ channel bound to PIP2 show how this lipid induces conformational changes and influences channel gate opening [Lipids including PIs are also known to modulate activities of various channels based on patch-clamp studies . Most sttivation ,75, and the gate . The clos family . Crystal opening ,79. High opening .E. coli and solubilization with DIBMA copolymer. The heptamer was resolved by cryo-EM to 3.0 Å, displaying a bilayer of lipid molecules including a contact with a key Lys residue [The structure of the mechanosensitive channel YnaI has been determined in several states following expression in  residue . The for residue , suggest2AR) is a prototypical and ubiquitous class A receptor, of interest in the pharmacological treatment of specific cardiovascular disorders, immune response and wound repair, cancer, and central nervous system disorders [2AR, ligand affinities are typically an order of magnitude lower in detergents than in liposomes or cell membranes. This implies that the cell or membrane environment plays a significant role in modulating receptor function. As GPCR reconstitution efforts have improved, it has become possible to directly interrogate the stabilizing effects of phospholipid mixtures over detergent [2AR with the alpha-subunit of the G protein is largely driven by electrostatic interactions which would likely be screened by the acidic nature of early-generation polymeric scaffolds.The adenosine A2A receptor  copolymer could be used to rapidly convert membranes into native nanodiscs (SMALPs) for structure–function analysis  has heraGrafting of different polar sidechains can enhance polymer solubility and may offer handles for visualization and purification , Table 1Another goal has been the unambiguous detection of copolymer concentrations in a manner that does not overlap other signals of protein cargo. A variety of fluorescent or affinity tags can be attached to a thiol-containing version such as SMA-SH in order to detect and purify polymers and native nanodiscs . In DIBMThe need to enhance resolution has led to efforts to reduce the dispersity in comonomer sequence distribution, composition and chain length (Ð) of copolymers. Comparative studies of compositionally defined SMA copolymers shows that the kinetics of lipid solubilization kinetics is dependent on the length of copolymer chains, with shorter copolymers solubilizing membranes faster than longer ones . AcrylicE. coli membrane protein PagP [The use of methyl-substituted stilbenes in copolymerization reactions (rather than styrene) along with maleic anhydride generates STMA copolymers that possess semi-rigid backbones ,101,102.ein PagP . FurtherA significant body of evidence of how native nanodiscs can be produced by a family of SMA-related polymers has emerged over the past decade. This is allowing researchers to make new inroads into the structural biology and drug discovery applications of native membrane assemblies. The range of polymer types is allowing structure–activity relationships to emerge, informing the development of new polymers that are zwitterionic, tagged, more homogeneous, conformationally restrained and/or circularized for giving optimal performance for demanding transmembrane targets. Such tools are allowing the preparation of endogenous complexes that are unsuitable for detergent extraction or reassembly for structure–function analysis and high-throughput screening. Challenges remain including the isolation of in vivo states of mammalian protein multimers that are poorly expressed or metastable as well as the transient complexes with signaling partners. Some membrane assemblies present technical issues due to reactivity or fragility and still require careful optimization in order to obtain sufficient material for biophysical analysis, and some polymers may influence activities of proteins and affinity tags due to spurious interactions. While the polydispersity of synthetic polymers may be useful for solubilizing the mixed populations of memteins found in cells, this property can limit the resolution of structural data. The sizes of discs may be limiting in the cases of very large targets, although they generally appear to be able to adapt to wrap variously shaped protein cargo bound to a bilayer of lipid. Polymers with integrated tags are being designed for much more convenient purification and visualization of membrane proteins with minimal impact of their function. Such tools could prove useful in screening campaigns for small molecule and antibody ligands, and may unlock a wide array of applications in fields ranging from proteomics, lipidomics and metabolomics. The recent effort of the SMALP community to design improved polymers to enable memtein structure determination is geared to support the discovery of therapeutic agents for native-state targets as well as ongoing advances in fundamental research into membrane biology.


Succinate dehydrogenase (Complex II) plays a dual role in respiration by catalyzing the oxidation of succinate to fumarate in the mitochondrial Krebs cycle and transferring electrons from succinate to ubiquinone in the mitochondrial electron transport chain (ETC). Mutations in Complex II are associated with a number of pathologies. SDHD, one of the four subunits of Complex II, serves by anchoring the complex to the inner-membrane and transferring electrons from the complex to ubiquinone. Thus, modeling SDHD dysfunction could be a valuable tool for understanding its importance in metabolism and developing novel therapeutics, however no suitable models exist.SDHD produced significantly less number of cells in culture. The mutant cells predictably had suppressed Complex II-mediated mitochondrial respiration, but also Complex I-mediated respiration. SDHD mutation also adversely affected glycolytic capacity and ATP synthesis. Mutant cells were more apoptotic and susceptible to necrosis. Treatment with the mitochondrial therapeutic idebenone partially improved oxygen consumption and growth of mutant cells.Via CRISPR/Cas9, we mutated SDHD in HEK293 cells and investigated the in vitro role of SDHD in metabolism. Compared to the parent HEK293, the knockout mutant HEK293ΔOverall, our results suggest that SDHD is vital for growth and metabolism of mammalian cells, and that respiratory and growth defects can be partially restored with treatment of a ubiquinone analog. This is the first report to use CRISPR/Cas9 approach to construct a knockout SDHD cell line and evaluate the efficacy of an established mitochondrial therapeutic candidate to improve bioenergetic capacity. Mitochondria generate the majority of adenosine triphosphate (ATP) through the electron transport chain (ETC). Succinate dehydrogenase  uniquely serves as a component of both the Krebs cycle and the ETC . As a coComplex II carries four protein subunits , all of We successfully used a CRISPR/cas9 approach to mutate the SDHD subunit of Complex II in HEK293 cells, and characterized its requirement for mitochondrial respiration, ATP synthesis, and cell growth in vitro. Furthermore, we demonstrate the efficacy of the mitochondrial therapeutic idebenone to improve mitochondrial dysfunction in cells with SDHD mutation. Our results demonstrate the necessity of SDHD for energetic homeostasis and suggests it as a viable target for therapies aimed to improve mitochondrial function.SDHD nucleotide sequence of Homo sapiens (GenBank locus ID KR710199.1) using Integrated DNA Technologies’ online tool . A 118-bp site of the forward strand of SDHD was predicted as the most reliable for mutation with an on-target specificity score of 68% and off-target score of 58%. The guide strand predicted was TCTGTTGCTTCGAACTCCAG, which is located right upstream of the protospacer adjacent motif (PAM) sequence TGG . Our mutant HEK293ΔSDHD was found missing both 17 kDa and 29 kDa protein bands  sequences were designed based on the 609 bp long SDHD region encompassing the CRISPR targeting site (at 118-bp) of the mutant. The results indicated that an INDEL mutation has been generated at a site two bases upstream of the GTT PAM sequence. Two bases (a C and an A) have been.We then PCR amplified and sequenced the deleted from this site and one base (a G) has been inserted at the deletion site new Fig. c. As a rUsing the online CRISPR prediction site of the Integrated DNA Technologies, we identified the top three potential off-target sites Table . The DNASDHD altered the expression of other subunits of the five ETC complexes. Expression of complex II subunit SDHB  suggesting that SDHD expression has direct impact on expression of other subunits of the complex II. Moreover, expression of complex I subunit NDUFB8  suggesting an association in protein expression between complexes II and other ETC complexes. Nevertheless, the expression of complex III subunit UQCRC2  and 72 h (p < 0.0001) after addition of the substrate. These observations suggest that disruption of SDHD results in subsequent decrease of ROS generation over time. It is possible that leakage of electrons due to impaired complex II caused increased ROS production at early stages of cell growth . Nevertheless, the severe impairment of electron transport may have caused decreased overall electron leak and subsequent ROS generation at late stages of growth . At all the time points, the mutant displayed significantly increased apoptosis . Subsequent to rotenone treatment, oxygen consumption of both parent and mutant decreased, but the oxygen consumption of mutant cells was significantly less than that of parent cells (p = 0.0101). In response to treatment with succinate, Complex II-mediated oxygen consumption was significantly repressed in mutant cells compared to parent cells (p = 0.0002), further confirming the effect SDHD mutation. The mutant also had decreased OXPHOS.Oxygen consumption was measured using Oroboros Oxygraph 2 k. Mutation in SDHD decreased oxygen consumption all along the ETC Fig. a and f. (p = 0.0002) and maximal respiration (p = 0.0017). Oxygen consumption of permeabilized cells was also assessed using an Agilent Seahorse XFe96 Analyzer  of intact cells was determined using an Agilent Seahorse XFe96 Analyzer Fig. a. Both gSDHD cells by Seahorse XFe96 and O2k respirometry. In Seahorse assay, treatment of permeabilized mutant cells with 1 μM idebenone significantly increased basal respiration , which peaked after the second dose of 5 μM idebenone   decreased significantly suggesting interdependency in expression among different subunits of this complex. Quite interestingly, the expression of a complex I subunit (NDUFB8), a complex IV subunit (MT-CO1), and a complex V subunit (ATP5A) also decreased significantly in the mutant suggesting an association in protein expression between complex II and other complexes. Nevertheless, the SDHD mutation did not alter expression of the complex III subunit UQCRC2 suggesting independency of this complex from complex II in terms of protein expression.Herein, we successfully used a CRISPR/Cas9 approach to mutate e region , 2, in HSDHD disrupted respiration all along the ETC, suppressed glycolysis and overall ATP synthesis, as well as limited cellular growth. Importantly, acute treatment with the synthetic ubiquinone analog idebenone was sufficient to improve Complex II-mediated mitochondrial respiration, OXPHOS capacity, and cell proliferation.Due to the role of Complex II in the ETC and Krebs cycle –3 as welPredictably, mutation of SDHD inhibited Complex II-mediated respiration. Interestingly, however, mutation in SDHD significantly reduced Complex I-mediated respiration as well. Since Complex II drives Krebs cycle in a clock-wise direction by converting succinate to fumarate, we hypothesize that mutation of SDHD slowed and/or disrupted Krebs cycle production of NADH, the substrate for Complex I. Mutation in SDHD also impaired OXPHOS capacity, maximal respiration, and ATP synthesis. Taken together, impairment in the SDHD subunit of Complex II is sufficient to impair overall ETC energy production.SDHD compared to parent cells. Reduced glycolysis may reflect a lower demand for pyruvate due to disrupted Krebs cycle as a result of mutation in SDHD, representing a potential negative feedback on glycolysis. Also, inhibition of mitochondrial respiration with oligomycin did not elicit a change in glycolysis in mutant cells, further reflecting impaired mitochondrial respiration. In addition to the suppressed metabolism, we also noted significantly slower growth in mutant cells compared to parent cells in culture media, which is likely reflective of the metabolic impairment in mutant cells.Glycolysis, as determined by ECAR, was significantly reduced in HEK293Δ2 to generate ROS [2, the two electron donors to the ETC. Thus, limited donation of electrons may have led decreased electron transport through the ETC as well as decreased electron leakage out of the ETC giving rise to decreased ROS generation.Mutation of SDHD increased apoptosis and necrosis. The impaired glycolysis and mitochondrial respiration may have weakened the cell and cell membrane of the mutant making the cells more apoptotic and susceptible to necrosis. Quite interestingly, the long-term ROS generation declined as a result of SDHD mutation. In general, electrons that do not follow the normal order of the ETC pathway and instead leaked out are eventually transferred directly to Orate ROS , 14. In SDHD cells whereas 5 μM did not. Idebenone is known to have cell-type specific effects on cell proliferation [SDHD cells with idebenone is likely a result of improved substrate utilization, cell type should be taken into consideration for mitochondrial targeted therapies as to how other process may be influenced.Finally, we explored whether SDHD mutation could be bypassed to improve some of the phenotypes observed. Idebenone, is a short-chain benzoquinone that is more hydrophilic than ubiquinone , and hasferation –25. WhilWe generated a novel mutant of the Complex II subunit SDHD via CRISPR/Cas9 that resulted in severe augmentation to mitochondrial respiration and cell metabolism as well as suppressed growth. This novel tool could be valuable for testing potential mitochondrial-focused therapeutics as well as elucidating other mechanisms regulated by Complex II function.2. A 0.25% trypsin-EDTA solution was used for detachment of cells. One Shot Stbl3 Chemically Competent cells of Escherichia coli (Life Technologies Corporation) were used for constructing the mutagenesis plasmid. Bacteria carrying the plasmids were maintained in Luria Bertani  agar or broth, and sustained in a humidified incubator at 37 °C.Human embryonic kidney cell line 293 (HEK293) was kindly provided by Dr. Joseph Ruiz at Enzerna Biosciences . Media and reagents for growing and maintaining cells were purchased from Life Technologies Corporation . The cells were maintained in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% (by volume) fetal bovine serum and 1% penicillin-streptomycin. Cells were sustained in a humidified incubator at 37 °C and 5% COSDHD were picked using procedures described elsewhere [SDHD . sgRNA oligonucleotides were cloned into plasmid px458  and HEK293 cells were transfected with recombinant px458 plasmid using transfection reagent Xfect . Clones with mutation in lsewhere . DisruptDHD Fig. .SDHD DNA region including the targeted mutation site was PCR amplified using the primers shown in Table The Expression of protein subunits representative to each of the five ETC complexes was examined by Western immunoblotting using Total OXPHOS Human WB Antibody Cocktail . Since the expression of complexes I and IV subunits was not clearly visible in Western blots with this antibody, the expression of these two complexes was examined separately by Western immunoblotting using antibodies to NDUFB8  and MT-CO1 .SDHD were introduced into 75 cm2 flasks each carrying 25 ml growth media. After 4 days of incubation, cell numbers were quantified in triplicates. Generation of reactive oxygen species (ROS) was measured using ROS-Glo H2O2 Assay . Apoptosis and necrosis were assessed using RealTime-Glo Annexin V Apoptosis and Necrosis Assay . Extracellular Acidification Rate (ECAR) was determined with an Agilent Seahorse XFe96 analyzer [Aliquots of 100,000 cells of the parent HEK293 and the mutant HEK293Δanalyzer , 28, andanalyzer , 31. CelSDHD was evaluated. In this procedure, mutant cells were cultured on 6-well plates at 2000 cells/well in DMEM medium supplemented 1, 5, or 10 μM of idebenone. Fresh media containing the drug was added on days 3, 6 and 9, and the cell counts in wells of triplicates was determined following 10 days incubation. Efficacy of idebenone in improving respiration of permeabilized mutant cells was measured by O2k respirometry as described above with the following modification: subsequent to succinate injection, idebenone was injected into chambers at 1 or 5 μM final concentration. The control groups received DMSO. Moreover, effectiveness of idebenone in improving OCR of permeabilized cells was assessed with the Agilent Seahorse XFe96 analyzer as outlined above with the following modification: cells grown overnight in XFe96 culture plates were washed, and glucose-free DMEM (pH 7.4) supplemented with 10 mM glucose, 10 mM sodium pyruvate, 2 mM glutamine, 20 μg/ml saponin and 1 μM of idebenone was added to the wells prior to incubating for 40 min at 37 °C in a CO2-free station. The control wells received the same medium added with DMSO but without idebenone.Effectiveness of the potential mitochondrial therapeutic idebenone in restoring the growth defects of the mutant HEK293Δp < 0.05.Student’s t-tests were performed using Microsoft Excel program  or GraphPad Prism 7  to compare the means of the parent versus mutant or vehicle control versus idebenone treatment. The ECAR data were analyzed by repeated measures ANOVA using GraphPad Prism 8. Mean differences between groups were considered statistically significant at