ABCC3 is associated with poor survival and impaired response to temozolomide. Importantly, high expression of ABCC3 is restricted to glioblastoma, with negligible levels in healthy brain tissue, and further correlates with tumor grade and stemness markers. We identified three immunogenic epitopes of ABCC3 which were used to isolate nanobodies from a glioblastoma-specific phage-display nanobody library. Two nanobodies targeting ABCC3 (NbA42 and NbA213) were further characterized and demonstrated in vivo selective recognition of ABCC3 in glioblastoma xenograft mouse models upon systemic administration. We designate NbA42 and NbA213 as new candidates to implement immunotargeted applications guiding a more personalized and precise diagnosis, monitoring, and treatment of glioblastoma patients.The cancer “omics” reveal many clinically relevant alterations that are transforming the molecular characterization of glioblastomas. However, many of these findings are not yet translated into clinical practice due, in part, to the lack of non-invasive biomarkers and the limitations imposed by the blood–brain barrier. Nanobodies, camelid single-domain antibody fragments, emerge as a promising tool for immunotargeted applications for diagnosing and treating glioblastomas. Performing agnostic bioinformatic analysis from glioblastoma patient datasets, we identified ATP Binding Cassette subfamily C member 3 (ABCC3) as a suitable target for immunotargeted applications. The expression of  Isocitrate Dehydrogenase (NADP( +)) (IDH)-wildtype diffuse and astrocytic grade 4 glioma in adults which presents microvascular proliferation or necrosis or mutation of Telomerase Reverse Transcriptase (TERT) promoter or amplification of Epidermal Growth Factor Receptor (EGFR) gene or changes in + 7/ − 10 chromosome copy number1. Advances in molecular biology and "omic" sciences  provide clinically relevant biomarkers for the further development of powerful tools to study the biology of glioblastoma and to identify novel therapies2.Glioblastoma is a highly aggressive and heterogeneous primary brain tumor. According to the WHO central nervous system (CNS) 5 classification, glioblastoma is an 4. The current standard of care of glioblastoma patients includes maximal surgical resection, followed by radiotherapy with concomitantly and adjuvant chemotherapy with the alkylating agent temozolomide (TMZ)4. TMZ is an alkylating prodrug that delivers a methyl group to purine bases of DNA generates O6-methylguanine in DNA, a cytotoxic lesion removed by O6-methylguanine DNA methyltransferase (MGMT). Methylation of the MGMT promoter results in gene silencing and correlates with better outcome in TMZ-treated glioblastoma patients5. Although a tissue sample from surgery or biopsies is often required, genetic alterations and epigenetic modifications have uncovered their relevance towards more personalized treatment strategies. The combination of IDH1 mutations and MGMT promoter methylation status offers a higher predictive potential of survival in glioblastoma patients than either alone6.Despite advances in the molecular classification of glioblastoma, there is an urgent need to designate novel molecular targets to improve patient management strategies. Overall survival has not changed in past decades and remains at 14.6 months for patients with primary glioblastoma. This life expectancy is reduced to 6.7 months after relapse7. Molecules showing both lipophilic solubility (formation of less than eight hydrogen bonds with solvent water) and MW < 400 Da may be transported through the BBB via lipid-mediated free diffusion8. Although drug reengineering contributes to BBB transcytosis, pharmacologically significant amounts of many CNS targeting drugs are unreachable, leading to treatment failure. The range of access of proteins in the blood to the CNS is only 0.01–0.4%, including antibodies with intrinsic therapeutic potential9. A wide variety of smaller protein scaffold-based drugs is being developed to circumvent BBB selectivity10.A hurdle in diagnosing, monitoring, and treating glioblastoma is imposed by the blood–brain barrier (BBB) by limiting the effectiveness of most of the strategies against this tumor. This highly selective filter restricts access from the bloodstream into the brain parenchyma for almost 100% for large molecules and more than 98% for all small-molecule drugs targeting the CNS11. Nanobodies are single-domain antigen-recognizing fragments from the heavy-chain-only antibodies of Camelidae. They contain three Complementarity Determining Regions (CDRs) with, on average, larger hypervariable H1 and H3 antigen-binding loops than human antibodies12. These characteristics enable nanobodies to recognize unique conformational epitopes, such as cavities or unstructured regions of proteins, with high affinity and stability14. Besides, various molecular mechanisms for delivering nanobodies through the BBB have been extensively described. Nanobodies targeting CNS molecules have been characterized to cross the BBB by receptor-mediated transcytosis and adsorptive-mediated transcytosis. In this regard, nanobodies have also been utilized not only as shuttle platforms for modeling BBB-permeable drugs but they also have served to improve the targeting of multifunctional therapies  into the brain parenchyma16.Nanobodies represent an innovative alternative due to their reduced size , constituting one of the smallest molecules with intact functional antigen-recognition capacity17. Nine of the ABCC subfamily members are designated multidrug resistance proteins (MRPs), as they mediate cancer multidrug resistance (MDR) by actively extruding chemotherapeutic agents out from tumor cells, constituting targets for cancer therapy18. Among them, ABCC3 is an organic anion transporter responsible for effluxing anticancer compounds20. ABCC3 has been involved in chemotherapy failure and reduced survival in various cancers22. Also, several reports have elucidated a plausible role of ABCC3 in the pathology and prognosis of glioblastoma patients24. ABCC3 may constitute a promising target for future molecularly targeted clinical intervention of glioblastoma.Performing agnostic bioinformatic analysis in multiple datasets of glioblastoma patients, we identified differentially expressed genes coding for proteins located in the plasma membrane. Among them, we selected ATP-Binding Cassette subfamily C member 3  as a biomarker of glioblastoma, suitable for further development of immunotargeted applications with clinical impact. The ABC transporter superfamily comprises the largest family of proteins for translocating substrates across membranes25. Five of these nanobodies showed in vitro specific binding of ABCC3 transporter expressed on the cell surface. Two nanobodies, NbA42 and NbA213, were further characterized and demonstrated selective recognition of ABCC3 transporter in vivo upon systemic administration in glioblastoma xenograft mouse models. Therefore, we propose that NbA42 and NbA213 could serve as molecular candidates for developing immunotargeted applications that could improve the diagnosis, monitoring and therapy of glioblastoma patients.We have isolated several nanobodies targeting ABCC3 by following a peptide-based strategy for biopanning of a previously constructed glioblastoma-specific phage-displayed library26 using limma 27. According to these criteria, 287 genes (Supplementary Table 2FC). 9 genes were identified to be the most differentially expressed in glioblastoma compared to healthy tissue (Log2FC > 4) encoding for membrane-associated proteins  their higher gene expression on glioblastoma compared with healthy tissue, and (2) the location of the encoded protein on the extracellular surface of the plasma membrane. To identify the most differentially expressed genes in glioblastoma, we performed a bioinformatic analysis of 142 glioblastoma and 5 normal brain samples (RNA-Seq) deposited in The Cancer Genome Atlas (TCGA) their hiABCC3 expression levels in MGMT methylated patient samples20, we found similar ABCC3 levels of expression independently of the MGMT methylation status ) with ABCC3 in glioblastoma . We also observe high levels of expression of ABCC3 in a panel of glioblastoma cell lines  with Aoma Fig. c,d,h,i. d FUT4 CD5) with Ales Fig. , and itsd FUT4 CD5) with Ales Fig. b,e. ABCCd FUT4 CD5) with Ad FUT4 CD5) with A8 transformants was previously constructed25. Screening and selection of nanobodies targeting ABCC3 were performed following a peptide-based strategy. We predicted immunogenic epitopes within the amino acidic sequence of ABCC3 (UniProtKB reference: O15438) using BepiPred-2.0 analysis software31. Candidate immunogenic epitopes were selected based on (1) their sequential nature, as linear B-cell epitopes are commonly used for immunizations and antibody production32, (2) their length, with limitation to a minimum of 11 and a maximum of 30 amino acids to approximate the size of current linear epitopes34, and (3) their outer surface location at the plasma membrane, restricted to extracellular regions of the Membrane Spanning Domains (MSDs) and N-terminus of ABCC3 35 and was selected as a positive control for the initial experiments. We knocked-out ABCC3 by CRISPR/Cas9 technology. The resulting cell line (A549ABCC3KO) was used as a negative control . The affinity of NbA42 and NbA213 was assessed by targeting the control cell line A549 WT, using A549ABCC3KO as a non-specific background control . We confirmed the specific recognition of NbA42 and NbA213 to peptide ABCC3-A among the other ABCC3-derived peptides  by indirect ELISA  at 15 mg·kg−1 and their specific binding was analyzed 1 h after systemic injection . NbA42 and NbA213 showed minimal residual signals in other tissues  which confers resistance to various chemotherapeutic anticancer compounds19. In agreement with other reports we found that ABCC3 is highly expressed in glioblastoma samples, correlates with tumor grade and worse prognosis of patients24."Omic" sciences hold the promise of precision medicine in cancer by translating molecular-based big data into novel clinical approaches41. Also, the specific inhibition of ABCC3 with MK571 increases apoptosis upon TMZ treatment in other cell types42. The higher expression of ABCC3 in NK cells modulates their chemoresistance to TMZ in patients of malignant gliomas43. Recently, ABCC3 has been included in gene signatures  that classify glioma patients in agreement with therapeutic response to chemotherapeutic agents, including TMZ. According to previous reports20, we found higher ABCC3 expression in patients with methylated MGMT promoter, correlating with a worse prognosis. A role of ABCC3 in cancer stemness is emerging, as ABCC3 knockdown reduced the expression of stemness genes and the CD44high/CD24low breast cancer stem-like subpopulation. Also, SOX2 mediates transcription of ABCC3 in glioblastoma cells45. We found a similar correlation between CD44, FUT4 (CD15) and ABCC3 expression across glioblastoma patient datasets. These data support the possible intervention of ABCC3 in TMZ resistance and cancer stemness. Then, ABCC3 could represent a new molecular target of glioblastoma which may facilitate the subsequent development of molecular imaging approaches for patient management.ABCC3 may have a plausible role in temozolomide (TMZ) resistance. The expression of ABCC3 increases following TMZ treatment in glioblastoma cell lines, in a concentration and time-dependent manner46. However, the access of scFvs into the CNS is limited and fusion to cell-penetrating peptides acting as BBB shuttles is often required47. Nanobodies can circumvent the BBB by taking advantage of molecular routes of transcytosis16. In recent years, several nanobodies have sprouted against glioblastoma targets of protein biosynthesis , DNA metabolism (NAP1L1), and cellular growth 50. Here, we describe two novel nanobodies targeting ABCC3, NbA42 and NbA213, isolated from a previously constructed glioblastoma-specific phage-display library25. The rational design of immunogenic-like peptides located on the extracellular surface of ABCC3 allowed a peptide-based strategy for biopanning. In vitro studies showed that NbA42 and NbA213 specifically target ABCC3 at the N-terminus (peptide ABCC3-A). NbA42 and NbA213 recognized ABCC3-expressing cells in glioblastoma heterotopic xenografted tumor-bearing mice. Detection of ABCC3 by NbA42 and NbA213 was further achieved in two orthotopic glioblastoma mouse models with different degrees of BBB disruption. While further characterization of the BBB penetrance is required, these results suggest that these nanobodies do not present a high dependence on a highly disrupted BBB. Future applications of both nanobodies targeting ABCC3 may allow the development of novel glioblastoma theragnostic procedures.Novel immunotargeted applications obtained by combining the selectivity of antibody derivatives with the resolution capabilities of a given imaging modality, have been implemented for glioblastoma theragnosis. Single-chain variable fragments (scFvs) against ABCC3 have been used in preclinical models of glioblastoma. Interestingly, scFvs M25, M58, and M89 recognized extracellular epitopes of the N-terminus of ABCC3 and showed specific targeting of ABCC3-expressing glioblastoma cells in vitro51. Among others, nanobodies have been exploited to deliver cytotoxic payloads specifically to tumors, image-guided surgery, and to improve chimeric antigen receptor (CAR)-T and photodynamic therapies56. Since the approval of nanobody ALX-0681 (Caplacizumab) by the regulatory agencies57, examples of radiolabeled nanobodies conducting immuno-positron emission tomography (immuno-PET)58 studies have reached clinical trials in cancer patients. Regarding glioblastoma, 89Zr-labeled nanobodies targeting HGF have demonstrated their theragnostic potential in preclinical models59. We highlight the potential of NbA42 and NbA213 as new candidate molecules for the further design of nanobody-based molecular imaging probes. The predictive role of ABCC3 in the survival and prognosis of glioblastoma patients reveals a newfangled translational diagnostic and/or therapeutic relevance of NbA42 and NbA213 in the clinics.Nanobodies constitute a versatile tool for cancer managementABCC3KO cell lines, and U-87 MG, U251, U373-MG, and T98G human glioma cell lines were grown with DMEM medium (Sigma-Aldrich) containing 10% FBS (Fisher Scientific), and maintained at 37 °C and 5% CO2. A549 and U373-MG were kindly provided by Dr. Martín-Duque (IACS). U-87 MG, U251, and T98G were kindly provided by the Holland and Joyce laboratories (MSKCC). Short tandem repeat markers performed cell line authentication at different passages.Human lung adenocarcinoma A549 and its ABCC3 knocked-out by CRISPR/Cas9 system derivative A549https://www.cancer.gov/tcga accessed on April 26th, 2017)26 were analyzed using limma 27 to identify the most highly expressed genes. The differentially expressed list of genes was mined for those that encode proteins located in the plasma membrane. The subcellular localization was determined by experimental immunohistochemistry evidence as cataloged in the Human Protein Atlas . Gene expression and survival of adult glioblastoma patient data from the TCGA Project (TCGA-GBM and TCGA-GBMLGG), the Chinese Glioma Genome Atlas 61, and the Repository for Molecular Brain Neoplasia Data 62 were analyzed using GlioVis data portal (http://gliovis.bioinfo.cnio.es/)28. Log-rank and Wilcoxon tests were applied for Kaplan–Meier survival curves. Pearson's product-moment correlation was applied to determine gene correlations between paired samples. Tukey's Honest Significant Difference (HSD) was applied to compare ABCC3 expression across tumor grades and glioblastoma vs. normal samples. Statistical analysis was obtained from GlioVis and completed using R (http://www.r-project.org).RNA-Seq data from 142 glioblastoma samples and 5 normal brain samples deposited in The Cancer Genome Atlas 31. A list of 57 potential epitopes was obtained (Supplementary Table ABCC3-A (ALCGSGELGSKFWDSNLSVHTENPDLTPCFHHHHHH),ABCC3-K (KAEGEISDPFRHHHHHH),ABCC3-L (HHHHHHSAWTNDAMADSRQNNTSL).Prediction of immunogenic epitopes of human ABCC3 (UniProtKB reference: O15438) was performed by submitting the amino acidic sequence to BepiPred-2.0 analysis software , with 1011 phages for 1 h at room temperature. Non-specific binding sites were blocked with 5% nonfat milk. Bound phages were eluted with 100 mM triethylamine and neutralized with 1 M Tris–HCl (pH 7.4). Phages were used to infect the amber-codon-suppressing TG1 E. coli cells for 30 min at 37 °C, which were later co-infected with 1015 M13K07 helper phages. Enriched phages were grown overnight at 37 °C in 2× Tryptone & Yeast extract medium supplemented with 100 µg/mL ampicillin and 70 µg/mL kanamycin. New produced phages were precipitated with ice-cold polyethylene glycol-6000/NaCl and resuspended in 1 mL PBS.These peptides were used for biopanning as described elsewhereE. coli by adding isopropyl β-d-1-thiogalactopyranoside (IPTG) to cultures at a final concentration of 1 mM and incubating overnight at 28 °C. Extraction of periplasmic proteins including hemagglutinin(HA)-tagged nanobodies was performed by osmotic shock with 4× Tris/EDTA/sucrose and ddH2O. ELISA was performed by adding bacterial periplasmic extracts to 96-well NUNC MaxiSorp plates (Thermo Scientific), previously coated with 2 µg/mL of ABCC3-derived peptide in 0.1 M NaHCO3 (5 µg/mL for measuring binding specificity). Absorbance at 405 nm was measured on a Synergy HT multi-mode microplate reader (BioTek), after incubation with mouse anti-HA  and goat anti-mouse IgG conjugated with alkaline phosphatase  antibodies, and alkaline phosphatase substrate (Sigma-Aldrich). Potential clones targeting ABCC3 rendered 1.5 higher signal than its negative control without antigen. Genetic sequences of specific nanobodies were amplified by colony PCR with primers RP (5'-TCACACAGGAAACAGCTATGAC-3') and GIII (5'-CCACAGACAGCCCTCATAG-3'), and sequenced at Macrogen (The Netherlands).Nanobodies were expressed in TG1 12 into the pHEN664 expression vector, to replace their HA-tag by a hexahistidine(His)-tag for further purification. Briefly, similar colony PCR with primers A6E (5'-GATGTGCAGCTGCAGGAGTCTGGAGGAGG-3') and 38 (5'-GGACTAGTG CGGCCGCTGGAGACGGTGACCTGGGT-3') yielded products of 400 bp, further cloned into pHEN6 with PstI and BstEII (Eco91I) (Thermo Scientific). Conservation of sequences was verified by PCR with FP (5'-CGCCAGGGTTTTCCCAGTCACGAC-3') and RP (5'-TCACACAGGAAACAGCTATGAC-3'), with further sequencing. Expression of nanobodies encoded in the pHEN6 vector was performed in WK6 E. coli as previously described64. Briefly, 1 mM of IPTG was added to cultures to induce nanobody expression overnight at 28 ˚C. Periplasmic extracts were obtained by osmotic shock and dialyzed on Spectra/Por dialysis membrane tubing of MWCO 3.5 kDa  in PBS. His-tagged nanobodies were purified using an increasing gradient of 0.5 M imidazole on immobilized metal affinity chromatography columns in an ÄKTA protein purification system . Nanobody concentration was measured on a Synergy HT multi-mode microplate reader (BioTek) after incubation with Bradford reagent .Nanobodies with complete sequences were recloned from pHEN45 cells were counted using a Neubauer chamber using Trypan Blue Solution 0.4% (Fisher Scientific). After incubation in blocking and permeabilizing buffer (PBS/saponin 0.1%/FBS 5%) for 15 min at 4 °C, cells were stained with anti-ABCC3 monoclonal antibody  and APC goat anti-mouse IgG antibody  for 20 min at 4 °C. To stain with nanobodies targeting ABCC3, cells were detached with 10 mM EDTA (Fisher Scientific). Upon incubation in blocking buffer (PBS/1% BSA) for 15 min at 4 °C, cells were stained with 5 µg of anti-ABCC3 nanobody followed by ReadyTag anti-6-His mAb  and APC goat anti-mouse IgG antibody  for 20 min at 4 °C. Cells were analyzed on a Gallios flow cytometer (Beckman Coulter) and results were visualized at Kaluza analysis software (Beckman Coulter). To estimate the equilibrium dissociation constant (KD), nanobodies were conjugated to fluorescein-5-isothiocyanate using FluoroTag™ FITC Conjugation Kit (Sigma-Aldrich), and total and non-specific binding was analyzed with GraphPad Prism v.6.01.To analyze the expression levels of ABCC3, 4 × 10nu/nu mice were purchased from Envigo (Spain). Mice were housed in animal experimental core facilities of the Aragon Institute of Health Sciences (IACS) at the Biomedical Research Center of Aragon . All animal experiments were approved and carried out according to the Ethical Committee for Animal Experimentation of the University of Zaragoza, under the protocol project No. PI60/20. Mice were cared for in accordance with the European 2010/63/UE and Spanish national RD1386/2018 legal statements.Athymic Nude-Foxn16 cells; 6–8 weeks athymic nude mice). Nanobodies were administered intraperitoneally (i.p.) at 15 mg·kg−1 in mice bearing tumors (1000 mm3). Mice were sacrificed 1 h after administration; tumors were mechanically disaggregated and incubated in 10 mM EDTA (Fisher Scientific). The targeting profile of nanobodies was evaluated by prospective ex vivo flow cytometry in collected tissues .Subcutaneous xenograft models were established . Injections were made to the right frontal cortex, approximately 1 mm caudal and 1.5 mm lateral from bregma, and at a depth of 2 mm using a Hamilton syringe (Hamilton) as described previously66. Tumor growth was monitored by bioluminescence. Mice were injected with D-Luciferin (150 mg/kg). 15 min after injection, images were acquired for 5 s using an IVIS Lumina XRMS equipment (Perkin Elmer). Bioluminescence analysis was performed using Living Image software, version 2.50. Nanobodies were administered as described above in mice with tumors in a positive growth phase determined by bioluminescence (106 to 108 p/s/cm2/sr of average radiance). Mice were sacrificed 1 h after administration, and tumors were processed as described above.For orthotopic xenografts, U-87 MG and 251 cells were transduced with a TK-GFP-Luciferase reporter plasmid (TGL) to monitor tumor growth by bioluminescence measurements. To develop intracranial tumors, 6–8 weeks athymic nude mice were anesthetized by inhalation of 3% isoflurane and were subcutaneously injected with 50 μL of the local anesthetic 0.25% bupivacaine at the surgical site. Mice were intracranially injected with 2 μL containing 5·102 inhalation and perfused with acidified fixative . Collected brain tumors and samples of their contralateral hemisphere were incubated in 700 μL of formamide (Sigma) to extract Evan's blue at 60 °C overnight. Absorbance was measured at 610 nm and 740 nm on a Synergy HT multi-mode microplate reader (BioTek). Non-injected or intracranially injected with 2 μL of PBS (mock surgery), 6–8 weeks athymic nude littermates were included as intact brain controls.To evaluate the BBB disruption, 6–8 weeks athymic nude littermates bearing similar intracranial tumors were injected i.p. with 400 μL of 4% Evan's blue dye (Sigma). One hour after injection, mice were sacrificed by COTo evaluate the bioavailability of nanobodies plasma and urine samples were obtained from mice sacrificed 15 min, 30 min, 1 h, 4 h, and 24 h after administration of nanobodies. Protein content was measured in plasma and urine samples using a Synergy HT multi-mode microplate reader (BioTek) after incubation with Bradford reagent . A volume containing 5 μg of protein for plasma and 1 μg for urine was used for immunoblot detection. ReadyTag anti-6-His mAb  and goat anti-mouse IgG IRDye800CW antibody  allowed detection of His-tagged nanobodies in an Odyssey CLx Imaging System (LI-COR). Vehicle-treated healthy mice were used as controls.All experiments and methods were performed in accordance with relevant guidelines and regulations.Supplementary Information.


The existence of dynamic recrystallization (DRX), i.e., recrystallization occurring during straining, has long been questioned  despite Although DRX was first imagined to take place exclusively in low to medium stacking fault energy (SFE) materials, it was later observed that high SFE metals, such as ferritic steels or aluminium alloys, also exhibit recrystallization-like microstructure transformations during hot working. In the first case, DRX occurs by nucleation and the growth of new grains, which has been termed discontinuous DRX (DDRX); in the second case, DRX results from the progressive “fragmentation” of the initial grains and is often referred to as continuous DRX (CDRX) . The aimNagira et al.’s paper  investigTwo contributions are devoted to near-β titanium alloys deformed in uniaxial compression in both the α and α + β domains: Zhou et al.  investigThree papers deal with the hot working of magnesium alloys, which are much less frequently mentioned in the literature. Two similar Mg-Gd-Y alloys are submitted to large strains by Wu et al.  and Liu Finally, a theoretical paper by Montheillet  points o


Free tissue transfer is widely used for head and neck reconstruction. In certain circumstances, vein grafting is required to elongate free flap pedicles to connect them to appropriate recipient vessels. Because of controversy regarding the use of interposition vein grafts in free tissue reconstruction, this paper reports vein graft indications, techniques, safety, and outcomes for head and neck microvascular surgery.Twenty-six patients (23 men and 3 women) who underwent interposition vein grafting concurrent with free tissue transfer were included in this study. The most common reason for head and neck reconstruction with vein graft was tumor recurrence, followed by flap salvage. The interposition vein grafts were applied in two manners as temporary arteriovenous (A–V) loop and conduit to extend the length of the free flap for venous drainage.p = 0.380). The free flap complication rate was 50.0% and 16.8% in patients with and without vein grafts, respectively (p < 0.001). Radiation therapy, chemotherapy, prior neck dissection, and prior free flap transfer were more common in the vein graft group . The hospital stay was significantly longer for the vein graft group than for the non–vein graft group .The most common reconstructions were anterolateral thigh flaps (15 cases), followed by vastus lateralis myocutaneous (3 cases) and radial forearm (2 cases) flaps. The common recipient vessels were superior thyroid artery, superficial temporal artery and external jugular vein. The free flap loss rate was 7.7% with vein grafts and 4.9 without vein grafts (Overall free flap survival rates of 92.3% and 95.1% in the vein and non-vein graft groups, respectively – indicating the reliability of the vein grafts in challenging head and neck reconstructions, particularly in salvage cases and patients with multiple reconstructions.Level 3. From functional and cosmetic perspectives, microvascular techniques are associated with substantial head and neck reconstruction defects. Currently, free tissue transfer is widely accepted as the method of choice for reconstruction in complex cases. Selecting an adequate recipient vessel is critical for efficient free flap reconstruction. However, due to anatomical variations or previous neck dissection, external irradiation, chemotherapy, or some combination of these, local recipient vessels may be unavailable. In addition, free tissue reconstruction can be complicated by a lack of healthy recipient vessels. In such circumstances, vein grafting is required to elongate free flap pedicles to connect them to appropriate recipient vessels.Since Crowell et al.From January 2013 to June 2018, a single academic medical center performed 756 free flap reconstructions. We collected data, including patient demographics, surgical history, prior irradiation and chemotherapy, flap type, diagnosis, specific vein graft use, surgical outcomes, flap compromise, morbidity in flap donor site, and length of hospital stay, were collected for analysis in this retrospective case series. We identified patients who underwent interposition vascular grafting concurrent with free tissue transfer and evaluated their data.After we conducted ablative surgery, debridement, or scar contracture release, we performed microvascular reconstruction for all vascular anastomoses immediately in a standardized manner without a coupling device. We performed all vascular anastomoses under an operating microscope, and 9–0 nylon was used. We performed conventional end-to-end microanastomosis with the interrupted suture technique. In each patient, according to the area and tissue composition of the defect after ablation, we selected a suitable donor site for free flap harvesting. We determined vein graft indications when recipient vessels in the neck were evaluated. We prepared a single interposition graft with consideration for the predicted disparity between the recipient and donor artery or vein. However, we prepared a long vein graft for bridging the gap between both an artery and a vein to the designated vessels when suitable recipient vessels on the ipsilateral neck were depleted. The interposition vein graft source was dependent on the availability of vessels at the donor site of the free flap. The Great Saphenous Vein (GSV) was the most frequently used vein graft. Postoperative care in an intensive care unit was arranged for each patient; hourly flap monitoring was provided by nursing staff for the first 48 h. Clinical monitoring included monitoring of flap skin color, surface temperature, capillary refill time, and acoustic cutaneous Doppler to detect intravascular arterial blood flow. When their condition was stable, the patients were discharged and asked to attend regular follow-ups provided by the relevant surgical teams. Morbidity at the donor site including wound dehiscence, poor wound healing, hematoma, seroma, and skin graft loss postoperatively requiring wound treatment or surgical intervention.U test was used to assess associations between baseline parameters and vein grafting outcomes. A p-value of <0.05 was considered significant. Within-patient correlations were assessed using univariate and multivariable generalized estimating equation models to measure the effect of vein grafting on take-back for vascular exploration, partial skin necrosis of the flap, and flap loss. We adjusted odd ratios by using a multiple logistic regression model adjusting for potential confounding factors. We employed a backward model selection approach to select the predictive factors. Variables with a value of p ≥ 0.05 were removed from the multiple logistic regression model. All tests were two-sided. We analyzed all data by using the Statistical Package for the Social Sciences, version 22.0 . Again, a two-tailed p-value of <0.05 denoted statistical significance. This study was conducted with the approval of the institutional review board .We used frequencies and percentages to summarize the clinical data and outcomes. The Chi-Squared, Fisher’s exact, or Mann–Whitney In this study, vein grafts were required in 26 patients (23 men and 3 women) with head and neck defects, accounting for 3.44% of all microvascular reconstructions (n = 756). Oral cavity reconstruction after surgical ablation of squamous cell carcinoma was the most common indication for free flap transfer (69.5%).Interposition vein grafting methods were used in four clinical scenarios. The most common scenario was vein graft use in a planned manner because appropriate recipient vessels were unavailable in patients with tumor recurrence . In the second scenario, 10 patients (38.5%) were given interposition vein grafts for flap salvage due to postoperative thrombotic events. In this scenario, first, second, and third microsurgical reconstructions were performed in four, three, and three patients, respectively. In the third scenario, three patients received flap reconstruction for the repair of a previously applied flap  due to complications such as osteoradionecrosis, scar contracture, and iatrogenic infection. Finally, the fourth scenario was the application of a vein graft in an unplanned intraoperative approach .In the vein grafting series, 15 flaps (57.7%) were anterior lateral thigh flaps; 3 (11.5%) were Vastus Lateralis Myocuataneous (VLMC) flaps, and two each were radial forearm flaps (7.7%), posterior medial thigh flaps (7.7%), and fibula osteocutaneous flaps (7.7%). The most common vein graft source was the GSV . We measured vein graft length after vascular anastomosis, and the average vessel graft length required was 16.1 ± 6.7 cm (range: 4–30 cm).We applied interposition vein grafts in two manners. First, we employed such grafts when suitable recipient vessels were completely depleted in the ipsilateral neck of patients with complicated conditions. The most common technique was the creation of an arteriovenous (A–V) loop; this was performed in 14 patients (53.8%). A selected recipient artery and vein at the remote site of the neck was connected using a long vein graft. The anastomoses were released to allow vein graft perfusion, creating a temporary A–V loop. The A–V loop was physiologically distended through arterial inflow. After an observation period, we divided the vein graft at the midpoint such that it became two interposition vein grafts, connecting it to the artery and vein of the flap. With this approach, the flap can reach the contralateral side of the recipient vessels A and B. In patients who received interposition vein grafts, the common recipient arteries were the superior thyroid artery  and the superficial temporal artery . The most common recipient vein was the external jugular vein ; we used contralateral vessels as recipient vessels in 12 patients (46.2%). In the two procedures with flap failure, one patient underwent subsequent free flap surgery, and the other was treated using pedicled pectoralis major myocutaneous flap. Complications included wound dehiscence 7 (26.9%), partial flap necrosis 3 (11.5%), wound infection 1 (3.8%), hematoma 1 (3.8%) and orocutaneous fistula 1 (3.8%). We performed skin graft operations for four patients with minor complications, and the remaining patients received wound care. The total complication rate  was 50%.p = 0.001) or chemotherapy  were higher in the vein graft group than in the group not given vein grafts. More patients in the vein graft group had prior neck dissection and prior free flap transfer   .Table 1Pp = 0.380; 7.7% with vein grafting and 4.9% without vein grafting). The free flap postoperative complication rate  was significantly higher in the vein graft group compared with the non–vein graft group . The take-back rate for vascular exploration was not significantly different between the two groups . Flap donor site morbidity of the two groups did not significantly differ . The hospital stay was significantly longer for the vein graft group than for the non–vein graft group  (The free flap loss rates of the two groups did not significantly differ (= 0.001) .Table 2SA univariate general estimating equation model was used to identify variables affecting take-back for vascular exploration and flap loss , Table 4p = 0.049).A univariate general estimating equation model was used to identify variables affecting partial skin necrosis of the flap . Only prTheoretically, interposition vein grafting offers benefits of increased vascular pedicle length to overcome problems of tension and gaps between free flaps and recipient vessels. However, vein grafting techniques in microsurgery are far more difficult than primary anastomosis of free flap vessels because the vessel caliber of the graft may vary, and more vascular anastomoses are required.Miller et al.p = 0.530) for free flap loss in vein-grafted flaps. In 2018, Maricevich et al.19,Furr et al.Schanzer et al.Vein graft handling and anastomosis techniques are other crucial factors affecting flap survival rate. In our study, a temporary A–V loop was created in 14 patients. Such a loop allows physiological distension of the vein graft, ensures no kinks or twists are present, and can be used to seal leaks of graft during observation periods.20Furr et al.This study had several limitations. First, the small number of included patients means that the results may not be generalizable to other populations,Interposition vein grafts can be used to salvage compromised free flaps; they are most often used in patients who have undergone multiple microvascular flap surgeries of the head and neck. Although an increased postoperative complication risk was noted compared with standardized surgical techniques, vein grafts have a high success rate, even in difficult cases. The selections of healthy recipient arteries and veins as well as meticulous flap monitoring are the key determinants of successful microvascular surgery using interposition vein grafts. It is a reliable option for challenging head and neck reconstruction and a valuable alternative for salvage cases.Study conception and design: Chih-Sheng Lai, Ching-Hui Shen, Yi-Ting Chang, Yi-Ling Lin. Acquisition of data: Chih-Sheng Lai, Yi-Ling Lin, Yueh-Chi Tsai. Drafting of data: Chih-Sheng Lai, Chen-Te Lu, Yueh-Chi Tsai, Jung-Hsing Yen. Critical revision: Chih-Sheng Lai, Yueh-Chi Tsai, Yi-Ling Lin, I-Chen Chen.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare no conflicts of interest.


Monkeypox is a self-limiting zoonotic viral disease and causes smallpox-like symptoms. The disease has a case fatality ratio of 3–6% and, recently, a multi-country outbreak of the disease has occurred. The currently available vaccines that have provided immunization against monkeypox are classified as live attenuated vaccinia virus-based vaccines, which pose challenges of safety and efficacy in chronic infections. In this study, we have used an immunoinformatics-aided design of a multi-epitope vaccine (MEV) candidate by targeting monkeypox virus (MPXV) glycoproteins and membrane proteins. From these proteins, seven epitopes  were finally selected and predicted as antigenic, non-allergic, interferon-γ activating and non-toxic. These epitopes were linked to adjuvants to design a non-allergic and antigenic candidate MPXV-MEV. Further, molecular docking and molecular dynamics simulations predicted stable interactions between predicted MEV and human receptor TLR5. Finally, the immune-simulation analysis showed that the candidate MPXV-MEV could elicit a human immune response. The results obtained from these in silico experiments are promising but require further validation through additional in vivo experiments. Monkeypox is a zoonotic viral infection with a 3–6% fatality rate and is caused by the monkeypox virus (MPXV) ,2. Two sviz. ground squirrels, mice and cynomolgus monkeys [Although monkeypox is self-limiting, people with comorbidities and compromised immunity, pregnant women, and the pediatric population are at higher risk. The protection from MPXV in humans could be provided by the smallpox vaccine . However monkeys ,13. As p monkeys ,15. Apar monkeys . Intrigu monkeys . Similar monkeys . Neverth+ and CD8+ T-cell epitopes in order to design a potential vaccine for the monkeypox virus.Interestingly, with the recent advances in the field of immunoinformatics, the pace of the vaccine development process has been relatively accelerated ,22,23,24The final predicted vaccine is a multiepitope vaccine (MEV) construct that comprises all identified epitopes fused altogether to elicit an optimal immune response. Afterwards, the structural, immunogenic and physicochemical parameters were evaluated for the designed MPXV-MEV. Additionally, molecular docking was performed to investigate the affinity of the vaccine construct towards the human toll-like receptor 5 (TLR5). Later, the molecular dynamics (MD) simulations confirmed the stability of the MPXV-MEV construct with the TLR5 receptor and the associated interactions. Further, immune simulations were carried out to ascertain the immune response profile of the vaccine candidate.The sequence of the MPXV strain W-Nigeria (Accession number: KJ642615.1) was obtained from the GenBank database. A total of 176 proteins in the proteome of MPXV strain W-Nigeria were characterized, where we filtered only glycoproteins and membrane proteins for further analysis, see + T cell epitopes, the HLA alleles selected were DRB1_0101, DRB1_0301, DRB1_0401, DRB1_0701, DRB1_0801, DRB1_0901, DRB1_1001, DRB1_1101, DRB1_1201, DRB1_1301, DRB1_1501, and DRB1_1602. Further, to predict CD8+ T cells, the HLA alleles selected were HLA-A0101, HLA A0201, HLA-A0301, HLA-A2402, HLA-A260, HLA-B0702, HLA-B0801, HLA-B2705, HLA-B3901, HLA-B4001, and HLA-B5801. Default parameters of the NETMHC 2.3 and NETMHC 4.0 webservers were used for the threshold for both the strong and weak binders. The IEDB B cell epitope prediction website’s Bepipred linear epitope prediction 2.0 approach was used to predict the multiple linear B cell epitopes [To predict T-helper cell epitopes that might bind to MHC class II molecules, the NETMHC 2.3 website was used, whereas for the identification of T- cytotoxic cell epitopes that might bind to MHC class I molecules and elicit a cellular immune response, we used the NETMHC 4.0 website . The NETepitopes . The Vaxepitopes ,33.The epitopes conservation was predicted in membrane proteins and glycoproteins of the three monkeypox virus strains, namely MPXV-WRAIR7-61, Sierra Leone, and COP-58, by employing the IEDB Epitope Conservancy Tool . The seqSalmonella typhimurium) and RS09—were also linked to form the multiepitope vaccine construct using the GGS linker [Both the B cell and T cell epitopes which fulfilled the selection criterion of properties, such as antigenic, non-toxic, non-allergic and interferon-γ activation, were linked together using the GSS linker. Two adjuvants—flagellin protein  is a position-specific scoring matrices-based machine learning approach and is used to investigate the immune response profile of a vaccine construct. This online webserver was used to predict the immunogenicity and immune behavior towards the candidate MPXV-MEV in the present study [The C-IMMSIM webserver  simulations . The com, 0.15 M . The sim of 2 ps . Then, t of 2 ps . Coulomb of 2 ps . At an i of 2 ps ,55. In-h of 2 ps ,58,59,60Out of 176 proteins, 18 proteins were identified. These proteins are classified as glycoproteins and membrane proteins in the proteome of the monkeypox virus strain W-Nigeria. + T cell epitopes, four CD8+ T cell epitopes and one linear B cell epitope) fulfilled the selection criterion and were chosen for designing the final MPXV-MEV (+ Tcell) epitopes, which are RIYFVSLSL and FSIGGVIHL and MHC-I (CD8+ Tcell) epitopes, which are IYFVSLSLL, LKHKYGCSL, AYTSISVVF and RYPIIDIKW, showed a world population coverage of 85.97% and 87.03% respectively.Strong binder epitopes of the MHC alleles, i.e., MHC I and MHC II, were first identified. The number of epitopes that were determined as strong binders for MHC I was 384 see . SimilarMPXV-MEV . All theThe epitopes identified were conjugated using adjuvants and Pan HLA DR-binding epitopes (PADRE) to develop the MEV construct. The final predicted vaccine construct has 390 residues . The MPXThe 3D structures of both MPXV-MEV and the immunogenic TLR5 receptor were predicted using the Alphafoldv2.0 program ,45. SincMD simulations were performed to study the structural stability of the docked complex of MPXV-MEV and TLR5 . GROMACSThe superimposition of complex structures extracted every 20 ns during the simulation time resulted in good overlap, with RMSD values below 1 nm E; a limi50 and C70 values of 0.87 and 0.51, implying that 87% and 51% of ICs were kept conserved for at least 50% and 70% of the frames. Hence, quite a high conservation of the complex interface during the simulation time was observed. The ICs remained stable over the simulation time and two such examples are shown in Further, the MDcons program was used to assess the stability of the complex between MPXV-MEV and TLR5, and the conservation of inter-residue contacts (ICs) at the interface along the simulation time was plotted ,67,68,69+ T l was also increased following every vaccination. Moreover, the population of memory T cells was increased following each vaccination  relative to the first dose of MEV A. The thcination C. Howeves of MEV C. Furthecination D. The reecreased D. Thus, ecreased E. Initiaecreased E. OveralMonkeypox is an orthopoxvirus, which is responsible for infectious diseases such as cowpox, smallpox, camelpox, and horsepox . The monThe currently available vaccines that have provided immunization against monkeypox include FDA approved ACAM2000 or NYCBH and LC16m8 which are under clinical trials. These vaccines are live, attenuated vaccinia viruses which face challenges of safety and efficacy in chronic infections ,77. Furt+ T cell population and the release of interferon-γ in rhesus macaques [The epitopes ISPDGCYSL and LTFDYVVTF, which were derived from the F8L protein of the monkeypox virus, have been reported to activate the CD8macaques . Interesmacaques ,95.Homo sapiens) in a protein–protein BLAST analysis, indicating that it could be safely used for humans.In the present study, the membrane proteins and glycoproteins were targeted for predicting antigenic epitopes to design a potential MEV candidate against monkeypox. These proteins are major constituents of pathogenic viruses and play a significant role in immunity and pathogenesis , and areThe immunogenic and non-allergic glycoproteins and membrane proteins for MPXV have been targeted in order to predict epitopes for T-cells and B-cells that could be used to design a candidate MEV against MPXV. The predicted MPXV-MEV is antigenic and non-allergic in nature and interacts strongly with human TLR5. In silico immune simulation of the MEV construct predicted that the vaccine candidate could elicit an immune response in humans.