Trametes species are efficient wood decomposers that are widespread throughout the world. Mitogenomes have been widely used to understand the phylogeny and evolution of fungi. Up to now, two mitogenomes from the Trametes genus have been revealed. In the present study, the complete mitogenomes of two novel Trametes species, Trametes versicolor and T. coccinea, were assembled and compared with other Polyporales mitogenomes. Both species contained circular DNA molecules, with sizes of 67,318 bp and 99,976 bp, respectively. Comparative mitogenomic analysis indicated that the gene number, length and base composition varied between the four Trametes mitogenomes we tested. In addition, all of the core protein coding genes in Trametes species were identified and subjected to purifying selection. The mitogenome of T. coccinea contained the largest number of introns among the four Trametes species tested, and introns were considered the main factors contributing to size variations of Polyporales. Several novel introns were detected in the Trametes species we assembled, and introns identified in Polyporales were found to undergo frequent loss/gain events. Large-scale gene rearrangements were detected between closely related Trametes species, including gene inversions, insertions, and migrations. A well-supported phylogenetic tree for 77 Basidiomycetes was obtained based on the combined mitochondrial gene set using 2 phylogenetic inference methods. The results showed that mitochondrial genes are effective molecular markers for understanding the phylogeny of Basidiomycetes. This study is the first to report the mitogenome rearrangement and intron dynamics of Trametes species, which shed light on the evolution of Trametes and other related species. Trametes species, belonging to Polyporales, Basidiomycota, are widely distributed throughout the world1. Trametes species are often distributed on standing dead or fallen hardwood trees2. Like other species from the order Polyporales, Trametes species are efficient wood decomposers that cause white rot in colonized wood. Their efficient lignin decomposition ability makes Trametes important decomposers in ecosystems and plays an important role in the natural cycle of earth forest ecosystems. In addition, Trametes species have also been used in ex situ and in situ biosynthesis and bioremediation studies6. A series of lignin-degrading enzyme systems and genes have been found to be closely related to the excellent degradation ability of Trametes9, including lignin peroxidases, manganese peroxidases, and laccases. To date, only two mitochondrial genomes (mitogenomes) of Trametes species have been reported11, including T. cingulata and T. hirsute, and the characterizations and differentiation of mitogenomes in Trametes have not been fully analyzed.13. The variation or mutation of mitogenomes can affect the growth, metabolism, and development of eukaryotes, leading to their aging and even death15. In addition, mitogenomes are also an effective tool to understand the evolution and phylogeny of eukaryotes17. Other characteristics of mitogenomes, such as genome size, intron type, tRNA genes, gene arrangement, and repeat sequences, have also become important references to reflect the evolutionary status of species21. The size, structure, and content of fungal mitogenomes vary greatly24, which has made it difficult to obtain complete fungal mitogenomes. When compared with the available fungal nuclear genome, the number of available fungal mitogenomes is very low (https://www.ncbi.nlm.nih.gov/genome/browse#!/overview/). The rapid development of high-throughput sequencing technologies, such as 454 pyrosequencing, Illumina (Solexa) sequencing, ABI SOLiD sequencing, Oxford Nanopore sequencing, and Pacbio SMRT sequencing, has promoted understanding of fungal mitogenomes.Most eukaryotes contain mitogenomes, which are thought to be derived from Alphaproteobacteria through endosymbiosisTrametes species, Trametes versicolor and T. coccinea, were assembled and compared. The goals of this study were to: (1) characterize Trametes mitogenomes; (2) reveal the variations or similarities in genome size, structure, and gene content of the Trametes mitogenomes; (3) reveal the dynamic changes of introns in Polyporales mitogenomes; and (4) shed light on the phylogenetic relationships of Trametes in the phylum Basidiomycota based on the combined mitochondrial gene set. The results of this study will promote improved understanding of the origin, evolution, and taxonomy of Trametes species and other related fugal species.In the present study, the mitogenomes of two T. versicolor and T. coccinea were circular with sizes of 67,318 bp and 99,976 bp, respectively  and large subunit ribosomal RNA gene (rnl) . The T. versicolor mitogenome contained longer rnl and shorter rns genes than the T. coccinea mitogenome.Both the l) Table . We deteTrametes mitogenomes we assembled each contained 27 tRNA genes  varied in length between the 4 Trametes mitogenomes tested  rate, whereas the nad4L gene had the lowest Ka mean value among the 15 core PCGs detected. We also found that the nad3 gene had the highest mean synonymous substitution rate (Ks), whereas the atp8 gene exhibited the lowest Ks mean value among the 15 core PCGs detected. The Ka/Ks values for all core PCGs were less than 1, indicating that the core genes in Trametes species were subjected to pressure of purifying selection.The largest K2P genetic distance between ion Fig. . The atpPolyporales  using the cox1 gene of Ganoderma calidophilum as a reference22, and introns belonging to the same Pcls were considered homologous introns. Overall, 153 introns were detected in the 14 cox1 genes of the order Polyporales, and the number of introns in each cox1 gene varied between 6 and 15, with an average of 11. Five of the 153 introns belonged to the group II, and the other introns belonged to the group I.Pearson correlation analysis showed that there was a high correlation coefficient between the number of introns and the size of the mitogenome in the order les Fig. . The dynPolyporales species  were only detected in one of the 14 Polyporales species. The rare introns in Polyporales, such as the P480 and P941, were also detected in distantly related species, such as Armillaria sinapina28 and Agaricus bisporus29 from the order Agaricales. These results indicated potential horizontal gene transfer events occurred during evolution. However, P218, P309, P276, P894, and P1114 were only distributed in Polyporales, and no homologous introns were found in other Basidiomycota species; accordingly, the origins of these rare introns need to be further investigated. P1107 was the most commonly distributed intron type in the four Trametes species we tested and was present in all four Trametes species. We detected two novel introns in our newly sequenced T. coccinea species, indicating diverse intron types in Trametes species. The varied classes and number of introns in Trametes species indicated that intron gain/loss occurred during evolution of the Trametes mitogenome.Overall, 32 Pcls were detected in the 14 ies Fig. , with P7T. coccinea was the largest mitogenome among the four Trametes species reported11  and Bayesian inference (BI) methods based on the combined mitochondrial gene set (15 core PCGs) Fig. . All majes Table . T. coccTrametes, T. coccinea and T. versicolor, were newly assembled. Comparative mitogenomic analysis showed that the novel mitogenome of T. coccinea was largest among the four Trametes mitogenomes reported11. Introns were considered the main factors contributing to the size expansion of the T. coccinea mitogenome, and the T. coccinea mitogenome was found to have the most introns among Trametes species. We also found a high correlation coefficient between the mitogenome size and the number of introns in Polyporales35. These findings further suggested that the dynamic change in introns is one of the main factors leading to the size variation of mitogenomes from the order Polyporales and other fungal species, which is consistent with the results of previous studies37. We also found that the mitogenome content of Trametes species varied. The four Trametes mitogenomes varied in AT and GC skews. According to the second parity rule, as long as there is no mutation or selection bias, each base in the complementary DNA strand exists at approximately equal frequencies38. The presence of AT or GC skews on the same DNA strand from different species indicated that mitogenomes of different Trametes species underwent different mutations or environmental selection39. In the present study, the core PCGs of Trametes species were found to vary frequently in length and base composition. Different core PCGs showed varied evolution rates, and some core PCGs were relatively conserved between Trametes species, whereas others showed large differentiation. All core PCGs were found to have been subjected to the pressure of purifing selection. In addition to these core PCGs, some non-conserved PCGs were also detected in Trametes species, including intronic ORFs, DNA polymerases, and RNA polymerases. Introns encoding endonucleases mainly mediate intron homing in Trametes mitogenomes40, while DNA and RNA polymerases are thought to be obtained from plasmids43. Several PCGs with unknown functions were also detected in Trametes species, indicating that there are still genes with unknown functions in Trametes mitogenomes that need to be revealed.In this study, two mitogenomes of the genus 45. Variations in the mitochondrial gene order can be used to infer the phylogenetic status and phylogenetic relationship of eukaryotic species47. The rearrangement of the animal mitogenome has been widely studied, and several models have been proposed to reveal the rearrangement of animal mitogenomes49. When compared with the mitogenomes of animals, the mitogenomes of fungi show greater variation in gene order50. In the present study, we detected large-scale gene rearrangements between Trametes species, including gene inversions, insertions, and migrations. Gene rearrangement events have even been observed between closely related Trametes species within the same clades, indicating that the mitochondrial gene arrangement of Trametes species is highly variable. However, the mechanism of mitochondrial gene rearrangement in fungi has not been elucidated to date. Previous studies showed that the accumulation of repetitive sequences was the main factor contributing to mitochondrial gene rearrangement in fungi50. We found a large number of repeat sequences in Trametes species, which may result in gene recombination and rearrangement in these species.Mitochondrial gene rearrangement was frequently detected in mitogenomes of animals, plants, and fungi53. Variations in mitochondrial introns among different eukaryote lineages vary greatly. Generally, animal mitogenomes do not contain any introns, whereas plant mitogenomes mainly contain group II introns and fungal mitogenomes mainly contain group I introns54. The cox1 gene was found to be the main host gene of introns in mitogenomes of Basidiomycetes56. We divided the introns of cox1 genes of 14 Polyporales species into different position classes (Pcls) according to their insertion sites in the protein coding region. The introns belonging to the same Pcl were considered homologous29. We found that the number and class of introns varied greatly between different Polyporales species, indicating that the loss/gain of introns occurred in the evolution of Polyporales. Several Pcls from common ancestors of Polyporales, such as P706, P807, and P1305, were found to be widely distributed in Polyporales species. However, several introns were found only in one of the 14 Polyporales species, such as P480 and P941, whereas homologous introns were detected in distant species29, indicating potential intron transfer events. We also found some novel introns in Polyporales that have never been detected in other Basidiomycetes, including P218, P309, P726, P894, and P1114. The origin, evolution, and function of these novel introns need to be further analyzed.Introns are considered mobile genetic elements in the eukaryote mitogenome, and their dynamic changes have a significant effect on the size and organization of mitogenomesITS)57, RNA polymerase II subunits (RPB), and elongation factor 1-ɑ (EF1ɑ) genes58 have been widely used to evaluate the phylogeny and reconstruct the early evolution of fungi. Mitochondrial genes have been widely used as molecular markers to analyze phylogenetic relationships of animals because of their unique advantages44. When compared with the mitogenomes of animals, the mitogenomes of fungi have been less studied, especially those of Basidiomycetes59. Currently, less than 130 complete mitogenomes of Basidiomycetes are available in the NCBI database (https://www.ncbi.nlm.nih.gov/genome/browse#!/overview/), which has limited comprehensive analysis of the origin and evolution of fungi. In this study, 77 Basidiomycete species were included in the phylogenic study. We have also obtained a phylogenetic tree with high support values based on the combined mitochondrial gene set, indicating that mitochondrial genes are a powerful tool for analyzing phylogenetic relationships of Basidiomycota species. With the rapid development of second and third generation sequencing technology, additional mitogenomes of fungi are needed to understand the origin and evolution of Basidiomycetes and other fungal lineages.Owing to the limited morphological characteristics, it is difficult to classify and identify Basidiomycetes accurately only by morphology. To date, only rRNA internal transcribed spacers (T. versicolor and T. coccinea mitogenomes assembly were downloaded from the Sequence Read Archive (SRA) database under the accession numbers SRR3927404 and SRR1588030, respectively. We conducted a series of quality control steps to generate clean reads from the raw sequencing reads, including removing adapter reads using AdapterRemoval v260 and filtering low-quality sequences using ngsShoRT 2.261. We used the clean reads to assemble mitogenomes of T. versicolor and T. coccinea using SPAdes 3.9.062. Several contigs were obtained using SPAdes 3.9.062, and gaps between the contigs were filled using MITObim V1.963. NOVOPlasty64 was also used to assemble or test mitogenomes of the two Trametes species. After these steps, we obtained the complete mitogenomes of T. versicolor and T. coccinea, and then annotated them according to previously described methods33. We initially annotated the protein-coding genes (PCGs), open reading frames (ORFs), introns, rRNA genes, and tRNA genes in the two Trametes mitogenomes using MFannot65 and MITOS66. PCGs or ORFs were also predicted based on the NCBI Open Reading Frame (ORF) Finder67 and annotated by BLASTP searches against the NCBI non-redundant protein sequence database68. Intron–exon borders of PCGs or ORFs were verified using Exonerate v2.269. We also used tRNAscan-SE v1.3.170 to predict or identify tRNA genes in the two Trametes mitogenomes. Finally, graphical maps of the two Trametes mitogenomes were drawn with OGDRAW v1.271.The raw sequencing data used for Trametes mitogenomes and other Polyporales mitogenomes were analyzed using DNASTAR Lasergene v7.1 (http://www.dnastar.com/). We calculated strand asymmetries of Polyporales mitogenomes using the following formulas: AT skew = [A – T]/[A + T] and GC skew = [G – C]/[G + C]72. We analyzed codon usages within the two Trametes mitogenomes using the Sequence Manipulation Suite73. We calculated the nonsynonymous (Ka) and synonymous (Ks) substitution rates for core PCGs in the four Trametes mitogenomes using DnaSP v6.10.0174. The genetic distances between each pair of the 15 core PCGs  were detected using MEGA v6.0675 based on the Kimura 2-parameter (K2P) substitution model.Base compositions of the two Trametes mitogenomes against themselves76 to determine whether there were intragenomic duplications of large fragments or interspersed repeats in the two Trametes mitogenomes. We also detected tandem repeats (> 10 bp in length) in the two Trametes mitogenomes using a Tandem Repeats Finder77 with the default parameters. In addition, BlastN searches of the two Trametes mitogenomes were conducted against their published nuclear genomes to determine whether there were gene segments that naturally transferred between mitochondrial and nuclear genomes79.We conducted BlastN searches of the two Polyporales mitogenomes were compared to identify variations and similarities between them. Introns of cox1 genes in the 15 Polyporales mitogenomes that have been published to date were classified into different position classes (Pcls) according to the method described by Ferandon et al.80. The cox1 genes of 15 Polyporales mitogenomes were first aligned with the cox1 gene of the medical fungus Ganoderma calidophilum22 using Clustal W81. Each Pcl was composed of introns inserted at the same position of the cox1 reference gene based on the insert sites (nt) in the corresponding reference gene. Identical Pcls were considered orthologous introns with high sequence similarity.The genome sizes, GC contents, base compositions, start and stop codons, gene numbers, and intron numbers of Trametes species in the phylum Basidiomycota using previously described methods82 based on the combined mitochondrial gene set (15 core PCGs). We used Annulohypoxylon stygium from the phylum Ascomycota as the outgroup83. Individual mitochondrial genes were first aligned using the MAFFT v7.037 software84, after which we concatenated the aligned mitochondrial genes into a combined mitochondrial gene set using the SequenceMatrix v1.7.8 software85. A preliminary partition homogeneity test was then conducted to detect potential phylogenetic conflicts between different genes. Best-fit models of partitioning schemes and evolution for the combined mitochondrial gene set were determined based on the PartitionFinder 2.1.1 software86. We constructed phylogenetic trees using both Bayesian inference (BI) and maximum likelihood (ML) methods, with MrBayes v3.2.687 used for the BI analysis and RAxML v 8.0.088 for the ML analysis.A phylogenetic tree of 77 Basidiomycota species was constructed to investigate the status of T. versicolor and T. coccinea were deposited in the GenBank database under accession numbers MT479165 and MT479166, respectively.The complete mitogenomes of Supplementary Information.


Anterior cruciate ligament (ACL) rupture is a common injury in young and active population and reconstruction is the mainstay treatment to maintain stability of the knee. Hamstring tendon (HT) is the most popular graft for ACL reconstruction. In certain patient, particularly in women, the size of tendon graft to reconstruct the ACL is unpredictable. In the case of unqualified donor, we need a combined graft from another source to match the size requirement.We present two cases of ACL reconstruction using combination of HT and PLT graft in adolescent female. We evaluate knee functional score and ankle donor site morbidity. In the second case we also did second look for the ACL graft integrity arthroscopically. This study has been reported in line with the Surgical Case Report (SCARE) criteria.One year after surgery, International Knee Documentation Committee (IKDC) score for both cases were improved and no ankle-donor site morbidity with good motoric power and 100% American Orthopedic Foot Ankle Score (AOFAS).Additional peroneus longus tendon can be considered as an alternative in undersized hamstring tendon graft without significant donor site morbidity. HamstriIn this report, we combined HT and PLT graft in two adolescence female patients with similar characteristic and evaluated the knee and ankle stability parameters. Written consent was obtained from parents for publication of this case report and accompanying images. The authors declare no conflicts of interest. This work has been reported in line with the SCARE criteria .2Two patients presented to our center with knee instability following a twisting injury. The first patient was 15-year-old female with 50 kg of weight and 150 cm in height with a confirmed ACL rupture on Magnetic Resonance Imaging (MRI). The second patient was 16-year-old female with 55 kg weight and 155 cm height with a confirmed ACL rupture and meniscus injury on MRI. We performed ACL reconstruction only in the first patient and ACL reconstruction with meniscus repair in the second patient.2.12.1.1After drawing the osseous landmark at patella and tibial tubercle, an oblique 2 cm incision was made along the pes anserinus between tibial tubercle and posteromedial border of tibia. Gracilis and semitendinosus tendons were identified and harvested . The dis2.1.2PLT was harvested through an incision along the posterior border of the distal fibula, above the superior peroneal retinaculum. The tendon was exposed on its posterolateral surface. The distal end was cut and held with suture and the proximal end was released by tendon stripper .Fig. 2PeIn the first patient, HT graft by combining gracillis and semitendinosus tendon was only 5 mm in diameter with 13 cm length after quadrupled . This 5 Meniscus was repaired in the second patient and ACL reconstruction was planned with PLT graft considering the activity level and the similarity of body characteristic as the previous patient. We harvest the PLT with the same manner as previous patient. The tendon was surprisingly insufficient 6 mm × 10 cm (after doubled). We decided to take the HT as augmentation and a final 7.5 mm × 10 cm six strand graft was obtained.In both patients, autografts were fixed to the femoral tunnel with Endo-button (Smith & Nephew) and to the tibial tunnel with interference screw (Smith & Nephew).Post operatively, the first patient had the knee immobilized in 0° extension in a week and gradually increase until 90° flexion in the second week. Afterwards the brace was set in flexible range so that patient could start the active and passive range of motion exercise. Weight bearing was increased as tolerated. The second patient had a combined ACL reconstruction and meniscus repair rehabilitation protocol. In both patients, gentle ankle stretch was encouraged since the first post-operation day by using a resistance band. Proprioceptive exercises were performed 3 months after surgery which were gradually increased in intensity until another 3 months.2.2Pain and stability at knee and ankle joint subjectively and objectively evaluated at one-year post operatively. We used the visual analogue scale (VAS) to measure pain before and after surgery. Knee and ankle stability were evaluated using the International Knee Documentation Committee (IKDC) 2000 Standard Evaluation Form and American Orthopedic Foot and Ankle Society (AOFAS) Ankle-Hindfoot Score respectively. The power of foot eversion and first ray plantar flexion was examined using Medical Research Council (MRC) scale for muscle strength and compared to the normal sides on contralateral ankles. Other donor-site morbidities at ankle region were also recorded such as numbness or proximal stump irritation. In the second patient ACL graft was re-evaluated on the second look 8 months after the reconstruction.33.1In the first patient, knee pain was VAS 3–4 before surgery and VAS 0 on one year follow up. IKDC Score 100% at one year follow up. In the second case, the knee pain was VAS 3 before surgery, and now only experienced mild discomfort. IKDC score was 95.4% at one year follow up.3.2Both of patient knees were unstable pre-operatively as observed by the positive measurement on anterior drawer test, pivot shift test and Lachman test (+3). Both patients had negative Lachman and anterior drawer test in one year follow up.3.3In both patients, no complaint regarding the ankle with good motoric power  and no limitation of ankle range of motion . The 1003.4The second patient agreed to undergo second-look arthroscopy 8-months after ACL reconstruction. Graft was taut on probing, completely covered by synovium, and no tear was observed .Fig. 4AC4Besides females have a more common ACL injury than males, they also have some special considerations regarding to the reconstruction . Female The common problem regarding the reconstruction in female is usually the graft size mismatch ,13. To ePLT is one of the important supportive structure to provide stability of the foot and ankle. Chayanin et al.  study noThis study used PLT as augmentation graft. IKDC scores were improved to normal. The AOFAS score were excellent, similar with previous result by Chayanin et al.  and RhatLimitations of this study include are small number of participants and the need of quantified data for the ankle muscle power with longer period of evaluation regarding the long-term ankle morbidity outcome.5Uncertainty in predicting graft size urged the preoperative planning to prepare graft augmentation. Combining hamstrings tendon with peroneus longus tendon could be an alternative for undersized graft without a significant ankle-donor site morbidity.The authors report no declarations of interest.The authors report no external source of funding during the writing of this article.Ethical approval was not required in the treatment of the patient in this report.Written informed consent was obtained from the parents for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.Dina Aprilya and Kanya Tania contribute to the study concept or design, data collection and writing the paper.Andri MT Lubis contributes in the study concept or design, data collection, analysis and interpretation, oversight and leadership responsibility for the research activity planning and execution, including mentorship external to the core team.Andri MT Lubis is the sole guarantor of this submitted article.Not commissioned, externally peer-reviewed.


Lightweight materials are finding plentiful applications in various engineering sectors due to their high strength-to-weight ratios. Hole-making is an inevitable requirement for their structural applications, which is often marred by thermal damages of the drill causing unacceptable shortening of tool life. Efficient cooling of the tool is a prime requirement for enhancing the process viability. The current work presents a novel technique of cooling only the twist drill between drilling of holes with no effect of the applied cryogenic coolant transferred to the work material. The technique is applied in the drilling of two commonly used high-strength lightweight materials: carbon fibers reinforced polymer (CFRP) and an alloy of titanium (Ti-6Al-4V). The efficacy of the cooling approach is compared with those of conventionally applied continuous cryogenic cooling and no-cooling. The effectiveness is quantified in terms of tool wear, thrust force, hole quality, specific cutting energy, productivity, and consumption of the cryogenic fluid. The experimental work leads to a finding that between-the-holes cryogenic cooling possesses a rich potential in curbing tool wear, reducing thrust force and specific energy consumption, and improving hole quality in drilling of CFRP. Regarding the titanium alloy, it yields a much better surface finish and lesser consumption of specific cutting energy. Hole-making in alloys and composites is a primary requirement of their structural applications. Drilling, by far, is the most commonly used method for creating holes. Just like other machining processes, drilling also generates process heat, which should be efficiently dissipated to keep the progress of tool wear in check and obtain cutting of high-quality holes . Unlike Ti-6Al-4V alloy and carbon fiber reinforced polymers (CFRP) are the most commonly used engineering materials possessing high strength-to-weight ratios. Their high resistance to corrosion makes them even more attractive for the structural applications. Both materials undergo cutting of holes at a huge scale, globally. The materials find extensive applications in automotive, aerospace, aircraft building, and marine engineering sectors. The high strengths and large-scale applicability of the materials make them ideal for carrying out investigations regarding effectiveness of cryogenic cutting fluids and their modes of application. A brief review of the published literature is provided below in respect of the application of cryogenic fluids in drilling of titanium alloys and CFRPs.2) whose working temperature is around −196.5 °C. An experimental investigation was carried out to study the effects of applying LN2 in drilling of a titanium alloy using an inserted carbide twist drill. It was found that the application of the cryogenic fluid resulted in temperature drop effectively on one hand but degraded the hole quality on the other [2 is reported to have significantly reduced tool wear leading to a positive gain in cutting speed [2, as a cryogenic fluid, is also reported to have positive impacts on machining viability [2 is caused by reduction in chipping and crack propagation. An increase in flow rate of LN2 is found to have prolonged the tool life [2 and compressed CO2 gas in side-and-end milling of Ti-6Al-4V is investigated [2) and emulsion cooling in respect of cutting temperature, thrust force, dimensional accuracy, and surface roughness when applied to the drilling of ASTM B265 Grade 2 titanium alloy [2-MQL and CO2-MQL hybrid lubro-coolants yielded highly favorable results regarding tool wear and surface finish, respectively.Cryogenic cooling involves application of a fluid operating at a temperature lower than −150 °C to the cutting zones in order to efficiently dissipate process heat. The most commonly utilized cryogenic fluid in the machining domain is liquid nitrogen , design of experiments, experimental setup, tooling, and measurements.3, 4.9 GPa, and 240 GPa, respectively. The densities of the epoxy resin and the resulting composite are 1.104 and 1.38 g/cm3, respectively. The resin is so normalized to yield 60% of fiber volume.The performance of the novel cryogenic cooling approach is tested upon drilling of a high-strength alloy and a composite material. Their pertinent details are provided below. The composite material is T700G, a bi-directional 0°/90° weaved carbon-fiber composite plate. Thirty three plies are stacked up to yield 22.5 mm thickness of the CFRP plate. T700S high-strength non-twisted carbon yarns of 7 µm diameter are packed into Rhino 1411 epoxy resin. The density, tensile strength, and tensile modulus of the carbon fibers are 1.8 g/cmA commonly used α + β alloy of titanium, Ti-6Al-4V is the alloy tested in this work. The dimensions of the work material plate are 100 mm × 200 mm × 19 mm. The annealed form of the work material is obtained by holding the soaking temperature of the plate between 777 and 783 °C for about 70 min followed by air cooling. The drilling tools used in the 12 runs are FIREX (multiple layers of TiAlN and TiCN) coated tungsten carbide stub drills. The ceramic coating imparts the features of extra heat resistance and shock resistance to the drills. The drills possess the number of flutes, point angle, cutting diameter, total length, and fluted length of 2, 140°, 8 mm, 79 mm, and 41 mm, respectively. Each drill is held in a collet 45 mm away from the chisel edge before being clamped in the tool holder.2; and (c) Between-the-holes cooling using LN2.Cooling option. The following three techniques are tested for each of the two work materials: (a) Dry drilling (no cutting fluid is used); (b) Continuous cooling using a jet of LNVc). The low and high levels of this parameter are 100 and 150 m/min and 13 and 20 m/min, respectively, for CFRP and Ti-6Al-4V.Cutting speed  experimental runs for each of the two work materials. The experiments are run in a single replicate. The two levels of cutting speed for each of the two work materials are finalized after performing preliminary runs. A cutting speed in excess of 20 m/min resulted in complete rupture of drills in hole-making of the titanium alloy. For CFRP, a cutting speed beyond 150 m/min caused a jump in tool’s abrasion and surface deterioration around the drilled holes. Therefore, the high levels of the predictor are fixed to these values of cutting speed so as to ensure viable production rates. Each of the 12 experimental runs involves drilling of five 8 mm diameter thru-holes using the same twist drill. A new twist drill is used for each run. The following parameters (response variables) are determined for each experimental run.VB (µm), to be evaluated after drilling five holes in the work material;Tool damage, measured as the average width of flank wear land developed on the lips of the twist drill, Fz (N), averaged for the five holes;Thrust force, Ra (µm);Average surface roughness of the holes, SCE (J/mm3), averaged for the drilling of five holes;Specific cutting energy, tp (sec);Production time for cutting five holes, FC (L).Volume of the cryogenic fluid consumed in cutting five holes, h, fV, 5, tRP, and st are depth of hole, drill’s feed speed, number of holes drilled per run, time for retracting and positioning the tool, and time consumed in jetting the cryogenic fluid on the tool before initiating cutting of each of the five holes, respectively. RPt is fixed to 0.6 s, whereas the values of st are equal to 4 and 8 s for the hole-making of CFRP and Ti-6Al-4V, respectively.The feed rate for all the runs is fixed to 0.1 mm/rev leading to various feed speeds for the different levels of cutting speed. The CNC machining center’s rapid traverse  is utilized to retract the drill when it completes the cutting stroke, lift it to the tool-only cooling position (for the runs involving between-the-holes cooling), and move to the next hole’s location. Equations (1) and (2) present the formulae for calculating the production times:2 is supplied from a storage dewar at a flow rate of 0.5 L/min through a thermally insulated pipe fitted with a nozzle of 6 mm diameter on its delivery end. For continuous cooling, the nozzle is so directed to have the maximum impingement of the fluid’s jet on that part of the drill’s body which is rotating just outside the surface of the in-process hole. The between-the-holes cooling technique is realized by rapidly lifting the rotating drill up to a height of 15 cm from the work surface and impinging it with the jet of LN2 for exactly 4 and 8 s related to the drilling of CFRP and Ti-6Al-4V, respectively. The lifting of the drill prior to the jet impingement is carried out to ensure complete avoidance of work cooling through the cryogenic coolant. The cooling process is followed immediately by a rapid displacement of the drill to a location close to the point of drilling next hole on the work surface. The regular feed speed of the drill commences thereon for cutting the hole. All the experiments are performed on a 5-axis CNC machining center, DMG Mori DMU 60 having maximum motor power and spindle speed of 25 kW and 20,000 rpm, respectively. All the runs involving CFRP are performed before those of Ti-6Al-4V. Although the thicknesses of the CFRP and Ti-6Al-4V plates are 22.5 mm and 19 mm, respectively, the tool is fed to the depths of 24.5 mm and 21 mm, respectively, to ensure complete exit of the cutting lips at the exit sides of the plates. For the runs involving continuous and between-the-holes cryogenic cooling, the LNVB is determined by averaging the width of the flank wear land measured at four different locations of each of the two lips. A portable roughness tester, Mahr Perthometer M1, is used to measure the average arithmetic roughness (Ra) of the holes. The stylus of the instrument scans the surface for a distance of 5.5 mm and the procedure is applied at three different locations of each of the five holes drilled in an experimental run. The Ra is then determined by averaging the 15 readings. The thrust force is measured using Kistler piezoelectric dynamometer 9265B utilizing a force plate 9443B. The range of force measurement for the instrument along the direction of drill’s feed (z-axis) is 0–30 kN. The force–time data obtained from the force measurement system is processed in Origin (OriginPro 2015 SR2), a computer program for interactive scientific graphing and data analysis. The processing returns a numbered value of the force data as Fz, which is most central to the force-time graph obtained for the length of actual cutting.The tool damage is measured using a camera-fitted optical microscope, ARTCAM 130-MT-WOM. The used drills are cut at a distance of about 15 mm from the chisel edges using wire electric discharge machining and are cleaned in an ultrasonic cleaner prior to the measurements. The V and PF represent potential difference (in volts) and power factor, respectively. I1, I2, and I3, quantified in Amperes, are the current readings of the three clamp meters. Non-cutting powers for the four levels of cutting speed  are measured by rotating and vertically moving down the twist drill in the air at the given combinations of cutting speed and feed speed. The drill is moved at the feed speeds of 51.7, 79.6, 398, and 597 mm/min against the cutting speeds of 13, 20, 100, and 150 m/min, respectively, ensuring the fixed feed rate of 0.1 mm/rev. The four feed speeds yield the material removal rates (MRR) of 43.3, 66.8, 333.4, and 500 mm3/sec, respectively. The average cutting power for each run is found by subtracting the respective non-cutting power from the corresponding total electric power consumed by the equipment. The average cutting power for each run is then divided by the corresponding MRR to get the SCE.Specific cutting energy is determined through the measurements of electrical current drawn in by the CNC machine tool. Three current clamp meters, Hantek CC 65, are applied to the three phases of the CNC machine’s power bus. The total power drawn in by the machine tool is determined by using the formula:This section presents the experimental results, analyses, and discussions regarding the response variables listed in the previous section.VB, categorized by the two work materials tested (CFRP and Ti-6Al-4V). Two inferences, common to both the materials, can be directly drawn from the data bars. Firstly, the high level of cutting speed causes rapid deterioration of the cutting lips. Secondly, the application of the cryogenic coolant (LN2) does inhibit the progress of tool wear.VB of the favorable mode related to the high level of cutting speed is lower than the VB of the non-favorable mode associated with the low level of cutting speed.It is clear from the data presented in 2, due to its extremely low working temperature, puts a curb on the progress of tool wear by quashing the heat flux and maintaining the hardness of the twist drill’s cutting lips. Even at the full depth of the drill inside the hole, as is the case with the continuous mode of cooling, a noticeably high thermal conductivity of tungsten carbide (110 W·m−1·K−1) causes efficient heat dissipation from the cutting area because of a steep temperature gradient existing between the high temperature cutting lips and the considerably colder drill’s body rotating outside the hole and being impacted directly with the LN2. The sustained hardness of the cutting edges and the adjacent faces keeps up the wear resistance against the abrasion of the hard carbon fibers of CFRP or hard phases of Ti-6Al-4V matrix. As is visible in images (b), (c), and (e) of −1·K−1). The higher conductivity of the titanium alloy quickly disperses any cooling/heating effect throughout the material’s body, thus, diminishing any localized hardening/softening effect. Hence, it can be safely said that the continuous cooling technique does more good than bad to the drilling process by effectively removing the process heat and maintaining the tool’s hardness during cutting while not causing any significant work hardening. The between-the-holes cooling technique in drilling of the titanium alloy does not provide much needed cooling during the cutting process. The pre-cutting cooling of the drill is not enough to take on the high rate of heat generation and associated softening and wearing of the tool during cutting of the high-strength titanium alloy. The discussion, thus, establishes that the between-the-holes mode of cryogenic cooling is not as effective as the continuous mode in drilling of Ti-6Al-4V.Application of LN2 jet impacting the work material close to the cutting area, in the continuous cooling mode, significantly cools it down because the cooling effect cannot be dispersed efficiently through the body. The resulting cooling effect raises the strength of the composite, especially of the carbon fibers, around the hole being drilled. Consequently, the hole is cut with a higher severity of abrasion leading to an increase in the VB. On the other hand, the between-the-holes mode does not offer any cooling to the work material that might otherwise cause it to gain strength. The cryogenically cooled drill, prior to the start of hole cutting, remains hard enough to cut the hole in a composite material whose major portion consists of a soft polymer matrix. Furthermore, as the process of hole-cutting carried out on CFRP is exceedingly faster than Ti-6Al-4V . Both forms of the tool damage seriously cut short the useful life of the twist drill.2 keeps a check on adhesion due to the effective lowering of working temperature around the cutting lips. Pre-drill cooling with a curtailed supply of the cryogenic coolant during cutting does not help in abating adhesion. Furthermore, it is also visible that the levels of abrasive wear endured by the drills in cutting the titanium alloy are higher than in cutting the composite material. Although, carbon fibers are much harder than the titanium-based phases, the presence of a soft epoxy matrix soothe things down for CFRP drilling. The three micrographs related to the drilling of CFRP show mild progressive wear and no signs of adhesion. Furthermore, the micrograph related to the dry cutting shows a wider flank wear land than the ones employing cryogenic cooling. The observation suggests that the application of the cryogenic fluid does help the cutting lips to maintain their wear resistance by effectively removing the process heat.Ti-6Al-4V is an alloy that possesses a high degree of adhesivity, especially at elevated temperatures, with most of the tool materials. Adhered particles of work material damage the tool’s geometry by taking minute flakes of the tool material with themselves as they chip off from the tool’s surface under the machining stresses. Considerably higher degrees of adhesion are visible in the first and the third images of Fx and Fy, are not taken as the process’s performance measures. The two are exceedingly smaller in magnitude than the thrust  component, Fz, and are, thus, not analyzed. As drilling is a uniaxial cutting process, the two planar and mutually orthogonal components of the cutting force, The thrust forces experienced in the drilling of the titanium alloy are exceedingly higher than in the composite material. The magnitude of cutting forces depends primarily on the shear strength of a work material; the higher the shear strength the higher is the magnitude of the cutting force required overcoming the strength and forming the chip. As can be observed from x and Fy) and the thrust component (Fz) is exceedingly larger in drilling of Ti-6Al-4V. This is why thrust force is solely taken for analyses as it is the component which gets most affected by a change in the work material’s shear strength. The Fz curves related to Ti-6Al-4V show a downward trend as the cutting progresses with more prominence in the one related to the continuous mode of cryogenic cooling. The trend depicts thermal softening of the material occurring due to the intensification of heat flux as the drilling process progresses. The trend is more prominent in the graph related to continuous cooling because the work material is already strengthened a bit due to the impingement of the cryogenic fluid at the start of the cutting. Thereafter, the cutting heat steeply brings the thrust force down as the material quickly starts to soften down.Ra measurements of the drilled holes for the 12 experimental runs. A few inferences can be easily drawn from the data bars. Firstly, the between-the-holes cooling approach is easily the best option for both the work materials as it collectively yields the lowest values of Ra. Secondly, for CFRP, dry drilling yields far worse Ra results than the cryogenic cooling based options whereas its results are comparable with those of the continuous cooling mode for Ti-6Al-4V drilling. Thirdly, the high level of cutting speed yields lower values of Ra for all the material-cooling combinations except Ti-6Al-4V–continuous cooling combination. Fourthly, the surface finish of holes produced in the titanium alloy is exceedingly better than in the composite material. As CFRP is a heterogeneous material comprising very hard carbon fibers blended in a very soft epoxy matrix, the machining process is, generally, expected to yield poor surface finish.Ra. The supply of LN2 in a continuous mode does reduce the magnitude of tool wear causing improvements in surface roughness of the drilled holes. Unfortunately, the application of a cryogenic fluid in a continuous manner has a side effect as well. In addition to cooling the tool, the fluid also cools the work material. The cooling effect induced on the work material varies in magnitude depending upon the distance from the cutting area, cryogenic fluid’s impingement location, volume flow rate of the fluid, and thermal characteristics of the material. The simultaneous heating (because of cutting) and uneven cooling effects produce complex expansion and contraction patterns in the work material with respect to time, resulting in deterioration of the holes’ surface. The between-the-holes cooling approach is free of such a side effect. It not only keeps a check on the wear of the cutting edges, as described in Dry drilling yields poor surface finish because of the blunt cutting edges of the associated drills. Dry cutting causes intense tool wear, leading to loss of cutting edges’ sharpness, which in turn leads to the high values of The high level of cutting speed is found to yield lower surface roughness. The improvement in surface quality is attributed to a faster cutting process which does not grant a time period long enough to allow a high proportion of process heat to be absorbed by the work body. Rather, a large proportion of the process heat is taken away by the chip. Consequently, a more mechanical and less thermal effect dispensed to the work surface results in the reduced values of surface roughness.SCE for the 12 experimental runs. SCE is the energy required at the cutting edges to remove a unit volume of work material. Its magnitude mainly depends on the shear strength of the work material. As the titanium alloy is much stronger in shear than the CFRP composite, the cutting of the former requires much larger consumption of specific energy than the latter. For both the work materials, an increase in cutting speed causes a reduction in consumption of specific cutting energy. The decreasing effect on SCE caused by a rise in cutting speed is attributed to the work material’s thermal softening, which is instigated by the resulting intensification of heat flux [MRR) [SCE is formulized as the total cutting power divided by the MRR, an increase in cutting speed causes a reduction in the SCE.eat flux . By incrux [MRR) . As SCE SCE is increased when the cryogenic coolant is applied in the continuous mode. On the other hand, the between-the-hole mode of cooling causes a reduction in specific cutting energy consumption in the drilling of CFRP whereas a marginal increase is recorded for Ti-6Al-4V. The obvious reason associated with the unfavorable outcome yielded by the continuous mode is the simultaneous cooling and, thus, hardening, of the work material in addition to the twist drill. The supply of the coolant during the cutting process raises the strength of the material making it consume more energy for its plastic deformation and conversion into a chip [SCE magnitude rise significantly. In a contrast, the same cooling technique has not yielded any SCE reduction in the drilling of Ti-6Al-4V when compared with dry drilling. The attributed reason is that although the work material’s mechanical behavior is same for the two cooling options (dry and between-the-holes cryogenic cooling), the drills’ deteriorated cutting lips in the case of between-the-holes cooling, as is obvious in SCE requirement.The data bars related to both the work materials reveal that the o a chip . The favAs described before, production time and volume of the cryogenic fluid consumed can directly be calculated from the settings of the input parameters. Their calculations for the 12 experimental runs are presented in With respect to production time, continuous mode of cryogenic cooling is as good as dry drilling because it does not need to halt the tool movement to ensure the requirement of tool-only cooling. The performance of the between-the-hole mode of cooling depends upon the cutting speed used for the particular run and the time duration for which the jet is applied to the drill before cutting each hole. The cooling technique takes 40%, 61%, 127%, and 179% more time than dry drilling (and continuous cooling) to process 5 holes against the cutting speeds of 13, 20, 100, and 150 m/min, respectively. This implies that the higher the cutting speed the larger is the percentage difference between the between-the-holes cooling technique and the other two options regarding production time. As the high levels of cutting speed are associated with the drilling of CFRP, the between-the-holes technique may badly affect the productivity of hole-making in the composite material.2), both of the non-dry drilling options incur an extra cost. Of the two, the better cryogenic cooling option is decided by the cutting speed and the time duration of the fluid’s application selected for the between-the-holes cooling mode. As described in 2 than the latter in the drilling of Ti-6Al-4V at the cutting speeds of 20 and 13 m/min, respectively. The analysis suggests that the between-the-holes mode of cryogenic cooling is an economical option as compared to the continuous mode when low-to-medium cutting speeds are involved, such as for the drilling of Ti-6Al-4V. Whether or not the between-the-holes technique of cooling is more economical than dry drilling needs a deeper cost analysis involving the acquisition costs of the twist drills and liquid nitrogen, electricity tariff, and tool life criterion.With respect to consumption of the cryogenic coolant  work material to be drilled in exceedingly shorter processing time. Moreover, the impact of the cryogenic coolant on the work, in the continuous cooling mode, hardens it significantly due to its poor thermal conductivity. Therefore, the between-the-holes mode of cryogenic cooling is better than the continuous mode for the drilling of CFRP but not for Ti-6Al-4V.The data on thrust force and specific cutting energy show similar patterns as both are dependent on work material’s shear strength. The latter is dependent also on cutting speed. Regarding the two performance measures, the between-the-holes cooling technique yields better results than continuous cooling for both the work materials. The underlying reason is that the cryogenic cooling of the work material during the cutting process enhances the strength of the work material causing it to draw more energy (and force) to get plastically deformed. The between-the-holes mode of cooling avoids such an anomaly because the work material is never impacted by the cryogenic fluid.The between-the-holes cooling technique outperforms continuous cooling in respect of surface finish of the holes drilled in Ti-6Al-4V and the holes drilled in CFRP at the high level of cutting speed. The complex work expansion-contraction pattern induced by the application of a cryogenic fluid on the work material, as is the case in continuous cryogenic cooling, compromises the surface finish of the holes. Between-the-holes cooling is also found to yield better results regarding hole roundness, burr generation, and fiber pullout in CFRP drilling.Finally, the continuous mode of cooling is better than between-the holes cooling regarding production time. The difference becomes more prominent at high levels of cutting speed, as is the case with hole-making of CFRP. On the other hand, between-the holes cooling is better than continuous cooling regarding consumption of the cryogenic fluid except when the cutting speed is very high. Reductions in production times and consumption volumes of a cryogenic fluid add to the economic sustainability of the machining process.In context of the discussion provided above, it can be safely concluded that the between-the-holes mode of cooling is a far more effective way of applying a cryogenic coolant than the traditional mode of continuously supplying the fluid in drilling of CFRP. It outperforms the conventional approach with respect to almost all the performance measures of a machining process such as tool damage, cutting forces, cutting energy consumption, work surface quality, and consumption of the cryogenic fluid. The only area of concern for the novel cooling technique is increased production time. It is expected that the weakness in this regard can be minimized by increasing the flow rate of the coolant while reducing the time of drill’s exposure to the cryogenic fluid’s jet before cutting each hole. Regarding drilling of the titanium alloy, the choice of the cryogenic coolant’s delivery is not as straightforward. Although the between-the-holes cooling technique yields better results than the traditional approach in respect of cutting forces, specific cutting energy, holes’ surface roughness, and cryogenic fluid’s consumption cost, it is humbled against the continuous mode of cooling in respect of controlling tool damage. Longer production time is, obviously, another matter of concern but its percentage difference is much lower than in CFRP drilling. In this situation, the selection of the coolant’s supply mode hinges on the proportion of tooling cost in the total processing cost. A small proportion would tilt the decision in the favor of the between-the-holes mode of coolant’s delivery.Between-the-holes cryogenic cooling effectively checks the progress of tool wear, more effectively than continuous cooling, in CFRP drilling. On the contrary, it underperforms in this regard in Ti-6Al-4V drilling owing to the higher shear strength of the titanium alloy and a much slower and coolant-less cutting process.Application of the novel cryogenic technique yields lowers thrust force and specific cutting energy consumption in comparison to the continuous mode of cooling due to avoidance of work strengthening caused by the cryogenic fluid’s impact on the work material.The between-the-holes cooling technique also leads to improvements in holes’ surface roughness, especially in the titanium alloy because of preventing complex work expansion-contraction patterns. It also yields significantly better surface quality at both sides of the holes in CFRP drilling.The novel cooling technique though compromises productivity because of the additional time required to cool the drill, it generally consumes lesser cryogenic fluid than the continuous mode of cooling. Consumption of the cryogenic fluid is the only measure in which dry drilling outperforms the two cryogenic cooling options.In a holistic view, the between-the-holes mode of coolant delivery is an unambiguous choice of heat dissipation for CFRP drilling. For Ti-6Al-4V drilling, the choice between the continuous and the between-the-holes modes of cryogenic cooling depends on the share of tooling cost in the total processing cost. A small share would lead to selection of the between-the-holes mode.The presented work establishes the efficacy of a novel between-the-holes technique of applying a cryogenic fluid to the tool, without impacting the work, in hole-making of two commonly used materials: Ti-6Al-4V and CFRP. Its effectiveness is compared with the traditional approach of continuously supplying the coolant during cutting and dry drilling. The following conclusions can be drawn from the experimental results, their analyses, and discussion provided beforehand:


Pseudomonas aeruginosa is a common pathogen that is responsible for serious hospital-acquired infections, ventilator-associated pneumonia, and various sepsis syndromes. Also, it is a multidrug-resistant pathogen recognized for its ubiquity and its intrinsically advanced antibiotic-resistant mechanisms. It usually affects immunocompromised individuals but can also infect immunocompetent individuals. There is no vaccine against it available till now. This study predicts an effective epitope-based vaccine against fructose bisphosphate aldolase (FBA) of Pseudomonas aeruginosa using immunoinformatics tools. The protein sequences were obtained from NCBI, and prediction tests were undertaken to analyze possible epitopes for B and T cells. Three B cell epitopes passed the antigenicity, accessibility, and hydrophilicity tests. Six MHC I epitopes were found to be promising, while four MHC II epitopes were found promising from the result set. Nineteen epitopes were shared between MHC I and II results. For the population coverage, the epitopes covered 95.62% worldwide excluding certain MHC II alleles. We recommend in vivo and in vitro studies to prove its effectiveness. Pseudomonas aeruginosa is a motile, nonfermenting, gram-negative opportunistic bacterium that is implicated in respiratory infections, urinary tract infections, gastrointestinal infections, keratitis, otitis media, and bacteremia in patients with compromised host defences  [P. aeruginosa is responsible for millions of infections each year in the community, 10–15% of all healthcare-associated infections, with more than 300,000 cases annually in the EU, USA, and Japan [P. aeruginosa infections despite supportive treatments. Vaccines could be an alternative strategy to control P. aeruginosa infections and even reduce antibiotic resistance; however, no P. aeruginosa vaccine is currently available [P. aeruginosa strains and within the same strain, grown in diverse environmental conditions [ibrosis) . Intensiibrosis) –4. This ibrosis) . Accordind Japan , 7 that nd Japan , 9 whichnd Japan –13, bothvailable . Döring P. aeruginosa [P. aeruginosa using computational approach through prediction of highly conserved T and B cell epitopes from the highly immunogenic protein fructose bisphosphate aldolase (FBA). This is the first study that predicts epitope-based vaccine from this moonlighting protein of P. aeruginosa. This technique has been successfully used by many authors to identify target vaccine candidates. These types of vaccines are easy to produce, specific, capable of keeping away any undesirable immune responses, reasonable, and safe when compared to the conventional vaccines such as killed and attenuated vaccines [Contemporary, integrated genomics and proteomics approaches have been used to predict vaccine candidates against ruginosa . Althougruginosa , 17. Theruginosa , 19. Thuvaccines .Pseudomonas aeruginosa FBA were retrieved in FASTA format from the National Center for Biotechnology Information (NCBI) database (https://ncbi.nlm.nih.gov) on May 2019. The protein sequence had a length of 354 with the name fructose-1,6-bisphosphate aldolase.A total of 20,201 strains of Pseudomonas aeruginosa FBA were subjected to multiple sequence alignment (MSA) using the ClustalW tool of BioEdit Sequence Alignment Editor Software version 7.2.5 to determine the conserved regions. Also, molecular weight and amino acid composition of the protein were obtained [The retrieved sequences of obtained , 22.Pseudomonas aeruginosa FBA was submitted to different prediction tools at the Immune Epitope Database (IEDB) Analysis Resource (http://www.iedb.org/) to predict various B and T cell epitopes. Conserved epitopes would be considered candidate epitopes for B and T cell [The reference sequence (NP_249246.1) of d T cell .http://tools.iedb.org/bcell/) to identify the surface accessibility, antigenicity, and hydrophilicity with the aid of random forest algorithm, a form of unsupervised learning. The BepiPred linear prediction 2 was used to predict linear B cell epitope with the default threshold value 0.533 (http://tools.iedb.org/bcell/result/). The Emini surface accessibility prediction tool was used to detect the surface accessibility with the default threshold value 1.00 (http://tools.iedb.org/bcell/result/). The Kolaskar and Tongaonkar antigenicity method was used to identify the antigenicity sites of a candidate epitope with the default threshold value 1.032 (http://tools.iedb.org/bcell/result/). The Parker hydrophilicity prediction tool was used to identify the hydrophilic, accessible, or mobile regions with the default threshold value 1.695 [B cell epitope is the portion of the vaccine that interacts with B lymphocytes which are a type of white blood cell of the lymphocyte subtype. Candidate epitopes were analyzed using several B cell prediction methods from the IEDB (ue 1.695 –28.http://tools.iedb.org/mhci/) to predict cytotoxic T cell epitopes . The presentation of peptide complex to T lymphocyte undergoes several steps. The Artificial Neural Network (ANN) 4.0 prediction method was used to predict the binding affinity. Before the prediction, all human allele lengths were selected and set to 9 amino acids. The half-maximal inhibitory concentration (IC50) value required for all conserved epitopes to bind was a score less than 500 [T cell epitope is the portion of the vaccine that interacts with T lymphocytes. Analysis of peptide binding to the MHC (major histocompatibility complex) class I molecule was assessed by the IEDB MHC I prediction tool (than 500 –35.http://tools.iedb.org/mhcii/) for helper T cell, which is known as CD4+ cell also. Human allele reference set was used to determine the interaction potentials of T cell epitopes and MHC class II allele . The NN-align method was used to predict the binding affinity. IC50 score values less than 100 were selected [Prediction of T cell epitopes interacting with MHC class II was assessed by the IEDB MHC II prediction tool (selected –39.http://tools.iedb.org/population/iedbinput). The appropriate checkbox for calculation was checked based on MHC I, MHC II separately, and a combination of both [The population coverage tool was selected to analyze the epitopes in the IEDB. This tool calculates the fraction of individuals predicted to respond to a given set of epitopes with known MHC restriction ( of both .http://raptorx.uchicago.edu), i.e., a protein structure prediction server developed by Peng and Xu's group, excelling at 3D structure prediction for protein sequences without close homologs in the Protein Data Bank (PDB). USCF Chimera (version 1.8) was the program used for visualization and analysis of molecular structure of the promising epitopes (http://www.cgl.uscf.edu/chimera) [The 3D structure was obtained using RaptorX (chimera) , 42.Pseudomonas aeruginosa FBA is illustrated in The amino acid composition for the reference sequence of The reference sequence of fructose 1,6-bisphosphate aldolase was subjected to BepiPred linear epitope prediction, Emini surface accessibility, Kolaskar and Tongaonkar antigenicity, and Parker hydrophilicity methods in the IEDB to test for various immunogenicity parameters –5. The tThe reference fructose 1,6-bisphosphate aldolase sequence was analyzed using the (IEDB) MHC I binding prediction tool to predict T cell epitopes which suggested interacting with different types of MHC class I alleles, based on Artificial Neural Network (ANN) with half-maximal inhibitory concentration (IC50) < 500 nm. 206 peptides were predicted to interact with different MHC I alleles.The most promising epitopes and their corresponding MHC I alleles are shown in The reference fructose 1,6-bisphosphate aldolase sequence was analyzed using the (IEDB) MHC II binding prediction tool based on NN-align with half-maximal inhibitory concentration (IC50) < 100 nm; there were 662 predicted epitopes found to interact with MHC II alleles. The most promising epitopes and their corresponding alleles are shown in  along wiAll promising MHC I and MHC II epitopes of fructose 1,6-bisphosphate aldolase were assessed for population coverage against the whole world .For MHC I, epitopes with the highest population coverage were LVMHGSSSV (60.41%) and QMLDHAAEF (31.7%) . For MHCP. aeruginosa is highly accredited due to the high mortality rates associated with the pathogen that spreads through healthcare areas. In addition, multidrug resistance of the pathogen demands the design of vaccine as an alternative [Vaccination against ernative . In thisernative . Thus, tP. Aeruginosa FBA was subjected to BepiPred linear epitope prediction 2 test to determine the binding to B cell, Emini surface accessibility test to test the surface accessibility, Kolaskar and Tongaonkar antigenicity test for antigenicity, and Parker hydrophilicity test for the hydrophilicity of the B cell epitope.As B cells play a critical role in adaptive immunity, the reference sequence of Out of the thirteen predicted epitopes using BepiPred 2 test, only three epitopes passed the other three tests  after segmentation. BepiPred version 2 test was used because it implements random forest and therefore predicts large epitope segments.The reference sequence was analyzed using the IEDB MHC I and II binding prediction tools to predict T cell epitopes. 28 epitopes were predicted to interact with MHC I alleles with half-maximal inhibitory concentration (IC50) < 500. Six of them were most promising and had the affinity to bind to the highest number of MHC I alleles . 19 predicted epitopes were interacted with MHC II alleles with IC50 < 100. Four of them were most promising and had the affinity to bind to the highest number of MHC II alleles . Nineteen epitopes  appeared in both MHC I and II results.The best epitope with the highest population coverage for MHC I was LVMHGSSSV (60.41%) with seven HLA hits, and the coverage of population set for the whole MHC I epitopes was 88.75%. Excluding certain alleles for MHC II, the best epitope was KVNIDTDLRLASTGA scoring 27.37% with two HLA hits, followed by GEIKETYGVPVEEIV scoring 24.27% with five HLA hits. The population coverage was 61.1% for all conserved MHC II epitopes. These epitopes have the ability to induce T cell immune response when interacting strongly with MHC I and MHC II alleles effectively generating cellular and humoural immune response against the invading pathogen. When combined, the epitope LVMHGSSSV had the highest population coverage percent 60.41% with seven HLA hits for both MHC I and MHC II.Many studies had predicted peptide vaccines for different microorganisms such as rubella, Ebola, dengue, Zika, HPV, Lagos rabies virus, and mycetoma using immunoinformatics tools –51. Limiin vivo and in vitro studies to prove its effectiveness along with formulation of appropriate adjuvants. Finding another immunogenic target and analyzing the associated epitopes support the vaccine formula.We hope that the world will benefit from these predicted epitopes in the formulation of the peptide-based vaccine and recommend further Pseudomonas aeruginosa for the first time. Three B cell epitopes have successfully passed the required tests. Six MHC I epitopes were found to be most promising, while four were found from MHC II epitope result set. These epitopes covered 95.62% worldwide excluding certain MHC II alleles.Vaccination is used to protect and minimize the possibility of infection leading to an increased life expectancy. The design of vaccines using immunoinformatics prediction methods is highly appreciated due to the significant reduction in cost, time, effort, and resources. Epitope-based vaccines are expected to be more immunogenic and less allergenic than traditional biochemical vaccines. We have illustrated different epitopes that have the ability to stimulate both B and T cells against fructose bisphosphate aldolase protein of


We aimed at investigating the effect of matrix stiffness on the phenotype of liver cells of rats with cirrhosis, assessing its influence on their response to antifibrotic strategies and evaluating associated molecular mechanisms.Liver stiffness is increased in advanced chronic liver disease (ACLD) and accurately predicts prognosis in this population. Recent data suggest that extracellular matrix stiffness Hepatocytes, hepatic stellate cells, and liver sinusoidal endothelial cells were isolated from healthy rats or rats with cirrhosis (carbon tetrachloride or thioacetamide), and cultured on polyacrylamide gels with different physiologically relevant stiffness for 72 h.vs. rigid matrix . Additionally, stiffness modified the antifibrotic effects of liraglutide in stellate cells of rats with cirrhosis. Finally, evaluation of nuclear morphology revealed that high stiffness induced nuclei deformation in all cell types, an observation confirmed in cells from human livers. Disconnecting the nucleus from the cytoskeleton by cytoskeleton disruption or a defective form of nesprin 1 significantly recovered spherical nuclear shape and quiescent phenotype of cells.All cell types of rats with cirrhosis cultured at low stiffness showed a significant phenotype amelioration per se modulates the phenotype of healthy rats and liver cells of rats with cirrhosis by altering the nuclear morphology through cytoskeleton-derived mechanical forces. The reversibility of this mechanism suggests that targeting the stiffness-mediated intracellular mechanical tensions may represent a novel therapeutic strategy for ACLD.The environment's stiffness tricking liver cells into believing they are in a healthy, soft liver improved their function and could potentially contribute to treat cirrhosis.During cirrhosis, the liver becomes scarred, stiff, and unable to perform its normal functions efficiently. In this study, we demonstrated that cells from diseased (stiff) livers recovered their functionality when placed in a soft environment . Furthermore, treatments aimed at   •Low stiffness directly improves the phenotype of hepatocytes, hepatic stellate cells, and liver sinusoidal endothelial cells.•Stiffness modulates sinusoidal crosstalk in advanced chronic liver disease (ACLD).•Stiffness may determine the efficacy of antifibrotic strategies.•Mechanical forces applied to the nucleus through the LINC complex mediate the effects of stiffness on liver cell phenotype. Fibrosis and associated tissue remodelling modify the mechanical properties of the liver, leading to a shift from an elastic, soft organ, well compliant to increases in blood flow, to a stiff organ, with high resistance to flow. As such, fibrosis in combination with liver microvascular dysfunction is the primary factor driving the development of portal hypertension in advanced chronic liver disease (ACLD).Liver fibrosis results from excessive extracellular matrix (ECM) deposition and formation of scar tissue in response to persistent liver injury .Male Sprague Dawley rats were kept at the animal facilities of the University of Bern, whilst male Wistar Han rats were used at the animal facilities of the University of Barcelona Medical School. All animals were maintained in controlled environmental conditions with 12 h light-dark cycles, and fed 4) model, cirrhosis was induced in male Wistar rats weighing 100 g by chronic CCl4 inhalation (thrice per week) whilst receiving 0.3 g/L phenobarbital in the drinking water. Rats were considered with cirrhosis after development of ascites, which takes 14–16 weeks, approximately.For the carbon tetrachloride (CClFor the thioacetamide (TAA) model, male Sprague Dawley rats weighing 250 g were injected with 250 mg/kg TAA i.p. twice a week for 12 weeks.Human tissues were obtained from remnant pieces from partial hepatectomy to excise tumour metastasis from colon carcinoma  or from explanted livers from patients with cirrhosis receiving orthotopic liver transplantation (NASH or chronic ethanol aetiology). HSCs were isolated from 3 different livers of patients with cirrhosis. The Ethics Committee of the Hospital Clínic de Barcelona approved the experimental protocol (HCB/2015/0624), and patients gave their signed informed consent.Immortalised human-activated HSC LX-2 cells  were used for transfection and toxicity experiments. Results using LX-2 cells were derived from at least 3 different batches per experimental condition.Isolation of primary cells was performed as previously described.Collagen-coated polyacrylamide gels were prepared as described in the ,The stiffnesses of the gels were established at 0.5, 13.5, and 30 kPa (Young's modulus),HSCs and liver sinusoidal endothelial cells (LSECs) were freshly isolated from rats with cirrhosis and directly plated either on gels with 0.5 or 30 kPa, or on transwell inserts with adjustable height (Thermo Fisher). After 12 h of monoculture, cells were washed with PBS and co-culture was started by placing the HSC-containing inserts on top of the LSEC-containing gels and vice versa (with inserts set at their medium hanging position), and allowed to communicate for additional 36 h. The mRNA expression of phenotypic markers was assessed in the cells contained in the insert (which were not in direct contact with the gels).KASH (kindly provided by Catherine Shanahan)Human HSCs (LX-2 and primary HSCs from patients with cirrhosis) were transfected with the dominant-negative plasmid EGFP-nesprin 1-Hepatocytes were washed twice with PBS and culture medium was changed every 24 h. Synthesis of urea and albumin was evaluated at the Zentrum für Labormedizin  in the supernatant corresponding to the time range of 48–72 h and normalised to the number of cells of the corresponding well.Four images per well (100× magnification) were taken using a standard phase contrast microscope. Differences in cell morphology were quantified as described in the LSEC fenestration was assessed by scanning electron microscopy.Please see the LSECs were incubated with Roswell Park Memorial Institute's medium without phenol red containing 3 ng/ml DAPI (Thermo Fisher) and 5 μg/ml diaminofluorescein diacetate (Thermo Fisher) for 20 min at 37°C. After 2 washes with PBS, 6 images per cover glass and channel  were taken. The same settings were used for gathering and quantification of all images. Intensities were normalised to the number of cells per field.Real-time PCR was performed using TaqMan probes, specified in the t test (2 samples) or ANOVA (>2 samples) followed by the Tukey post hoc analysis. Otherwise, means were compared using the non-parametric Kruskal-Wallis test followed by the Mann-Whitney U test. Differences were considered significant at p <0.05.Statistical analyses were performed using the IBM SPSS 23 statistics software for Windows. Normality of sample distribution was assessed using the Kolmogorov-Smirnov test. For samples fitting a normal distribution, means were compared by the Student The effect of matrix stiffness on the phenotype of healthy hepatic cells was evaluated in freshly isolated hepatocytes, HSCs, and LSECs from healthy rat livers, and plated on polyacrylamide gels with increasing stiffness. Hepatocytes, HSCs, and LSECs plated at higher stiffness (30 kPa) became apparently larger and flatter than on physiological stiffness (0.5 kPa). Moreover, quantification of texture descriptors confirmed a shift in cell morphology at 72 h of culture  and albumin mRNA expression , middle.Culture of quiescent HSCs on high stiffness (30 kPa) gels did not change the mRNA expression of the activation markers α-smooth muscle actin (α-SMA) and collagen I , middle.vs. low stiffness matrices.Finally, 72 h culture on different stiffnesses also modified the phenotype of LSECs. When cultured on high stiffness surfaces, LSECs displayed higher levels of the capillarisation marker laminin b1 (Lamb1) at the mRNA level. In addition, LSECs cultured on high stiffness gels showed an upregulation of endothelial nitric oxide synthase (eNOS) mRNA , middle,Collectively, our observations confirm previous reports showing activation of HSCs and hepatocytes in response to high stiffness, and reveal that LSECs become capillarised when cultured in high stiffness matrices.4-exposed rats with cirrhosis, cultured at decreasing stiffness for 72 h.We evaluated the effects of substrate stiffness in cells isolated from CClin vitro urea and albumin synthesis and release (Similar to cells isolated from control (without cirrhosis) rats, the phenotype of liver cells of rats with cirrhosis was also affected by stiffness. Hepatocytes, HSCs, and LSECs isolated from rats with cirrhosis cultured on low, healthy stiffness (0.5 kPa) displayed morphological changes when compared with cells cultured on stiffer substrates B and in  release C.Fig. 1LLow stiffness surfaces ameliorated HSC over-activation, displaying lower mRNA levels of the activation markers α-SMA and collagen I compared with stiffness of rats with cirrhosis B, withouSimilarly, LSECs isolated from livers of rats with cirrhosis and cultured on low stiffness (0.5 kPa) exhibited a decreased expression of the capillarisation marker Lamb1. Moreover, whilst their eNOS mRNA expression decreased B, their Improvement of the phenotype in low stiffness conditions was additionally validated in primary HSCs isolated from TAA-injected rats with cirrhosis. Also in these experiments, HSC activation markers α-SMA and collagen I were significantly decreased in cells cultured on low stiffness substrates compared with stiffness conditions of rats with cirrhosis .In addition to the direct effect on cellular function, we assessed whether stiffness may influence crosstalk between sinusoidal cells of rats with cirrhosis. For this, we isolated HSCs and LSECs from rats with cirrhosis and plated 1 cell type on stiffness-modulated gels at the bottom of the well, which was allowed to communicate paracrinally with the other cell type, attached on the permeable membrane of a transwell , top. HS4-exposed HSCs of rats with cirrhosis cultured on high stiffness matrices and treated with 50 μM liraglutide (a drug with described antifibrotic effects)CClNuclear deformation in healthy hepatocytes, HSCs, and LSECs cultured on high stiffness matrices was significantly greater than on low stiffness gels D. By con,To study the link between matrix stiffness and nuclear shape, human HSCs (LX-2 and primary HSCs of patients with cirrhosis) were transfected with a vector coding for a dominant negative nesprin peptide, containing a KASH domain (DN-KASH), inhibiting the coupling of the nucleus with the cytoskeletonTo further assess whether mechanical deformation of the nucleus induced by increased matrix stiffness determines the phenotype of liver cells, we used a pharmacological approach to uncouple the nucleus from the cytoskeleton using cytoskeletal disruptors  and analysed the effects on cell phenotype. Cd proved effective in uncoupling the nucleus under high stiffness conditions in LX-2 and primary rat liver cells, as observed by broken actin filaments , and ame,With 844 million cases and 2 million deaths per year, chronic liver disease represents 1 of the major public health problems worldwide.,,,Mechanical forces, including ECM stiffness or mechanical stretch, can regulate the cellular phenotype.per se negatively influence hepatic cell biology in cirrhosis. Therefore, in the present study, we investigated whether physiological and increased matrix stiffness, to a level similar to that found in advanced liver fibrosis and in decompensated cirrhosis of the liver, could modulate the phenotype of the main hepatic cell types and represent a possible additional mechanism for the well-known chronic activation of liver cells occurring in cirrhosis. Such a stiffness-induced continued activation of liver cells might contribute both to the progression of liver disease and to prevent its regression after eliminating the cause of liver injury.As increased liver stiffness is characteristically observed in ACLD and as its measurement by non-invasive tools has proven to provide accurate risk stratification in this population, we postulated that this mechanical property could ,per se to modify the expression of activation markers and to impair the morphology and functionality of hepatocytes, HSCs, and LSECs. Therefore, our data are in accordance with the hypothesis that liver cells in an ACLD environment would be continuously receiving activation signals just because of the mechanical properties of the liver.In this regard, recent studies suggested that increased matrix stiffness induces the activation of quiescent HSCs into myofibroblast-like cells, whilst hepatocytes isolated from healthy animals display reduced functionality in these conditions.[7]4-exposed livers of rats with cirrhosis and validated in an additional rat model of ACLD (the TAA-injected cirrhosis model)  ; Fig. S2et al. suggests that HSCs have distinct paracrine secretions in response to varying stiffness.Additionally, we hypothesised that activation of liver cells caused by increased stiffness also leads to impaired cell-to-cell communication, further enhancing changes in phenotype. Indeed, previous studies from our group have demonstrated that crosstalk between hepatic cells is determined by their activation state,In addition, we studied whether the ECM stiffness may be an additional factor contributing to the cellular response to antifibrotic strategies. Indeed, the reported beneficial effects of liraglutide on HSC phenotype,After characterising the effects of matrix stiffness on the biology of liver cells during ACLD, we studied the underlying mechanisms. This is relevant for the design of specific strategies targeting the stiffness-related molecular pathways. In that regard, it is worth noting that mechanical forces are transmitted from the ECM to the nucleus through the cytoskeleton (including F-actin and microtubules).in vivo environment with controlled stiffness. Collagen-coated acrylamide substrates with fine adjustable stiffness may have certain limitations regarding their physical properties,in vivo in cirrhosis by analysing the nuclei of liver cells in situ in human liver tissue  in ACLD by altering nuclear shape. Increased matrix stiffness appears to act tiffness , suggest10.13039/501100001711Swiss National Science Foundation (SNF 320030_189252/1), the 10.13039/100008273Novartis Foundation, the Swiss Foundation against Liver Cancer, the 10.13039/501100004837Spanish Ministry of Science and Innovation  (FIS PI17/00012), the European Union Fondo Europeo de Desarrollo Regional Funds, ‘una manera de hacer Europa’, and the 10.13039/501100003030Agència de Gestió d'Ajuts Universitaris i de Recerca 2017-SGR-517 and Centres de Recerca de Catalunya programme from the 10.13039/501100002809Generalitat de Catalunya. Part of the work was funded by own research funds of the Hepatology group, Universitätsklinik Viszerale Chirurgie & Medizin, Inselspital, Bern. Centro de Investigación Biomédica en Red en el Área temática de Enfermedades Hepáticas y Digestivas is funded by 10.13039/501100004587Instituto de Salud Carlos III. SG-M has a postdoctoral fellowship from the Stiftung für Leberkrankheiten and Asociación Española Para el Estudio del Hígado. MO-R has a fellowship from the Instituto de Salud Carlos III (iPFIS IFI16/00016). SS has a Juan Rodés Fellowship from the European Association for the Study of the Liver.This project was supported by the SG-M and MO-R designed the research, performed the experiments, analysed the data, and wrote the paper. CW and SS performed experiments and analysed data. IA and JZK performed experiments. CF provided essential materials and revised the paper. PR-C, J-FD, and JB critically revised the paper. AB conceived ideas, obtained funding, and critically revised the paper. JG-S conceived the study, designed and directed the research, wrote the paper, and obtained funding. All authors edited and reviewed the final paper.The authors declare no conflicts of interest that pertain to this work.Please refer to the accompanying