We report a novel orthohantavirus, putatively named Ozark orthohantavirus, in hispid cotton rats captured within the Ozark Plateau in Arkansas, USA. This virus phylogenetically clusters with other orthohantaviruses that cause severe human disease. Continued orthohantavirus surveillance and virus sequencing are needed to address the potential public health threat of this virus. Orthohantavirus) are a group of zoonotic viruses primarily found in muroid rodents; many of the viruses are pathogenic in humans  in Arkansas, USA. Hispid cotton rats are a reservoir host of a notable pathogenic orthohantavirus, Black Creek Canal virus (BCCV) , in hispid cotton rats  according to manufacturer guidelines. We used the NEBNext rRNA Depletion Kit (human/mouse/rat) to remove host rRNA, then the NEBNext Ultra II RNA Library Prep Kit  to construct libraries. We performed next-generation sequencing by using the Illumina NovaSeq system (https://www.illumina.com). We quality filtered and de novo assembled the raw data and annotated the contigs by using LazyPipe  to detect ORFs and the Expasy translate tool (https://www.expasy.org) to translate ORFs to amino acid sequences. We compared corresponding nucleic acid and protein phylogenies of each OZV genome segment with BCCV and other related orthohantavirus sequences obtained from GenBank by using IQ-TREE2 (http://www.iqtree.org). We then used the Sequence Demarcation Tool version 1.2 program (http://web.cbio.uct.ac.za/~brejnev) to compare protein sequence pairwise identities of each OZV segment with those of closely related orthohantaviruses. Finally, we performed pairwise evolutionary distance (PED) analyses by using TREE-PUZZLE version 5.2 (http://www.tree-puzzle.de) with a PED cutoff value of 0.1 for species classification , medium (M), and large (L) segments from 2 rat samples and partial genome sequences from 6 other rat samples that included 3 additional complete S and 4 additional complete M segment sequences  Table. WOZV nucleotide sequences most closely clustered with other sigmodontine-borne orthohantaviruses, particularly BCCV and Bayou virus (BAYV), which are pathogenic to humans, and Catacamas virus (CATV), which is not known to cause human infections  Figure 7OZV is the second definitive orthohantavirus species identified in hispid cotton rats. This discovery also expands the geographic distribution of orthohantavirus-carrying hispid cotton rats in the United States, previously limited to Florida and Texas; because of OZV’s similarity to BCCV and BAYV, which cause severe disease, this discovery provides crucial public health information. OZV identification also informs broader orthohantavirus evolution, especially for within-host evolution and divergence. Although uncommon, multiple orthohantaviruses in a single reservoir host species have been observed, particularly in cricetid-borne orthohantaviruses in the Americas , Arkansas, USA.Additional information for novel Ozark orthohantavirus in hispid cotton rats (


This verified that such a ligand effect is generally applicable in anhydride/epoxide ROCOP catalyzed by aluminum complex and provides hints for future catalyst design.Previous work has indicated that aluminum (Al) complexes supported by a bipyridine bisphenolate (BpyBph) ligand exhibit higher activity in the ring-opening copolymerization (ROCOP) of maleic anhydride (MAH) and propylene oxide (PO) than their salen counterparts. Such a ligand effect in Al-catalyzed MAH-PO copolymerization reactions has yet to be clarified. Herein, the origin and applicability of the ligand effect have been explored by density functional theory, based on the mechanistic analysis for chain initiation and propagation. We found that the lower LUMO energy of the (BpyBph)AlCl complex accounts for its higher activity than the AlCl counterpart in MAH/epoxide copolymerizations. Inspired by the ligand effect, a structure-energy model was further established for catalytic activity  predictions. It is found that the LUMO energies of aluminum chloride complexes and their average NBO charges of coordinating oxygen atoms correlate with the catalytic activity  of Al complexes (R Aliphatic polyesters have drawn a lot of attention as a potentially viable choice for petroleum-based polymers because of their numerous renewable sources, hydrolytic degradability, and excellent biocompatibility ,2,3. In β-diiminate zinc acetate [(BDI)ZnOAc] complex is an effective catalyst for various epoxide/cyclic anhydrides copolymerization and terpolymerizations of epoxide/cyclic anhydrides/CO2 , which have been successfully used in epoxide/CO2 copolymerizations Cl. Owing to the fact that the weakly nucleophilic Cl− is an easier leaving group than alkoxide with a strong nucleophilicity, the resulting six-coordination aluminate species INT2 is feasible to release the chloride anion, leaving a vacant site available for coordination of the coming PO. A coordination of PO to INT3 generated the neutrally 6-coordinated INT4 which could be converted to the bis Al complex INT5 via TS2 by the attack of another nucleophilic anion. It is noteworthy that the ring-opening of PO in the chain initiation stage proceeded at the two sides of the [ONNO-Al] plane, and the bis Al complex INT5 was the real active species that could initiate the subsequent chain propagation. As shown in G1‡) of the first PO insertion were very similar for complexes A and B; however, the ∆G2‡ for complex A  is higher than that for B , suggesting the slower chain initiation by complex A.The computed energy profiles for the chain initiation step mediated by complexes A-mediated polymerization, the distortion/interaction analysis was comparatively performed for the key TSs, A_TS2 and B_TS2, respectively  equatorial ligand plane in fragment A of A_TS2 (13.3° for the fragment A of A_TS2 relative to B_INT3) is much bigger than in the case of B_TS2 (6.3° for the fragment A of B_TS2 relative to B_INT3). These geometrical changes mainly account for the larger distortion of A_TS2 and thus its lower stability.As shown in INT5, the enchainment of MAH could take place through TS3 and yield anionic six-coordination INT6 with alkoxide at one side and carboxylate at the other side of the ONNO-Al plane. The release of the carboxylate anion  resulted in five-coordination neutral INT7 with an alkoxide and a vacant coordination site in the axial position, which could coordinate with either PO or RCOO− (dissociated from INT6) and thus has two possible reaction pathways. One possible route is the formation of INT8, in which PO was activated upon coordination to INT7. Then, the bis Al complex INT9 was generated by the nucleophilic attack of RCOO− via transition state TS4. INT9 is structurally similar to the bis aluminate complex INT5, and the repeat of the process of INT5 to INT9 could grow the polymer chain. The alternative route is an association of RCOO− with INT7 and the formation of the anionic six-coordination INT10. Upon the approach of MAH, the alkoxide in INT10 could nucleophilically attach the carboxyl carbon of the MAH and generate the bi(carboxylate) Al complex INT11, which could convert to INT12 via the release of a carboxylate anion. The RCOO− anion (dissociated from INT11) is easily coordinated with INT12 in another fashion and forms more stable intermediate (INT12’). Similar to INT7, PO could coordinate to INT12 and was ring-opening under the action of a dissociated carboxylate anion from INT11 to afford INT14 . The chain propagation could occur via a similar process from INT10 to INT14.To further explore the effect of the ligand backbone on the polymerization activity, the chain propagation was also calculated, as shown by the following process . Based oINT12’ → TS4) is the highest  among the reaction steps. Therefore, the ring-opening of PO serves as a rate-determining step of the copolymerization reaction. This is in agreement with the experimental finding that the polymerization rate is first-order in [PO] and zero-order in [MAH]  . Meanwhi1 and B1 .A_TS4 and B_TS4) were conducted as well, in order to elucidate the ligand framework effect on catalytic activity. As shown in A_TS4, the total deformation energy ∆Edist is 42.4 kcal/mol, which could be balanced out by its ∆Eint  leading to an ∆E of −3.7 kcal/mol. By contrast, the less negative interaction energy  in B_TS4 could not compensate for its total deformation energy , thus producing a higher ∆E‡ . Therefore, the stronger interaction between three fragments could account for the greater stability of A_TS4. Meanwhile, it has been found that the interaction  between the fragment A and B in A_TS4 is stronger than that in B_TS4  and mainly contributes to the stability of A_TS4. Such a discrepancy probably originates from the higher electrophilic ability of A_INT7 compared with B_INT7. As expected, the calculated LUMO energies for A_INT7 and B_INT7 were −3.21 and −2.73 eV Al analogues (B1).Furthermore, distortion/interaction analyses for turn-over limiting TSs  analysis. The catalytic activity toward PO and MAH copolymerization was estimated by the turnover frequency  value. Seven parameters of each aluminum chloride complex were chosen to characterize the copolymerization activity of these complexes , Wiberg bond index of Al-Cl (WBI), dihedral angle of [ONNO] , the NBO charges of central metal (qAl) and coordinating heteroatoms, viz., chloride atom (qCl), nitrogen atoms , and oxygen atoms .To further validate the above ligand effect, we further explored the six complexes A1~F1,  that havs A1~F1, , includiA1~F1 as the training set, we constructed the catalyst activity regression model for CHO and MAH copolymerization, with the help of the stepwiselm function in Matlab to perform multivariate linear regression and remove the insignificant descriptors . The TOF and parameters  are shown in 2) of 0.98 and the ‘3-fold’ cross-validation determination coefficient (Q2) of 0.88. As expected, the predicted TOF values derived from the MLR model are consistent with the experimental ones.Taking the catalysts ELUMO) and the average NBO charges of oxygen atoms (qO) were retained  of the corresponding metal complexes. This demonstrates that the electron-withdrawing group or conjugated backbone is beneficial to decreasing such a LUMO energy or the average NBO charges of oxygen atoms. Such an electronic effect therefore increases the activity of the corresponding metal complex toward PO/MAH copolymerization. This strategy was promising to utilize in the future design of metal-based catalysts and expand their application by advancing catalytic properties.In the regression model, only the LUMO energy  of 0.88. Strong correlations were found between the catalyst activity of aluminum chloride complexes and their LUMO energies (ELUMO), as well as the average NBO charge of coordinating oxygen atoms (qO). When the ELUMO is lower and the ligation oxygen atoms are more negatively charged (more negative qO), the Al complexes have a higher catalytic activity and lead to more rapid PO/MAH copolymerization. This is in agreement with the experimental results in which the complexes (F1) with an electron-withdrawing group or conjugated backbone demonstrate the highest TOF value, compared with other aluminum analogues (A1~E1). The mechanistic insight gained in this study could help to better interpret the catalytic activity and contribute to the rational design of novel metal catalysts.The ligand effect in the copolymerization of maleic anhydride (MAH) and epoxides (PO) catalyzed by Al-based complexes has been investigated by DFT calculations. Having achieved an agreement in activity discrepancy between BpyBph and salen-ligated Al complexes, it is found that the BpyBph ligand endows its Al complex with a lower LUMO energy than its salen counterpart, thus leading to the higher catalytic activity. Inspired by this ligand effect, a quantitative structure-activity relationship has been constructed via multivariate linear regression (MLR) analysis. The trained MLR model features an RCatalysts are the core competence of determining the industrial production efficiency and advanced product development of polyester. The development of traditional catalysts relies on chance encounters, chemical intuition, and large-scale experimental behavior. Meanwhile, the measurement of catalytic performance is high cost and needs a lot of resources as well. In order to meet the requirements of current social and economic development, it is urgent to change the outmoded development pattern to a predictable and designable model. With the rapid evolution of high-performance computing technology, the era of artificial intelligence is inexorably surging. Relying on the rapid development of different algorithms and computer hardware, it is the right time to harvest the potential of machine learning in the field of catalysis across academy and industry. Despite a substantial number of successful applications of machine learning, this exciting topic is still largely in its nascent stage and it is believed that machine learning will play an increasingly important role in accelerating the development of various kinds of functional materials in the foreseeable future. Herein, we will focus on the development of novel polyester and polyolefin catalysts by using machine-learning methods combined with essential DFT calculations, and provide insight into the underlying mechanism of the relationship between the micro-structure of the catalyst and its polymerization performance by constructing the prediction models of catalytical activity and selectivity at the molecular and electronic level in the near future. This may provide a catalyst design guideline for developing high-performance polymerization catalysts and accelerate new high-performance catalyst development and materials discovery.


Trigeminal neuralgia (TN) is defined as paroxysmal, sharp pain within the trigeminal nerve (CNV) distribution lasting up to 2 minutes.Hypoglossal nerve stimulation (HNS) is a modality of obstructive sleep apnea (OSA) treatment wherein an Inspire device  is surgically implanted to stimulate the extension of the tongue via branches of the hypoglossal nerve (CNXII) during inspiration. This keeps the pharynx from collapsing and, thus, improves OSA.A 63‐year‐old male with body mass index 27.2 presented for evaluation of OSA with apnea–hypopnea index of 22.9/hour and positive airway pressure intolerance. His medical history included right‐sided TN following lightning strike 35 years ago and refractory to medical therapy. He underwent GKR 3 years prior, with improvement in TN symptoms. He was deemed an appropriate candidate for HNS therapy. Placement of and recovery from HNS implantation was without incident.After device activation, the patient reported reduced snoring and improved daytime energy, but increased right facial discomfort. Over the subsequent weeks, he noted progressive aching right facial pain, small shocks in the corner of the eye, worm‐like crawling in his cheek, and hypersensitivity and numbness in the lower lip. Magnetic resonance imaging (MRI) was notable only for asymmetric enhancement of the right CNV in Meckel's cave, consistent with GKR changes.The HNS device was discontinued for 4 weeks, then retried with lower‐voltage settings. His discomfort initially improved, but reintroduction precipitated symptoms. It was subsequently discovered that prior to HNS implantation, he had reported subtle, but increasing right‐sided facial pain. Following several conversations between sleep surgery, sleep medicine, neurology, and neurosurgery, it was concluded that he developed ATN prior to device placement and, unfortunately, usage led to symptom exacerbation. Advanced titration of the device was suggested to see if altered electrode configurations or pulse width or rate could help, but the patient declined. Patient desired explantation to eliminate MRI restrictions, which was performed without issue. He was ultimately treated medically for ATN with improved symptoms.There is no known physiologic connection between CNXII and CNV, which controls facial sensation and provides motor branches to the muscles of mastication, mylohyoid, tensor veli palatini, and digastric muscles. Thus, a CNXII stimulator would not be expected to exacerbate trigeminal pain. Despite this, HNS device usage clinically exacerbated symptoms and disuse improved them. TN and ATN have multiple triggers, including touching the face, eating, and talking.Multidisciplinary collaboration was key to understanding the patient's underlying facial pain disorder and the contribution of the HNS device.There is no neurophysiologic connection between the trigeminal and hypoglossal nerve, yet this patient's ATN was exacerbated by HNS use and resolved only with disuse. Further evaluation of facial pain in patients treated with HNS is warranted, as is patient counseling in this regard.Jonathan L. Vincze, manuscript writing and editing, presentation of research; Megan E. Falls, manuscript writing and editing, conceptualization; Noah P. Parker, manuscript editing, conceptualization.Dr Parker is on a physician advisory council for Inspire. The other authors have no commercial associations or financial disclosures that might pose or create a conflict of interest.No external funding was used for this study.


It is often challenging to distinguish cancerous from non-cancerous lesions in the brain using conventional diagnostic approaches. We introduce an analytic technique called Real-CSF (repetitive element aneuploidy sequencing in CSF) to detect cancers of the central nervous system from evaluation of DNA in the cerebrospinal fluid (CSF). Short interspersed nuclear elements (SINEs) are PCR amplified with a single primer pair, and the PCR products are evaluated by next-generation sequencing. Real-CSF assesses genome-wide copy-number alterations as well as focal amplifications of selected oncogenes. Real-CSF was applied to 280 CSF samples and correctly identified 67% of 184 cancerous and 96% of 96 non-cancerous brain lesions. CSF analysis was considerably more sensitive than standard-of-care cytology and plasma cell-free DNA analysis in the same patients. Real-CSF therefore has the capacity to be used in combination with other clinical, radiologic, and laboratory-based data to inform the diagnosis and management of patients with suspected cancers of the brain.  •Limited DNA in the cerebrospinal fluid (CSF) poses challenges for genetic techniques•Copy-number alterations are a feature of nearly all CNS cancers•Real-CSF works in limited starting material and identifies copy-number alterations•Real-CSF can detect neoplasia in CSF across a heterogeneous set of CNS cancers Douville et al. introduce Real-CSF—a molecular technique designed to identify copy-number alterations spread throughout the genome from limited starting material in cerebrospinal fluid. Even though central nervous system cancers comprise a heterogeneous set of tumors, nearly all will contain copy-number alterations, making this a putative multi-cancer biomarker. Central nervous system (CNS) neoplasms comprise a heterogeneous class of tumors that are either primary, i.e., originate in the brain or the spinal cord, or metastatic, i.e., cancers that spread to the CNS from another organ. Approximately 24,500 cases of primary brain cancers occur a year in the United States, with the most common being glioblastoma in adults and medulloblastoma in children.,,,The small number of reliable, quantitative biomarkers for the diagnosis and monitoring of cancers in the CNS poses challenges for diagnosis of patients with suspected brain cancers. The current gold standard is cytology of cerebrospinal fluid (CSF), which has a sensitivity that ranges from 2% to 50% depending on the cancer type.,,,,,,,,,,,There have been several promising types of biomarkers proposed to identify cancers of the CNS. Given the relative lack of brain-derived analytes in the blood,In the current study, we describe our efforts to develop a simple strategy, called Real-CSF (repetitive element aneuploidy sequencing in CSF), for the evaluation of several of the most common and debilitating forms of brain cancers: glioblastomas, metastatic lesions, lymphomas, and medulloblastomas. These assays were performed on DNA purified from 1 mL CSF, including all cells and cell-free fluid within the CSF aliquot, and evaluated chromosome abnormalities. We also compared whether cell-free DNA from plasma could as easily detect such chromosome abnormalities as CSF.Two independent cohorts of patients were evaluated in this study: a training set and a validation set. The training set was composed of CSF samples from 85 patients: 31 with glioblastoma (GBM), 13 with metastasis from primary tumors outside the brain, 7 with lymphoma, and 34 without cancer. The validation set was composed of CSF samples from 195 patients: 27 with GBM (five of which were pediatric H3K27M diffuse midline gliomas), 52 with metastasis from primary tumors outside the brain, 27 with CNS lymphoma, 23 with medulloblastoma, and 62 without cancer. Information with respect to the timing of the CSF sample collection (prior to diagnosis or during management of patients with known cancers) is recorded in ,,CNS neoplasms comprise a heterogeneous class of tumors and an equally diverse landscape of genetic alterations. Identifying the optimal combination of genetic markers that could encompass all CNS cancers is difficult. There is often insufficient starting material in CSF to query all somatic mutations and translocations across all potential driver genes. Aneuploidy or the presence of an abnormal number of chromosomes is a feature of most CNS cancer cells.Here, we evaluate aneuploidy as a potential biomarker with a simple PCR assay that uses a single primer pair to amplify ∼350,000 short interspersed nuclear elements (SINEs) throughout the genome.w scores for each of the 39 non-acrocentric chromosome arms in each sample were calculated  could detect cancerous lesions with high specificity. To assess the degree of aneuploidy, ZBased on cross-validation in the training set, we established a global aneuploidy score threshold of 0.25 for subsequent validation. This threshold correctly identified 63%  of the 85 CSF samples from patients with cancer—58% of patients with GBM, 92% of patients with metastases to the brain, and 29% of patients with lymphomas. Of the 34 patients with brain lesions but without cancer, none had global aneuploidy scores <0.25, yielding a specificity of 100% .MDM4, EGFR, CDK4, and HER2, with genomic coordinates listed in We next sought to determine whether the evaluation of focal amplifications of oncogenes, i.e., those involving only a small region surrounding an oncogene rather than the entire chromosome arm on which the oncogene is located, could detect other CNS cancers using data generated with RealSeqS . For thiFor each of these four genes, we calculated a focal amplification score and a threshold for positivity in an analogous way to that described above for the global aneuploidy score. We found that 31%  of the 85 CSF samples from patients with CNS cancers scored positively . This included 74% of patients with GBM, 73% of patients with metastatic lesions, 41% of patients with lymphomas, and 78% of patients with medulloblastomas . Of the There were sufficient follow-up data to analyze progression-free survival (PFS) and overall survival (OS) in subjects with GBM treated at one of the institutions, JHU. Of the 14 patients with newly diagnosed GBM, 10 had detectable levels of CSF-tDNA, while 4 did not. The individuals with detectable levels of CSF-tDNA had an OR of 5.1  and 126 losses (Z < −7.5). Nearly all of these gains (96%) and losses (90%) were identified with WGS. The majority of the chromosome arms gains or losses (9 of 17) that were identified with Real-CSF, but not with WGS, had Z scores (Z > 5 or < −5) just below the Z score of [7.5] required for positivity. Of the 28 CSF DNA samples from patients without cancer, 1,091 of 1,092 of the chromosome arms evaluated (39 arms × 28 patients) were identified as euploid by WGS. The one arm that was aneuploid in one patient was chromosome (chrom) 19p, which has been reported to have a relatively high false positive rate with WGS.To orthogonally validate the copy-number alterations identified by Real-CSF, we performed conventional whole-genome sequencing (WGS) on the CSF DNA from 43 patients with CNS cancers and from 28 without cancer. The sequencing depth averaged ∼34.3 M read pairs, and copy-number alterations were identified with WisecondorX see .32 AmongOf the 121 patients with cancer from either the training or validation sets in whom cytology was available, only 28  were detectable by cytology. The sensitivity of Real-CSF in the same 121 patients was 69%, considerably higher than that of cytology B. TogethGiven that plasma is much more easily accessible than CSF, it was of interest to determine whether plasma could substitute for CSF in RealSeqS assays for aneuploidy or focal amplifications. We have previously described Z score for 2 population proportions).Thirty-five of the 65 patients with brain cancer who donated plasma had also donated CSF . In thesBiomarkers for distinguishing non-neoplastic from neoplastic lesions can help resolve the diagnostic conundrum that is posed for the thousands of patients with space-occupying lesions of the brain each year. There are many types of non-neoplastic processes that can present with an imaging abnormality that mimics a brain tumor. Correctly identifying these abnormalities as non-malignant is important because such patients rarely benefit from surgical biopsy and can almost always be managed non-operatively, and a diagnosis other than CNS malignancy can alleviate much of the anxiety experienced by patients without cancer after imaging. In contrast, patients with cancer could benefit from correct diagnosis by spurring immediate consultation with appropriate neurosurgical and oncology specialists.,,The ideal biomarker would not require brain tissue, would require only a small amount of CSF, would be simple to interpret, would be relatively inexpensive, could identify a myriad of cancer types, and could be readily automated. Real-CSF satisfies all of these criteria and appears to be more informative than cytology, the current gold standard. Previous studies have demonstrated that low-pass WGS can identify chromosomal copy-number alterations in the CSF from individuals with select brain cancers.The ability to identify amplifications and deletions is of increasing importance in neuro-oncology. They can identify potential therapeutic targets and help distinguish different categories of brain cancers. For example, based on imaging and clinical findings, GBM and lymphoma can have overlapping presentations but face drastically different clinical approaches. Real-CSF has the potential to distinguish between these entities based on patterns of chromosomal alterations and apparent somatic mutations. GBM frequently has gains on 7p and 7q and losses on 10p and 10q—all infrequently observed in lymphoma. Conversely, lymphoma often has a gain on 18q and very few chromosome arm losses. These chromosomal alterations alone could accurately distinguish 73% of the Real-CSF-positive GBM and lymphomas in the current cohort. With additional samples, we anticipate that the performance will improve and will allow for accurate identification and classification of other cancer types beyond just those tested in the current study. For example, though we did not examine samples derived from individuals with oligodendroglioma, we anticipate being able to identify the canonical 1p/19q co-deletion.The standard-of-care treatment for GBM includes concomitant chemo- and radiation therapy. Discerning true disease recurrence from treatment-related changes (pseudoprogression) on imaging can be very challenging.One of the strengths of our study is that it is the first to report a relatively large number (96) of CSF samples from patients without cancer but with a variety of other neurological conditions that can mimic cancer. These included patients with inflammatory, autoimmune, degenerative, congenital, and vascular conditions affecting the brain and CNS. Even so, the specificity in the validation set was 94%, comparable to CSF cytology.,In summary, Real-CSF is a simple molecular assay that can assess the presence of aneuploidy using a single primer pair to aid in the management of patients with suspected cancers of the brain. We envision several clinical applications for Real-CSF use. For maximum sensitivity, Real-CSF could be combined with mutation or methylation markers while retaining specificity. Samples with limited input DNA, however, may not have sufficient material for mutation or methylation analysis let alone querying multiple analytes. Real-CSF works in ultra-low concentrations as little as a few pg and could aid in diagnosis when there is insufficient material for other analytes. In some clinical settings such as pediatric patients, Real-CSF could act as a stand-alone test given the relative paucity of somatic mutations in many brain cancers of childhood.Our study has several limitations. Although the total number of samples is reasonably large compared with previously published CSF biomarker studies, confidence limits for subtypes of cancers could be narrowed by studying more patients. Second, we defined thresholds based on the training set and then used them to assess performance in an independent validation set derived from several different institutions. The optimal way to define thresholds for Real-CSF would be to use the training set to develop the model, use the validation set to define the thresholds, and then use a third, independent cohort (a “test set”) to establish sensitivity and specificity. We did not have a sufficient number of samples to achieve this in the current study. However, the similarity in performance of the training and validation sets suggests that we will achieve similar sensitivities and specificities in independent cohorts in the future. A third limitation is that this study was retrospective in nature. A large, prospective study will be required to document that Real-CSF can become a useful diagnostic tool. The results reported here establish the conceptual and practical foundation for such a future study, which could have an impact on standard of care.Cbetteg1@jhmi.edu).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Chetan Bettegowda ) are tertiary centers that care for patients referred for management of central nervous system tumors. In general, patients underwent sampling on the day of enrollment and only tumors with radiographic confirmation with contrast enhanced MRI were included in the study. Radiographic findings of disease were based on the findings of a board certified-neuroradiologist at each site. In total there were 280 samples collected for this study. Pathologic diagnosis for all cases was verified by board-certified neuropathologists at the site of enrollment. Patients whose diagnosis was GBM, CNS lymphoma, medulloblastoma or metastasis from outside the brain comprised the true positive subset. Patients who were not diagnosed with any neoplastic disease comprised the true negative set. We were able to assess plasma samples from 65 cancer patients of which 35 had matched CSF for comparison purposes.Samples were pre-specified into training and validation cohorts based simply on the time at which the sample became available for evaluation in our laboratory at Johns Hopkins. An initial batch of samples from Johns Hopkins were labeled as training samples. To reduce potential cohort biases and overfitting from machine learning, all samples from the Penn State University, CBTTC, and the University of Michigan were labeled as validation samples. The remaining Johns Hopkins samples not evaluated in the initial batch of samples were included in the validation set.CSF was frozen in its entirety at -80°C until DNA purification, and the entire volume of CSF (cells plus fluid) was used for DNA purification. The amount of CSF used for purification ranged from 0.5 to 1 mL. CSF using Biochain reagents according to the manufacturer’s instructions . Note that the K5011625MA workflow is designed for plasma, but we used it both for purifying CSF and for purifying plasma.A single primer pair was used to amplify ∼350,000 short interspersed nuclear elements (SINEs) spread throughout the genome.https://zenodo.org/record/3656943#.YaZZCdDMKUk).Sequencing was performed on an Illumina HiSeq 4000. The sequencing reads from the 8 replicates of each sample were summed for bioinformatic analysis. The average number of the summed, uniquely aligned reads was 10.5 million . The bioinformatic methods and pipeline used to process the raw sequencing data are available at .w). The training examples were 3,999 previously published plasma samples. The negative class of 1348 presumably euploid samples were taken from individuals without cancer. The positive class was taken from 2651 aneuploid samples across 8 different cancer types. We specifically built a support vector machine (SVM) and trained the model with the e1071 package in R, using a radial basis kernel and default parameters. The full bioinformatic pipeline is available at https://zenodo.org/record/3656943#.YaZZCdDMKUk.After generating a reference panel, copy number analysis could be performed on test samples. For each test sample, read depths were aggregated across the 5,344 non-overlapping autosomal 500-kb intervals. PCA normalization was performed. Chromosome arms were segmented using the circular binary segmentation algorithm (CBS).To identify copy number alterations in plasma we repeated the steps from above but made one key change. We reconstructed the euploid reference panel using a set of 1,500 euploid plasma samples. We then repeated the same protocol as above to calculate the statistical significances for each arm and generate Global Aneuploidy Scores.gene) across these amplicons were then determined for each sample. We estimated the expected read depth for a particular gene of interest using our reference panel (μgene).RealSeqS amplicons overlapping the genomic coordinates of the gene of interest, plus 1 Mb on either side of the gene, were identified. The summed read counts (Observedgene) which was calculated from the reference panel by the observed coverage in our test sample to estimate the expected number of reads across the gene of interest (λgene) for the given coverage. We assumed that the count data followed a Poisson distribution. Then, we aggregate the read depth across the gene of interest (Observedgene) for this test sample. We calculated the statistical significance for each gene of interest using the following equation.For each test sample, we calculated the total autosomal sequencing depth (Coverage). We multiplied (μThis protocol was followed for both CSF and plasma samples. The only difference between CSF and plasma was the euploid reference panel used to generate the expected depth for each gene, as noted above. Scores for both CSF and plasma are detailed in We developed a custom library preparation workflow that can efficiently recover input DNA fragments and simultaneously incorporate double-stranded molecular barcodes. Conceptual and practical details of this strategy are discussed in Wang et al.Z Score for 2 Population Proportions. The survival statistics were assessed using the log rank test.Performance comparisons between the training and validation sets were assessed with the


Tetanus is a major public health problem caused by clostridium tetani. Although it is vaccine-preventable, the case fatality rate among neonates in areas with poor immunization coverage and limited access to clean deliveries reaches 80-100%. Vaccination of pregnant mothers with the tetanus toxoid (TT) vaccine is the most effective way to protect against neonatal tetanus. This study aimed to examine the spatial distribution and determinants of tetanus toxoid immunization among pregnant mothers using the 2016 EDHS data.Secondary analysis of the Ethiopia Demographic and Health Survey 2016 was done to assess the spatial distribution and determinants of tetanus toxoid vaccine among pregnant women in Ethiopia. Spatial autocorrelation analysis and hot spot analysis were used to detect spatial dependency and spatial clustering of the tetanus toxoid vaccine in Ethiopia. Spatial interpolation was used to predict the tetanus toxoid vaccine coverage in unsampled areas. The multilevel binary logistic regression model was fitted to identify factors associated with tetanus toxoid vaccination. An adjusted odds ratio with 95% CI was calculated and used as the measure of association and a p-value less than 0.05 were considered statistically significant.From the total of 7043 pregnant women, 42.4% of them have taken at least two doses of tetanus toxoid immunization. Spatial clustering of TT immunization was observed in the Northern, Southwestern and Southwestern parts of Ethiopia. Whereas, low TT coverage was observed in the Eastern and Western parts of the country. Increased ANC visits and the richest economic status favored TT immunization, whereas living in Addis Ababa and Dire Dewa cities decreased the TT immunization coverage.The finding of this study reveals that TT immunization had spatial dependency, with the highest immunization coverage observed in the Northern, Southwestern and Southeastern parts of the Country. Thus, geographically targeted interventions should be implemented particularly in the eastern and western parts of the country. Tetanus is a vaccine-preventable disease caused by clostridium tetani. Vaccination of pregnant mothers with the Tetanus toxoid (TT) vaccine is one of the most effective ways to protect against neonatal tetanus disease , 2.Maternal and neonatal tetanus (MNT) remains a major public health problem,with 80-100% case-fatality rate among neonates, especially in areas with poor immunization coverage and limited access to clean deliveries , 4. GlobOne of the elimination strategies of neonatal tetanus is achieving ≥ 80% coverage with ≥ 2 doses of tetanus toxoid-containing vaccine (TTCV) among women of reproductive age through routine immunization of pregnant women and supplementary immunization activities .In many parts of low-income countries, however, the efforts made to improve maternal and neonatal protection through TT immunization programs with valid doses of TT (two and more) immunization coverage remains low , 3, 8.In addition, a systemic review conducted in different countries attained at least 80% coverage for women of reproductive age receiving at least two doses of tetanus toxoid-containing vaccine or protection at birth .BarriersIn Ethiopia, the prevalence of tetanus toxoid injection is different in different regions . The varDifferent studies conducted on TT immunization revealed that socioeconomic, and health-related factors were associated with TT immunization –15. EvenTherefore, this study aimed to determine the high-risk area (hotspot) of TT immunization among pregnant mothers through spatial analysis and identifying factors associated with it.This study was conducted in Ethiopia using the Ethiopian Demographic and Health Survey (EDHS) 2016. The 2016 EDHS is the fourth national representative cross-sectional survey, which is conducted by the Ethiopian Central Statistical Agency (CSA). The Ethiopian Public Health Institute (EPHI) surveyed in collaboration with the (CSA). The survey was conducted based on a nationally representative sample that provided estimates at the national and regional levels and for urban and rural areas as well.Ethiopian 2016 demographic and health survey includes nine regions and two administrative cities in Ethiopia. It used sampling frame and multistage sampling technique. This survey includes a total of 645 selected enumeration areas. The survey collects data on the characteristics of the household and lists all household members including TT immunization of pregnant mothers. The target population is all pregnant women in Ethiopia.The study population was pregnant mothers in the randomly selected enumeration areas (EAs) of Ethiopia.www.measuredhs.com. The survey covered all nine regions and two administrative cities in Ethiopia.Secondary data analysis from the 2016 Ethiopian demographic health survey was employed to identify the spatial distribution of TT immunization among pregnant mothers. An authorization letter for the use of this data was obtained from measure DHS and the data set was downloaded from the website; A two-stage stratified cluster sampling was employed to select study participants. From 645 enumeration areas, 15,683 reproductive-age women were included in the survey. About 7193 women were pregnant at the time of the survey or gave birth five years preceding the survey. But 150 women who did not remember TT immunization were excluded from the survey. Therefore the current study includes 7043 pregnant women who gave birth in five years preceding the survey , ANC visit, wealth index and region were considered independent variables.Individual (women’s) record was used from the 2016 EDHS. For the spatial analysis, data management was done by using STATA version 14 software and Microsoft Excel 2007. Mapping was done using ArcGIS version 10.8.A multilevel logistic regression model was the best-fitted model for the data after LR and ICC tests were checked. In this study, deviance was used for model comparison and the final model was the best-fitted model with the lowest deviance value. The ICC value was 0.258 in the null model, which indicates that about 25.8%% of the overall variability of TT immunization coverage was due to cluster variability. A two-level logistic regression model was fitted to identify factors associated with TT immunization coverage of pregnant mothers in Ethiopia. ANC visits, region and individual wealth index were statistically significant in the multivariable multilevel logistic regression.Spatial autocorrelation  was done to test for spatial clustering of tetanus toxoid immunization among pregnant mothers in Ethiopia. Moran’s I statistics is used to measure whether TT coverage in Ethiopia was distributed randomly, clustered, or dispersed, by taking the entire dataset and producing a single output value, which ranges from − 1 to 1. Moran’s I value close to -1 indicates TT immunization is dispersed in the area. On the other hand, Moran’s I value closer to 1 indicates TT immunization coverage is clustered in the area. Whereas 0 Moran’s I value means the data is randomly distributed. A statistically significant Moran’s I(p < 0.05) leads to the rejection of the null hypothesis (TT immunization is randomly distributed) and accepting the alternative hypothesis that indicates there is spatial dependence of TT immunization in Ethiopia.Hotspot analysis (Getis-Ord Gi*) was used to identify the spatial clustering of TT immunization in Ethiopia.Spatial Kriging interpolation was used to estimate the distribution of TT immunization across the region.www.dhsprogram.com and the information obtained was not disclosed to the third party.Permission was obtained from measure demographic and health survey through an online request at http//All methods were performed following the relevant guidelines and regulations.In this study, 7043 pregnant mothers were involved. The majority (32.58%) of the study participants were Orthodox Tewahido religion followers whereas the least were traditional religion followers. Related to residency, three-fourths (79.24%) were rural dwellers. The highest number of participants were from the Oromia regional state14.15%) and most (60.72%) were illiterate. Regarding maternal age, 28% were in the age groups of 25–29 years and 35.9% of mothers had ≥ 4 antenatal visits  and a z-score of 15.16. This indicates TT immunization coverage in Ethiopia has spatial dependence Fig. .In the mapping of TT immunization, significant spatial clusters were observed in the Northern, western and central parts of Ethiopia. TT immunization spatial dispersion was observed in the Northern, northeastern, and western parts of the country Fig. .Kriging interpolation was used to map the predicted TT immunization coverage in the observed areas. The highest TT immunization coverage was observed in the Tigray, Amhara, Addis Ababa and Oromia. But, low TT vaccine coverage was observed in the Afar, Somali and Gambela Fig. .A multilevel logistic regression model was the best-fitted model for the data after LR and ICC tests were checked. In this study, deviance was used for model comparison and the final model was the best-fitted model with the lowest deviance value. The ICC value was 0.258 in the null model, which indicates that about 25.8%% of the overall variability of TT immunization coverage was due to cluster variability.Moreover, the PCV valve in the fnal model indicates the variation in the TT immunization coverage among study participants was clarified by both the individual and community level factors. Model ftness was done using loglikelihood and deviance test, then the final model (Model IV) has the highest loglikelihood and the lowest deviance and was taken as the best-ftted model.Therefore number of ANC visits, region and individual wealth index were statistically significant in the multivariable multilevel logistic regression. The odds of protective TT immunization among pregnant mothers with no ANC visit and less than 3 visits were 48%  and 41%  respectively compared to those with ANC visit of ≥ 3. Only 35% of mothers with the poorest income status took a protective dose of TT vaccine  as compared to those with the richest income level. Based on the regions of the participants, 37%  and 59%  reside in Addis Ababa and Dire Dewa respectively took protective TT immunization (Table The overall TT vaccination coverage among pregnant women in Ethiopia was 42.4% which is lower than the study conducted at the University of Gondar Comprehensive Specialized Hospital, Ethiopia 69.8% , in SoutHowever, the current finding is higher than the study conducted in Turkey 27.8% . The posIn our study ANC follow-up, geographic region and individual level wealth status were statistically significant in the multivariable multilevel logistic regression model.In agreement with previous studies conducted in Ethiopia, Sudan, Cameroon, Egypt and five African countries pregnant mothers who have ANC follow-up had 3 times more likely to be immunized for TT , 21–23 aThe poorest income level of the participants negatively affected TT vaccination in the current study as compared to the richest. This finding is in agreement with the studies conducted in different regions of the world , 24. ThiRegionally, participants who live in Addis Ababa and Dire Dewa didn’t get adequate TT immunization as compared to those live in Tigray. We expect dwellers of Addis Ababa would get adequate TT immunization as most health facilities are concentrated to this area, but it is not true in the current research finding.The strength of the current study was based on EDHS national data, which is more representative for the total population in the nation and the model of data analysis was spatial and multilevel analysis. As a limitation, the data was only from EDHS 2016 data which doesn’t indicate the trend of TT immunization. In addition, the cross-sectional design of the 2016 EDHS data limits the ability to establish a causal relationship between the studied exposures and tetanus toxoid immunization.The finding of this study reveals that TT immunization had spatial dependency. Therefore policy makers could give attention for eastern and western part of the country which has low TT immunization. Additionally, lower ANC visit, poorest income level and living in Addis Ababa and Dire Dewa decreased the utilization of protective dose of TT immunization among pregnant mothers. Therefore, the health administrators of these two cities shall create awareness of mothers towards TT immunization using different mechanisms of communication like mass medias.


Polyorchidism, or supernumerary testis, is a rare congenital abnormality of the genitourinary system. In this paper, we present the case of triorchidism in a seven-year-old asymptomatic child with a suspect left scrotal mass detected on routine physical examination. Imaging studies revealed a third testicle in the left hemiscrotum, with comparable dimensions, signal intensity on MRI, and Doppler flow on ultrasound with the ipsilateral testis. We also discuss the clinical presentations, classifications, and current diagnostic and therapeutic strategies of this condition. Polyorchidism, also known as supernumerary testis, is a rare congenital abnormality of the genitourinary system characterized by the presence of more than two testicles. It is a sporadic phenomenon whose etiology remains unclear , with leOur urology clinic received a referral from the patient's pediatrician for a left scrotal asymptomatic mass in a 7-year-old male patient, detected during a routine physical examination. The patient's medical and surgical history was unremarkable, and he was observed to be developing normally in all other aspects.Upon physical examination, the patient presented a visible swelling on the left hemiscrotum. On the right side, the testicle and epididymis had normal consistencies and volumes, the vas deferens was palpable, and no inguinal hernia or varicocele veins were identified. However, the patient had a grade IV varicocele on the left side, accompanied by atrophic testicle and epididymis. Additionally, the left vas deferens was palpable and more prominent than its right-sided counterpart. A medial para testicular mass was palpated on the left side, exhibiting comparable volume and consistency with the atrophic testis. The levels of serum tumor markers for testicular cancer, including alpha-fetoprotein, human chorionic gonadotropin, and lactate dehydrogenase, were found to be within the normal ranges.The ultrasound examination revealed a somewhat echogenic, oval-shaped entity measuring 0.9 × 0.6 × 0.7 cm, positioned between the left epididymis and the left testicle. The mass was distinct from the left testis and exhibited slight intralesional Doppler flow with posterior reinforcement . To enhaThe collective findings were consistent with the presence of a third testicle. The varicocele on the left scrotum was surgically addressed through laparoscopic vein ligation, and the supernumerary testis was conserved. The potential hazards of testicular torsion and subfertility were thoroughly deliberated with the parents, and a comprehensive follow-up plan was outlined.Supernumerary testis or polyorchidism is a rare congenital anomaly. Since its first description by Lane et al. in 1895, less than 250 cases have been reported in literature . It is oMore recently, Bergholz et al. suggested a new anatomical classification based on the functional taxonomy of polyorchidism to standardize diagnosis and management based on the reproductive potential of the supernumerary testis, as outlined in The supernumerary testis typically shares epididymis and cord with the ipsilateral testis . While tPolyorchidism can potentially imitate various pathologies such as varicocele, hydrocele, spermatocele, and testicular neoplasms. Therefore, the physical examination may not be sufficient to diagnose polyorchidism. On ultrasonography (US), a supernumerary testis is identifiable as an oval structure with the same echogenicity as the normal testis. Magnetic resonance imaging (MRI) can provide supplementary information if the diagnosis cannot be made using US. The supernumerary testis exhibits identical signal intensities to the normal testis on MRI, including intermediate and high signal intensities in T1 and T2 sequences, respectively. Some authors suggested using serological markers and histologic confirmation when imaging cannot differentiate polyorchidism from other intrascrotal pathologies [The management of polyorchidism remains controversial, as no evidence-based approach is currently available. Over time, management strategies have evolved, with advancements in imaging techniques enabling surveillance to replace surgical interventions . When deciding on the optimal management option for supernumerary testis, it is crucial to consider the heightened risks of testicular torsion and malignancy  while prOur patient presented with a supernumerary testis in the scrotal region, a feature commonly observed and documented in literature . It sharPolyorchidism should be considered a potential diagnosis when assessing scrotal or inguinal masses or pain. It may imitate various pathological conditions, and imaging is helpful for differential diagnosis. Removal of the supernumerary testis must be considered when there is a concern for malignancy (cryptorchid supernumerary testis) and when the testis is not drained. Further research is needed to understand the factors contributing to this disorder and to establish an appropriate management plan.


We investigated the diagnostic values and artifact severities of 3D-T1W sequences in the diagnosis of cerebrospinal fluid (CSF) leakage.We retrospectively reviewed 3-tesla contrast-material enhanced MR cisternography exams of 22 patients with suspected CSF leakage in 4 years. The presence of the artifacts on 3D-T1W data was evaluated using a 4-point scale . Agreements between CSF leakage results of the 3D-T1W sequences and consensus decisions were evaluated via kappa values. Artifact scores were analyzed by Fisher’s exact test.The most compatible techniques with the consensus diagnoses were fat-saturated 3D-T1W-SPACE and 3D-T1W-VIBE sequences. The most artifact containing the 3D-T1W sequence was 3D-MPRAGE.3D-SPACE and 3D-VIBE are more successful in evaluating CSF leakages compared to 3D-MPRAGE. 3D-SPACE has lower artifact scores compared to 3D-VIBE and 3D-MPRAGE sequences. Contrast-material enhanced MR cisternography (CE-MRC) provides physiologic and morphologic details of the skull base without radiation, and it has quite a high accuracy for cerebrospinal fluid (CSF) leakages . RoutineGenerally, CE-MRC examinations are made in the prone position. Therefore, more motion artifacts are observed in CE-MRC images compared to routine MR exams obtained in the supine position. All isotropic 3D-T1W sequences still lack clinical validation for CE-MRC in patients with CSF leakages. In this retrospective study, we compared CSF leakage detection capabilities and artifact scores of three commercially available 3D T1W MRI sequences at 3-tesla (3T) in patients with suspected CSF leakage. According to our knowledge, there is no comparative study about the artifacts and diagnostic roles of 3D T1W MRI sequences at 3T in patients with suspected CSF leakage.According to respective institutional guidelines, an institutional review board approval was not required for this type of retrospective study. All the procedures being performed were part of the routine care. Informed consent was obtained from all subjects before the exams. In pediatric cases, informed consent was obtained from both parents and the patient. Patients who have (or suspected) otorhinorrhea and underwent 3T CE-MRC in 4 years period were included in this retrospective study. After a retrospective analysis of these patients and the CE-MRC images, 22 patients with CE-MRC obtained with 3D-T1W sequences were included in the study.Patients who have CSF leakage or otorhinorrhea suspicion based on neurosurgeon, otolaryngologist, or neurologist evalualtion, and patients with CE-MRC exam and 3T 3D-T1W  images were included in the study.Patients without detailed clinical and follow-up information were exluded from the study.All CE-MRC exams were acquired on a 3T MRI unit  with a 20-channel head coil. After obtaining noncontrast material-enhanced 3D-T1W and heavily 3D-T2W (noncontrast MR cisternography) images, lumbar puncture was performed by using a 22-gauge needle at the L3–L4 level under sterile conditions. Subsequently, 2 mL of saline mixed with 0.5 mL of gadobutrol was injected intrathecally for all patients. During transfer to the 3T MR unit, the patient is kept in bed in a prone position. Three hours after the gadobutrol injection, CE-MRC images were obtained in a prone position . All patients were clinically observed in the hospital for 24 h after the procedure. Details of CE-MRC acquisitions are given in MRC images were evaluated on a PACS workstation by two neuroradiologists (6 and 15 years of neuroradiology experience) that were blinded to clinical data using a Leonardo workstation . Curved, freehand, or oblique multiplanar reconstructions and maximum intensity projection images were generated from 3D datasets. An extension of spontaneously hyperintense CSF on 3D heavily T2W images (for NCE-MRC) and/or hyperintense contrast-material-enhanced CSF on postcontrast 3D T1W images (for CE-MRC) from the cerebral cistern into the paranasal sinuses, nasal cavity, or middle ear cavity was regarded as the positive finding for CSF leakage for each sequence, as stated in the literature . Pre- anThe final or definite diagnosis of the presence of CSF leak was established according to the physical exam, clinical, beta-2-transferrin test, thin-section CT, CT cisternography, and CE-MRC findings. If present, surgical and endoscopic results were used to confirm the diagnosis. Any disagreement related to the final diagnosis was resolved by consensus of the authors. All cases were followed during regression of the symptoms after CE-MRC exams and surgical and endoscopic therapies to verify the CSF leakage presence/localization and complications.Moreover, the presence of the artifacts on 3D-T1W data was evaluated using a 4-point scale . Follow-up clinical exam and imaging data were used to evaluate changes after the intrathecal gadolinium administration.Data were analyzed using SPSS v.21.0 software . Age and follow-up time related to the cases were given as mean, standard deviation, median, and minimum-maximum values. Agreement between techniques was evaluated via kappa values. Artifact scores of sequences were given as number and frequency. Artifact scores of the sequences were compared via Fisher’s exact test. Some 3D-T1W images or sequences of some patients were not available related to the PACS or hospital information system updates. These sequences were not included in the statistical analysis. p < 0.05 was accepted as statistically significant.CSF leakage was not detected in 5 cases. The arachnoid cyst was detected in 2 cases. In 11 cases, there were also CTC examinations obtained in the same week with CE-MRC. Indication of CE-MRC exam was rhinorrhea in 19 cases and otorrhea in 3 cases. CSF leakage was detected in 17 cases based on consensus sessions . Eight of these had trauma history, and one of them had postoperative leakage. The most common leakage locations were cribriform plate (in 6 cases) and ethmoid sinuses (in 5 cases) . The ageAll cases were followed for at least 3 years. No major complication was detected in the patients. Minor complications  were observed in three patients. None of the patients with negative CE-MRC reports had a new finding or recurrence of rhinorrhea during this follow-up period.In patients with CSF leakage, determination of the leakage site in the preoperative period is crucial for increased surgical success rate and reducing the possible complications ,7. Altho3D-T1W data were relatively newly introduced at 3T. 3D-T1W sequences still lack validation for clinical use in patients with suspected CSF leakage. We observed that the multiplanar reconstruction capabilities of 3D-T1W sequences were very useful for the demonstration of CSF leaks in complex-shaped tiny regions.Three-tesla isotropic 3D-T1W-SPACE has a shorter acquisition time and a larger coverage area (including the whole cranium) than multiplanar 2D-T1W spin-echo-based acquisitions . IsotropIt was reported that 3D-T1W-SPACE and 3D-VIBE have higher contrast-to-noise ratio values and visual conspicuity compared with 3D-MPRAGE . Subtly This study has several limitations. Our first and most important limitation is the low number of study cases. The other limitation is the absence of surgical or beta-2-transferrin test results in all cases of CSF leakage. These limitations are also present in many studies about cisternography or CSF leakages . Future In conclusion, 3D-T1W-SPACE and 3D-VIBE are more successful in evaluating CSF leakages compared to 3D-MPRAGE. 3D-T1W-SPACE is less susceptible to movement or susceptibility artifacts compared to 3D-VIBE and 3D-MPRAGE.


One well-documented risk of spinal surgery is cerebrospinal fluid (CSF) leak in the immediate postoperative period. While the majority of CSF leaks occur due to an obvious intraoperative dural tear, several reports have documented delayed CSF leakage from occult intraoperative dural tears. There is a paucity of published literature regarding the true incidence of dural tears in minimally invasive spinal surgery. Furthermore, the types of dural tears that require closure are poorly understood. According to the limited existing literature available, the recommended treatment of dural tears includes primary repair, subarachnoid drainage catheters, and blood patches. However, there are no distinct treatment guidelines between the different etiologies of CSF leakage. The most important aspect in the management of CSF leakage is prevention, including preoperative risk assessment and meticulous intraoperative manipulation. One emerging treatment strategy is to alter the pressure gradient in a manner that stops CSF leakage. This method is based on one of two mechanisms: direct suture or augmented closure with dural substitute material and either reducing the subarachnoid fluid pressure or increasing the epidural space pressure. Bed rest is a key element in the treatment of persistent CSF leaks, as it can reduce the lumbar CSF pressure, thereby preventing CSF leakage. We describe the challenging case of a persistent CSF leak despite multiple attempts at direct repair, as well as our management strategies. Understanding the proper positioning techniques to reduce leakage is crucial for proper management, and orthopedic surgeons, neurosurgeons, and neurointensivists may consider being more aggressive in treating persistent CSF leaks. Cerebrospinal fluid (CSF) leakage is a dangerous complication of some spine surgeries because it can lead to meningitis, intracranial hypotension, impaired wound healing, and other complications. Current literature suggests that the incidence of intraoperative dural tears is somewhere between 0.5% and 18% . IntraopManagement of persistent CSF leakage remains controversial. Many surgeons recommend for primary repair, while others advocate for a trial of CSF diversion with lumbar drainage . ReoperaA 31-year-old morbidly obese male with a past medical history of intervertebral disc herniation status post left L4-L5 hemilaminectomy and discectomy performed at another hospital three days prior presented to the emergency department with headache, radicular symptoms, and low back pain. The patient reported severe pain since his surgery, which had significantly limited his activity. His pain was initially localized to the lumbar spine and was more severe on the left than on the right side. He reported pain that radiated down the left leg and was refractory to analgesia.Outside records indicated that the initial laminectomy procedure was complicated by an intraoperative CSF leak, which underwent primary repair. Postoperatively, there was no immediate evidence of active CSF leakage, and the patient was discharged home. However, over the course of the next day, he developed worsening pain and headaches, prompting him to return to the outpatient office of the orthopedic surgeon who performed his operation. At that time, his physical examination revealed a fluid collection at the wound site. The patient was sent back to the hospital for magnetic resonance imaging (MRI) of his lumbar spine, which was concerning for a dural spinal fluid leak along with a moderate, centrally extruded disk between L4 and L5 Figure .As a result, the orthopedic surgeon opted to return to the operating room for repair of the CSF leak in addition to a minimally invasive L4-L5 fusion.The patient underwent a successful surgery and reported no pain or headaches for the first full day after surgery. However, after sitting up in bed for an extended period, his headache and lower extremity pain returned. In addition, the nursing staff noticed fluid leaking from his back. A repeat MRI of the lumbar spine showed a fluid collection posterior to the decompression extending through the paraspinal musculature all the way through the fascia. The decision was made to proceed with evacuation of the fluid collection, re-closure, and placement of a lumbar drain, followed by four days of bed rest limited to the supine position to allow for healing of the CSF leak and soft tissues. After the surgery, the lumbar drain yielded CSF at approximately 20 mL per hour. On postoperative day 4, the drain was removed by the orthopedic surgeon. Shortly after the lumbar drain was removed, the patient was found in the upright position, and there was evidence of repeat CSF leakage with saturation of several dressings. Subsequent MRI demonstrated persistent fluid in the subcutaneous fat, which tracked down to the level of the epidural space  was performed at the sites of the lumbar drain entry sites in an attempt to prevent CSF leakage from the lumbar drain dural entry points. On postoperative day 13, the patient was extubated and reported no headaches or lower extremity pain. The patient was observed for several more days, and there was no evidence of continued CSF leakage. At this time, the patient was cleared for discharge from a neurology and neurosurgery perspective.Dural tears are the most common complication in spine surgery, with reported incidence ranging anywhere from 0.5% to 18% . HoweverWhile published literature regarding the incidence of dural tears with minimally invasive spinal surgery exists, the types of dural tears that require closure remain poorly understood. According to the limited amount of existing literature available, recommendations for the treatment of dural tears include primary repair, closed subarachnoid drainage through a subarachnoid drainage catheter, grafting , application of Gelfoam® to the tear, and bed rest -12. HoweOne of the most widely accepted therapies for dural tearing is direct suture repair, although many reports estimate a failure rate of 5%-9% . One treThe other method involving the alteration of pressure gradients to stop CSF leakage is increasing the epidural fluid pressure. An epidural CSF seroma may develop secondary to a dural tear, which can eventually seep into the tissue surrounding the incision. When the CSF outflow exceeds the strength of the sutured tissue, it will create communication between the intradural space and the external wound. Therefore, a tight fascial closure, which increases the epidural fluid pressure, thereby stopping the CSF flow and facilitating adherence of the dural flaps, is required to avoid CSF leakage. While awaiting healing of the dural tear, a subfascial drain can be placed to clear excess CSF and thereby eliminate dead space formation, allowing the fascia to heal, thereby preventing CSF leak from the incision . One othRegarding this case, conservative measures ultimately resulted in the cessation of the CSF leak. Prolonged bed rest is a useful adjunct in the treatment of CSF leaks, and patient positioning to control intracranial pressure is imperative. The lumbar subarachnoid CSF pressure is highest in the sitting and standing positions. However, the subarachnoid pressure is lower in the supine position with foot elevation. Given the overall patient comfort in this position in conjunction with its efficacy, the supine position is commonly used in the treatment of CSF leaks. However, the patient had difficulty maintaining this position, which may have negatively impacted his treatment course. Therefore, we opted to utilize prone and lateral positioning. The subarachnoid CSF pressure is lowest in the prone position. To maintain patient positioning, sedation and intubation were used .In the present report, we have described the challenging case of a persistent, refractory postsurgical spinal CSF leak, as well as our management. Most patients with postoperative CSF leaks experience spinal-type headaches, which prevent them from laying in the supine position for extended periods of time. Most patients are able to lay supine in a bed for 1-2 weeks, allowing for appropriate repair, but our patient was repeatedly found sitting and standing with a persistent CSF leak. The decision was made to keep the patient intubated and lateral/prone to allow for continuous monitoring of the dressing for CSF leak as well as optimal reduction of pressure on the operative site. Thus, understanding suitable positioning techniques to reduce leakage is crucial for proper management.


Heart transplantation remains the conventional treatment in end-stage heart failure, with static cold storage (SCS) being the standard technique used for donor preservation. Nevertheless, prolonged cold ischemic storage is associated with the increased risk of early graft dysfunction attributed to residual ischemia, reperfusion, and rewarming damage. In addition, the demand for the use of marginal grafts requires the development of new methods for organ preservation and repair. In this review, we focus on current knowledge and novel methods of donor preservation in heart transplantation. Hypothermic or normothermic machine perfusion may be a promising novel method of donor preservation based on the administration of cardioprotective agents. Machine perfusion seems to be comparable to cold cardioplegia regarding donor preservation and allows potential repair treatments to be employed and the assessment of graft function before implantation. It is also a promising platform for using marginal organs and increasing donor pool. New pharmacological cardiac repair treatments, as well as cardioprotective interventions have emerged and could allow for the optimization of this modality, making it more practical and cost-effective for the real world of transplantation. Recently, the use of triiodothyronine during normothermic perfusion has shown a favorable profile on cardiac function and microvascular dysfunction, likely by suppressing pro-apoptotic signaling and increasing the expression of cardioprotective molecules. In recent years, an increase in the incidence of heart failure has been observed, a phenomenon attributed to the population aging. Despite progress in pharmaceutical and interventional treatment, heart transplantation remains the gold standard therapy for the treatment of end-stage heart failure. Heart transplantation is a life-saving strategy with favorable outcomes for most recipients despite its technical challenges. By the application of this method, the annual graft survival is 85–93%, while long-term survival is up to almost 70% in a ten-year period . HeterogBrain-dead donors (DBD) are the main source of hearts that are currently used for transplantation, affecting, in a negative way, the graft response. Organ dysfunction, structural alterations, inflammation, and infections are the main complications that can be seen . On the Nevertheless, technological advances have led to the establishment of DCD heart transplant programs with encouraging outcomes. The beneficial results of using these grafts in kidney transplantation is the bright example for their utilization in heart transplantation as well. In recent years, an attempt has been made to overcome the problems that arise in the post-transplantation period by using post-conditioning pharmacological agents on an ex situ perfusion platform, allowing, at the same time, for the ability to determine potential biomarkers that may be used to predict primary graft dysfunction (PGD). Thus, preliminary evidence support that the use of DCD grafts may benefit heart failure patients, with at least equivalent results to DBD heart transplantation .The limited number of available grafts and the need to extend donor selection forces the acceptance of a growing number of marginal grafts. On the other hand, discordance between the increasing need for transplants and organ availability, as well as population aging, extensive resuscitation, and shift in the causes of death, which are mirrored in the organ pool, elucidated the need to focus efforts on extending the latter as well as better preserving grafts . As a reOne of the main limitations in heart transplantation is the problem of viability of the donor heart since there is a certain transport time between donor explantation and recipient implantation, as the use of the donor heart has been limited by the ischemic time of 4 to 6 h . SeveralWhile cold ischemic storage has been the most widely used technique for preserving the donor heart, there are specific limitations. Prolonged ischemia is one of the main risk factors of early graft dysfunction and therefore primary graft failure. The quality of the donor graft highly depends on the cold ischemic time, which should not be longer than 6 h. Thus, the reduction in this period and the development of better formulations of heart preservation solutions seem mandatory. Prolonged ischemia time has been responsible for early graft dysfunction and primary graft failure, which are the major causes of death during the first month after heart transplantation, with the risk being higher after 180 min of the beginning of cold storage . The durCurrent methods of myocardial preservation using cold ischemic storage have multiple negative effects, among which cellular swelling, extracellular edema, and cellular acidosis are the most prominent. In addition, the depletion of metabolic substrate, reperfusion injury, and endothelial damage may be seen. Cellular swelling is triggered by the suppression of the Na-K-ATPase pump and the decrease in the plasma membrane potential, leading to the intracellular entrance of sodium and chloride with water following and causing cell swelling. This phenomenon may be limited by the addition of colloids, anions, and saccharides. Extracellular edema is the result of hydrostatic changes in the organ vasculature. In addition, tissue acidosis is caused by lactic acid production in the context of anaerobic glycolysis attributed to ischemia. Finally, reperfusion injury can be mediated by the production of harmful oxygen free radicals (ROS) and intracellular calcium overload .As mentioned above, cold ischemic storage may be harmful on the coronary endothelium, as both high-potassium-content blood cardioplegic solutions and intracellular crystalloid solutions, which are of ionic composition and are characterized as depolarizing reflecting its high potassium content, can cause chemical damage to the coronary endothelium . Thus, eThe optimization of the cardiac donor management through utilization of donor management goals (DMGs) is a key response to the growing need for organs. In fact, this has been shown to improve both the quantity and quality of transplantations through the increased yield of transplants per donor and improved transplant function, respectively . TowardsAs per the Canadian Council’s for Donation and Transplantation (CCDT) recommended DMGs, the monitoring and treatment of reversible organ dysfunction should be performed in the ICU environment for 12–24 h . SpecifiDuring brain death, both autonomic disturbances and neurohormonal axis failure may be seen, resulting in the rapid decline of circulating hormones. On the other hand, the depletion of myocardial energy stores leads to the predominance of anaerobic metabolism in combination with hemodynamic instability and myocardial dysfunction. Thus, hormonal supplementation, based on the administration of thyroxine (T4), methylprednisolone, vasopressin, and insulin, is widely used as one of the main interventions to improve cardiac function . HormonaRecently, the potential beneficial role of thyroid hormone (TH) in cardioprotection has come to the fore, dispelling the long-held belief that its administration may be detrimental to the ischemic myocardium. First, Pantos et al., showed that TH can be protective in the setting of ischemia–reperfusion injury in a pattern similar to ischemic preconditioning. The possible mechanisms, through which T3 exerts this effect, are the suppression of the pro-apoptotic p38 mitogen-activated protein kinase (MAPK), as well as the increased expression of cardioprotective molecules, such as the heat shock proteins HSP27 and HSP70. Experimental evidence of the administration of T3 after myocardial infarction in rat hearts, showed reduced cell apoptosis, which appears to be mediated by the activation of protein kinase B (Akt) and the miR30a/p53 axis .In addition, T3 may promote the restriction of myocardial injury in the post-ischemic period, contributing decisively to the recovery of graft function, through its binding to the nuclear receptor TRα1, on which T4 has no effect. This feature of T3 is remarkable, since it works as a molecular switch, thereby determining the activity of the receptor. TRα1 receptor expression is increased in the remaining viable myocardium after the ischemic period to levels like those seen in fetal life, in which T3 is very low. The increased concentrations of the hormone and the subsequent binding of the receptor results in the strengthening of the function of the myocardium. This is due to the induction of the maturation and growth of cardiomyocytes, in such a way that T3 can be characterized as a ‘’regeneration hormone’’ . This poReducing occurrence of ischemia reperfusion injury (IRI) of the donor heart is the cornerstone of effective preservation. Current preservation practice is static cold storage (SCS) in heart transplantation solutions (HPSs) following cardioplegia. There is a wide gamut of HPSs currently in use. At least 167 different types of solutions are used, and all of them are classified according to the sodium and potassium anion concentrations, as extracellular and intracellular. Intracellular solutions contain high levels of sodium, potassium, and magnesium, whereas extracellular solutions contain low electrolyte levels . The mosDespite SCS in HPSs being the main preserving technique in clinical use, research is active in new methods as well as augmenting current ones. Donor heart perfusion via perfusion systems, a method that was used in the early days of heart transplantation, is re-emerging and provides two ways of maintaining oxygenation. One method is performed by providing oxygenated, cold cardioplegic solution (CPS) to the arrested heart. Another, more arduous method is preservation of the beating heart with normothermic oxygenated blood-based buffers . Adding The sufficient preservation of donor hearts remains a critical issue in cardiac transplantation. The high metabolism of the heart, compared to the other organs, is considered responsible for the rapid depletion of ATP, leading to acidosis, and necrosis of the myocardium, respectively. Ex vivo perfusion appears to be a promising, novel, organ preservation approach to address some of the limitations of cold cardioplegia. This method uses similar solutions to cold cardioplegia, with some modifications, including blood products. Machine perfusion (MP) has the clinical advantage of reducing cold ischemic time significantly in long-distance procurements or complex recipient operations, such as explantation of mechanical circulatory support. Furthermore, MP allows for donor heart assessment before implantation, and this may facilitate the use of marginal donors. More importantly, MP may become a platform for cardioprotective approaches, enabling the repair and reconditioning of donor hearts . In hearWarm perfusion has demonstrated the enhanced myocardial protection of the donor heart and is used as an alternative option in donor graft preservation. The presence of buffer solutions, metabolic substrates, and hemoglobin (necessary for the transport of oxygen to the tissues) in the blood, makes this method of preservation particularly attractive. As a result, the protection of ischemic myocardium can be provided in a relatively simple way utilizing a nature product, such as blood.Maintaining donor’s heart through a perfusion platform advocates a plethora of advantages. First, it reduces cold ischemia time, which, if it exceeds 4–6 h, unpreventable increases to the risk of primary graft dysfunction occur, leading to 8% mortality in the first 30 days and post-transplantation mortality at 5 and 15 years, respectively ,36,37. AKeeping cardiac allograft perfused may potentially provide valuable time for surgeons to optimize their surgical plan and improve coordination. Patients required explant of left ventricular assist device or redo sternotomy, might benefit from EVHP limiting cold ischemic time, as long as surgeons optimize the recipient for implantation and copying any unanticipated circumstances ,42. The The Organ Care System  is the only ex vivo heart perfusion platform with warm, oxygenated, and nutrient-enriched donor blood-based buffer currently in use . With thMachine perfusion may become a platform for applying cardioprotective approaches, such as ischemic preconditioning or post-conditioning, and pharmacological cardioprotective agents. In fact, erythropoietin, glyceryl trinitrate, zoniporide, and antioxidants have been used in porcines for ischemic postconditioning and protection against primary graft dysfunction in DCD allografts with positive results . Church Utilizing the Organ Care System for cardiac allograft preservation demands several resources, leading inevitably to higher cost than standard cold storage techniques. Trained personnel, appropriate equipment, technical support, and harvesting donor blood for priming perfusion platform point to that direction. Nevertheless, this is counterbalanced with expanding of donor pool and the potential cost of primary graft dysfunction. Decreasing cold ischemia time translates to better survival rates. It has been reported that patients had a 25% increased risk of death during first year after heart transplantation for every extra hour of ischemia after 1 h . In addiProlonged ex vivo heart perfusion has the disadvantage of the development of myocardial edema, although it is not certain that this worsens the patient’s outcome . Attemptn = 5), cardioplegic arrest, 12 h of storage at 4 degrees C with modified Belzer solution, and 2 h of sanguineous reperfusion in the working state; for group II (n = 6), 12 h of continuous perfusion in the beating working state, 30 min of arrest (to simulate re-implantation time), and 2 h of reperfusion, as above; and for group III (n = 7), coronary ring control hearts. The results revealed that the perfusion group had better left ventricular developed pressure (LVDP) at 2 h of reperfusion, less myocardial edema, and significantly less myocardial acidosis during preservation and reperfusion. This initial evidence supported the hypothesis that a perfusion device could prolong the preservation of the donor heart, by reducing ischemia reperfusion damage and expanding the donor pool.Hassanein et al. (1998) were the first who proved that the preservation period of a donor heart could be prolonged by normothermic blood-perfusion in the beating working state, preventing ischemia reperfusion injury compared to standard hypothermic arrest and simple storage in a cardioplegic solution . They dePROTECT I was the first clinical trial that evaluated the safety of the Organ Care System in Germany and the United Kingdom. This European study was a prospective single-arm non-randomized safety and performance study conducted between January 2006 and February 2007. A total of 25 donor hearts were included in this trial, with three of them having been excluded due to the assessment criteria. All 20 patients met the primary endpoint of 30-day survival. Following PROTECT I, PROTECT II was conducted in Europe as well but was soon merged into commercial use and no full data collection was published . PROCEEDn = 63) in the Organ Care System group and 97% (n = 61) in the standard cold storage, with p = 0·45. The secondary results were cardiac-related serious adverse events, severe rejection rates, and the median length of stay in the intensive care unit. Likewise, the results revealed the non-inferiority of the Organ Care System group. The results showed that the donor hearts for heart transplantation were adequately preserved and had similarly short-term clinical outcomes in both groups. The authors acknowledge the potential advantage of the Organ Care System for the expansion of the donor pool with the prolonged adequate preservation of the donor heart for distant areas, but their main aim was the initial observation that Organ Care System is as safe and effective as cold storage for preservation. A remarkable observation of this trial was that the despite total preservation time (out of body) was longer for the Organ Care System group (324 min VS 195 min attributed to the extra time needed to instrument the donor heart into the Organ Care System circuit and optimize the perfusion characteristics), and the mean total cold ischemia time was significantly shorter in the Organ Care System group than in the standard cold storage group (113 min vs. 195 min). Further studies for the metabolic assessment of the allograft are suggested in Organ Care system group. PROCEED II is the first randomized clinical trial to assess the safety and efficacy of an ex vivo heart transplant platform (OCS) in human heart transplantation [In 2015, Ardehali A. et al. published in Lancet the results of PROCEED II, a prospective multicenter randomized non-inferiority trial between 2011 and 2013, in Lancet. In total, 130 patients who were to receive donor hearts were randomized into the Organ Care System group or standard cold storage group, correlating with the preservation technique to be used. Primary outcomes were 30-day patient and graft survival with a 10% non-inferiority margin . The 30-antation .p = 0.65), 81% (p = 0.28), and 79% (p = 0.21). The secondary end points were primary and chronic allograft failure, non-cardiac complications, and the length of hospital stay, and only revealed not statistically significant difference. Nevertheless, the authors concluded that normothermic ex vivo allograft blood perfusion in adult clinical orthotopic heart transplantation contributes to more beneficial outcomes regarding recipient survival, the incidence of primary graft dysfunction, and the incidence of acute rejection [Koerner MM et al. (2014) published the results of a prospective, non-randomized, single-institutional clinical study of 159 cardiac allografts transplantations. A total of 29 were assigned for normothermic ex vivo allograft blood perfusion with the Organ Care System and 130 for cold static storage. The primary outcome of the recipient’s survival at 30 days and 1 and 2 years after transplantation suggested similar results , whereas in the CSS group, the survival after oHTx was 95% , which analyzed high-risk transplants with anticipated prolonged ischemic times (>4 h) or marginal donor heart features . The short-term results showed excellent short-term outcomes in 75 patients transplanted from a total of 93 hearts recovered and perfused on the OCS .Sponga et al., in a single-center retrospective study of patients who underwent heart transplantation, also used marginal donors preserved via cold static or warm perfusion. The criteria for marginal donor hearts were age ≥ 55 years; expected ischemic time > 4 h; left ventricular ejection fraction ≤ 50%; interventricular septum thickness ≥ 14 mm; drug abuse history; episodes of cardiac arrest; and the presence of mild coronary artery disease. Ex vivo perfusion allowed for the continuous evaluation of marginal donor hearts, favoring the exclusion of unsuitable grafts and resulted in a reduction in complications and optimal survival up by to 5 years compared to cold cardioplegia. Notably, in this study, histopathological and ultrastructural examination revealed that, even with normothermic perfusion, pathological changes in cardiomyocytes, and endothelium may be seen. Cardiomyocyte degeneration at the end of organ transportation was more prominent in organs preserved with warm perfusion. However, following reperfusion, a significantly higher apoptotic rate was observed in cold-preserved hearts. Morphological changes of endothelial vascular damage were observed when cells were exposed either to cardioplegic or organ preservation solutions. Warm perfusion resulted in a significantly larger proportion of intramyocardial areas of hemorrhage at the end of the perfusion period .Recently, Langmuur et al. published the results of a meta-analysis, including 12 studies of HTx, wherein 260 out of 741 donor hearts were preserved with OCS. Outcomes in the OCS group were equivalent to static cold storage (SCS), regarding either early or late survival outcomes (primary endpoints). Notably, OCS outcomes concerned heart donation both after brain death (DBD) and circulatory death (DCD). The 30-day survival rate was 97.4% for the OCS-DBD group, 96.6% for the OCS-DCD group, and 95.7% for the SCS-DBD group. No differences in short-term survival between OCS-DBD and OCS-DCD, compared to SCS-DBD techniques, were detected in the pooled ORs for 30 d survival. As for the secondary outcomes, no significant differences in the length of hospital stay, primary graft dysfunction, or rejection were present. Interestingly, the pooled 1-year survival was 84.2% for the OCS-DBD group, 89.3% for the OCS-DCD group, and 87.0% for the SCS-DBD group, whereas rates for the pooled 4-year survival was 82.2%, 85.3%, and 80.3%, respectively. However, the main limitation of this meta-analysis was that it mainly involved observational retrospective studies, the majority of which were lacking a randomized and fully comparable control group, likely leading to bias. The authors concluded that the Organ Care System is a safe and an efficient platform for both DBD and DCD donation, especially in advanced surgical procedures or in cases where long-distance transport is required . MachineNon-ischemic heart preservation (NIHP) is an alternative to the Organ Care System heart-graft transport device, providing the ability to procure distant donors while simultaneously rejuvenating marginal heart grafts. In contrast to the OCS, in which a non-cardioplegic warm preservation solution is used, in NIHP, heart perfusion relies on a continuous supply of oxygenated cold cardioplegic solution. The perfusion is enriched with hormones, red blood cells, and nutritional components, maintaining a hematocrit of 8%. With the use of this device, continuous perfusion with oxygenated blood is achieved, while at the same time, the metabolic demands of storage are limited .Data from preclinical studies in porcine hearts have shown that using NIHP, heart preservation can be safely achieved for 24 h, with the advantage of maintaining endothelial function for at least 8 h. In 2020, Nilsson et al., published the results of the first-in-human study, a non-randomized, open-label, phase 2 trial, which showed the safety of NIHP in heart transplantation without heart-related complications or early mortality. When compared to static cold preservation, NIHP appeared to be beneficial in reducing myocardial damage to a greater extent, as well as allograft rejection at 6 months .The main disadvantages of the NIHP method are the appearance of edema, which is often irreversible, as well as the increased incidence of kidney damage in the postoperative period. The possible beneficial effects of NIHP on transplant rejection are also reinforced by Critchley et al., who showed that the use of non-ischemic heart preservation via hypothermic cardioplegic perfusion reduces donor heart immunogenicity. This is ensured mainly through the loss of resident leukocytes, whereas the limitation of the inflammatory response induced by IFN-γ is thought to play a central role . HoweverExperimental and clinical evidence suggests that early administration, above normal doses, of triiodothyronine (T3) after myocardial infarction appears to be cardioprotective and prevent cardiac remodeling. It is noteworthy that this action of T3 does not entail the worsening of myocardial damage as a sequela of the differential effect of the hormone on healthy and injured myocardium, respectively. The time of administration, as well as the dosage regimen that will be chosen, seem to be decisive parameters in the action of thyroid hormone (TH) in the ischemic myocardium . This maThyRepair was the first trial of its kind to show the potential of thyroid hormone to repair the infarcted myocardium. This may signal a new era for pharmaceutical organ repair. Notably, TH has also been used in cardiac transplantation as an inotrope for supporting donor heart hemodynamics. A meta-analysis of the data provided by several studies showed that TH may not have a significant effect on donor hemodynamics. However, the observation that a favorable TH effect was evident on unstable donors  indicates that TH effects are beyond its inotropic action. In accordance, in the analysis of the data obtained from 66.629 donors, the T3/T4 treatment of cardiac donors was associated with the procurement of significant greater numbers of hearts. Furthermore, the effect of the TH treatment was independent of other factors and associated with improved post-transplantation graft survival .In the era of machine perfusion, the cardioprotective effects of TH could be exploited for cardiac donor preservation and repair. Mourouzis et al. showed that the administration of T3 during normothermic perfusion with non-blood crystalloid buffer was shown to have favorable effects on cardiac function and perfusion pressure, and switched death to pro-survival kinase signaling. Especially, the improvement of coronary perfusion pressure with the addition of T3 in the perfusate indicates a favorable effect on microvascular function during ex vivo normothermic perfusion ,69. ExpeMitogen-activated Protein Kinase (MAPK) cascades are key signaling pathways that are involved in the modulation of cellular responses to a wide range of intracellular stimuli, including apoptosis, vascular permeability, cardiac function, immune cell activation, and cytokine production. Thus, MAPKs are responsible for the regulation of multiple cellular functions, such as gene expression, proliferation, and apoptosis. Thyroid hormone seems to promote the inhibition of p38-MAPK, consequently limiting inflammation and apoptosis. In particular, p38-MAPK activation is believed to be implicated in the inflammatory cascade through the stimulation of immune system, resulting in cytokine production ,71. AccoIn addition, T3 facilitates the activation of pro-survival signaling pathways, which in turn are involved in the blockade of cellular apoptosis, as well as the stimulation of angiogenesis. The PI3K-Akt signaling cascade is a signal transduction pathway that enhances survival, angiogenesis, and regulated calcium cycling in cardiomyocytes. T3 may stimulate PI3K signaling by reinforcing the interaction of both TRα and TRβ with the PI3K regulatory subunit p85. Thus, T3 may promote cell survival and the better handling of calcium via Akt activation. Furthermore, enhanced PI3K/AKT/mTOR activity may result in the increased expression of the transcription factor HIF-1α ,77. HypoMicrocirculation is impaired after reperfusion in both acute myocardial infarction and heart transplantation, and this dysfunction is associated with postischemic cardiac remodeling and impaired healing ,82. T3 sMechanical perfusion is now considered as a novel approach in donor heart preservation, contributing significantly to donor’s pool expansion. Experimental evidence supports that the adjunctive use of cardioprotective agents, such as T3, may provide beneficial outcomes in graft preservation, potentially preventing the cardiac remodeling of the donor’s heart. Thus, the addition of T3 to the perfusate may be a promising strategy to optimize donor heart preservation and repair. However, the benefits of T3 treatment are mainly based on animal data, whereas clinical trials testing its use in organ preservation are needed. Despite significant clinical and therapeutical importance, further research is needed on perfusion solutions and pharmacological agents, like T3, to maximize the potential of machine perfusion modalities.Despite the advancements in organ preservation, ischemia–reperfusion injury remains an unresolved problem in cardiac transplantation. Using different types of solutions, we have not achieved an ischemia time beyond a few hours. Static cold storage remains the method of choice for organ preservation. However, the method is associated with increased risk of early graft dysfunction, as well as reperfusion and rewarming injury. In an effort to find alternative ways in terms of heart protection and preservation, including extending the donor pool, new methods have been developed. Machine hypothermic or normothermic perfusion devices have been recently used as promising, novel, preservation methods. EVHP seems to be comparable to cold cardioplegia regarding donor preservation and allows for potential repair treatments to be employed and the assessment of graft function before implantation. It is also a promising platform for using marginal organs and increasing donor pool. However, complex logistics, the use of blood-based solutions, and cost-effectiveness remain the serious constrains of this method. In addition, donor selection criteria need to be better defined. Thus, larger randomized, prospective studies are required to validate utility in extended criteria donors. New pharmacological cardiac repair treatments, as well as cardioprotective interventions have emerged and could allow for the optimization of this modality, making it more practical and cost-effective for the real world of transplantation. T3 is known for its favorable effects in donor heart preservation and repair. Cardioprotective features of triiodothyronine are likely to be achieved by its implication in intracellular signaling pathways involving kinases. Nevertheless, the molecular mechanisms of T3′s actions require further elucidation. Therefore, more mechanistic studies would help optimize its clinical use.