The current COVID-19 pandemic has brought to public attention the conceptual difference between a test for COVID-19 derived RNA or proteins indicating the presence of an active infection, versus COVID-19 serology testing indicating pathogen infection. Only the former test for molecules derived from COVID-19 provides reliable evidence of a current active infection. As such, nucleic acid amplification methods, and COVID-19 antigen immunoassays, are used to diagnose SARS-CoV-2 active infection  and to gBorrelia, possible co-infection with other tick-borne pathogens, and the host response [Borrelia, the only existing direct test, is restricted to synovial fluid for Lyme arthritis and skin biopsy PCR for acrodermatitis chronica atrophicans [Borrelia alters the repertoire of self-peptides bound to MHC II class molecules in professional antigen presenting cells, and induces overexpression of self-antigen presenting MHC II molecules [Borrelia can persist in affected tissues in humans for months or years [Borrelia induces lymph node architecture disruption, alters normal lymph node T/B cell ratios, prevents the formation of stable germinal centers, and hinders the establishment of long-term memory cell populations [Clinical manifestations of Lyme Borreliosis are heterogeneous, depending on different strains of response . Erythemresponse . A two-tresponse , with a response . PCR tesophicans . Otherwiophicans . Objectiophicans ,11. Wideophicans , 2) persophicans  in systeophicans , has beeophicans . New expolecules . Authorsolecules . In paraor years . Animal ulations . The truSensitive and accurate molecular direct tests hold the promise to settle the controversies and confirm pathogen presence in treated Borreliosis patients with long term symptoms. A direct test can also be used to diagnose disease before seroconversion, and monitor therapy efficacy .ClinicalTrials.gov Identifier: NCT03505879). Using a combination of mass spectrometry and immunoassay methods, peptidoglycan persistence was demonstrated in synovial fluid of Lyme arthritis patients [Unbiased high throughput molecular methods hold the promise to identify pathogens in the face of strain and antigenic variability. Next generation sequencing has been proposed for vector characterization and surveillance studies and has led to new pathogen identification and tick microbiome characterization . The usepatients .Borrelia in a cohort of 158 patients under clinical evaluation for tick borne illnesses [Proteomics can provide functional information on the pathogen and its interaction with the host. We have applied an unbiased proteomics approach to identify tick borne pathogen peptides in the urine of patients under consideration for tick borne illnesses at different stages. In a cohort of 408 cases and controls, we have identified 2 pathogen derived peptides in 9/10 acute EM cases, and 0 false negatives in 250 asymptomatic and symptomatic controls . We founllnesses . ProteinBorrelia cell wall [Borrelia undergoes a peptidoglycan remodeling process to expand its cell wall during bacterial growth [Borrelia sheds 40–50% of its peptidoglycan per generation [Peptides derived from 6 enzymes involved in peptidoglycan biosynthesis and degradation were identified . Peptidoglycans are an essential component of ell wall . Borrelil growth . It has neration .Borrelia is deficient in fundamental metabolic pathways such as carbohydrate metabolism, phospholipid metabolism, and fatty acid metabolism. The pathogen therefore relies on the host to obtain its nutrients [Borrelia is carried on through the glycolysis pathway alone and not through tricarboxylic acid cycle or oxidative phosphorylation [Borrelia slow growth rate, as Borrelia glycolysis yield of ATP is 3 molecules versus 30 molecules in a cell with functioning TCA and OXPHOS [Borrelia glyceraldehyde-3-phosphate dehydrogenase, which is the sixth step in the glycolysis pathway. It has been shown that Borrelia induces human PBMCs to shift to an increased glycolytic activity bypassing the TCA cycle with an increased production of lactate, which can be utilized by Borrelia to sustain its metabolism. PBMC shift to anaerobic glycolysis accommodates an increased energy demand, and contributes to the inflammatory response both innate and adaptive immune system [Borrelia CoA-disulfide reductase, which plays a central role to regenerate NAD+ to sustain glycolysis [Borrelia PTS glucose transporter, which supports the established notion that Borrelia is expected to rely primarily on glucose as a carbon source in the mammalian host. The spirochete, however, is considered capable of using other carbon sources including mannose, N-acetylglucosammine, maltose, chitobiose, and glycerol. Our identification of Borrelia PTS mannose transporter in patient urine suggests that Borrelia might use mannose as an alternative carbon source in the human host [Due to its small genome, utrients . Genomicrylation . Sole red OXPHOS . In our e system . Resultsycolysis . In relaman host .Borrelia has limited fatty acid metabolism capabilities. It lacks the ability to synthesize new fatty acids, to extend existing fatty acid chains, and to catabolize fatty acids to derive energy through beta oxidation [Borrelia cellular membrane, however, is rich in fatty acids, mainly palmitate, covalently linked to lipoproteins (outer surface proteins and VlsE). Unusually for a prokaryotic organism, the Borrelia membrane also contains polyunsaturated fatty acids, which are likely derived from the host environment. Clinical and preclinical studies show that Borrelia infection triggers eicosanoid production in the vertebrate host [Borrelia and host cell membrane lipids. A Borrelia lipase like enzyme has been discovered [Borrelia lipase detection in the urine of Borreliosis patients in our clinical proteomics study supports the notion that Borrelia interacts directly with lipids in host cell membranes to aid the production of unsaturated fatty acids that become assimilated in lipids and lipoproteins incorporated in its cellular envelope. Polyunsaturated fatty acids in the cell envelope are a known target for reactive oxygen species produced by immune cells of the infected host [xidation . The Borate host . Eicosanscovered , which cted host .Borrelia has all the homologs of the mevalonate pathway, which leads to the synthesis of isopentyl-5-phopsphate (IPP) and dimethylallyl pyrophosphate (DMAP). However, Borrelia is unable to use IPP and DMAP to synthesize cholesterol [Borrelia growth [Borrelia type 2 isopentenyldiphosphate delta-isomerase which belongs to the mevalonate pathway and mediates the synthesis of IPP.lesterol . Consequlesterol . The impa growth . In our Borrelia contains only two main membrane phospholipids: phosphatidylglycerol (PG) and phosphatidylcholine (PC). PG biosynthesis in Borrelia proceeds through the enzyme phosphatidylglycerolphosphate synthase , which uses CDP-diacylglycerol and glycerol-3-phosphate as substrate. In our proteomics study, we identified the Borrelia enzyme phosphatidate cytidylyltransferase (2.7.7.41), which mediates the synthesis of CDP-diacylglycerol from a-1,2 –diacyl-glycerol 3-phosphate and it is the fourth step in the PG synthesis immediately before PGs [Borrelia phosphatidylcholine synthase BB0249 in conjunction with exogenous choline to yield PC, the other major phospholipid in the Borrelia membrane [fore PGs . CDP-diamembrane .Borrelia nicotinate phosphoribosyltransferase and nicotinate (nicotinamide) nucleotide adenylyltransferase NAD+ homeostasis is vital for many cellular processes, including glycolysis. A Borrelia encoded nicotinamidase that converts nicotinamide in nicotinic acid for NAD+ synthesis is essential for bacterial replication and infectivity.Our analysis revealed two enzymes involved in the biosynthesis of nicotinamide adenine dinucleotide NAD from nicotinic acid: Borrelia; thus, no reactive oxygen species (ROS) are expected to be produced endogenously [Borrelia, however, is exposed to ROS in the host, during phagocytosis and inflammatory response. Iron mediated, DNA oxidative damage, typical of most bacteria, is limited in Borrelia because it has evolved to function with very low levels of intracellular iron [Borrelia suffer from limited oxidative damage [There are no oxidative pathways known to be active in genously . Borrelilar iron  and lacke damage . We idenBorrelia lipid membrane, rich in polyunsaturated fatty acid, is considered an alternative target of ROS-mediated damage [Borrelia is superoxide dismutase [Borrelia in 1997 [Borrelia superoxide dismutase is a metalloenzyme that uses manganese as co-factor [Borrelia magnesium transporter MgtE which aids the import of magnesium to support metalloprotein functions. An additional protein found in our clinical study that indirectly protects the spirochete against ROS mediate membrane damage is Borrelia CoA-disulfide reductase. CoA-disulfide reductase reduces CoA-disulfides to CoA in an NADH-dependent manner to maintain thiol-disulfide homeostasis [Borrelia to establish infection in the human host [d damage . The keyismutase , which w in 1997  and its o-factor . In patieostasis . It has man host .Borrelia lacks the cellular pathways for de novo purine synthesis and is required to salvage purines and pyrimidines from the host environment to synthesize its nucleic acids [Borrelia burgdorferi, Borrelia hermsii and Borrelia turicatae have a complete pathway for purine salvage, which is thought to be one of the reasons why relapsing fever spirochetes achieve higher densities in blood [B. hermsii and B. turicatae and is absent in B. burgdorferi is adenylosuccinate synthase, which was detected in the urinary peptidome of patients in our clinical study. Purine metabolism is essential for Borrelia growth and virulence in mammals [Borrelia burgdorferi has a distinct pathway for purine salvage [B. burdgorferi imports adenine and hypoxanthine from the tick and vertebrate host. In our clinical study, human urine yielded the detection of a Borrelia uracil-xanthine permease that imports xanthine and a range of nucleobases that can be catabolized. A series of enzymatic reactions convert hypoxanthine to guanosine monophosphate (GMP) and deoxyguanosine monophosphate (dGMP), the building block for RNA and DNA synthesis, respectively. The last enzyme in the pathway is glutamine-hydrolyzing GMP synthase (guaA), which we found in our clinical study, GuaA, is essential for Borrelia survival in the tick-vertebrate infection cycle [ic acids . In contin blood . One of  mammals . Borreli salvage . B. burdon cycle .Borrelia glycoside hydrolase family 3 protein (BB_0620), which is an enzyme involved in sugar metabolism and catalyzes the reaction of chitobiose to N-acetyl-D-glucosamine (Kegg bbu00520). This enzyme has been associated to a pathway of Borrelia resistance to beta-lactams. As a consequence of inhibition of peptidoglycan biosynthesis and increase of muropeptides, BB_0620 is thought to participate in the induction of expression of beta-lactamases .Our clinical study identified Borrelia Bax inhibitor-1/YccA family protein a transmembrane protein thought to regulate mammal host cell survival and apoptosis. It has been demonstrated that expression of the gene coding for this protein is implicated in the shift from a cell survival to apoptotic state in neuroblastoma cells co-cultured with B. burgdorferi and primary microglia [We further identified icroglia .Borrelia Acriflavine resistance protein is a transmembrane protein involved in the resistance-nodulation-division (RND) family of drug efflux systems [ systems . A multi systems .Borrelia turicatae phage portal protein (A0A172XCQ6) was identified in our clinical study. B. burgdorferi bacteriophages have been identified and partially characterized [Borrelia burgdorferi genome [Borrelia strains, introduce new virulence or fitness factors, or lyse competing strains through prophage induction [Borrelia burgdorferi encodes, among other proteins, for a BBL01, Putative bacteriophage portal protein. Portal proteins are incorporated in the growing phage and are required for DNA packaging. Other bacteria  carry putative bacteriophage portal proteins [Borrelia burgdorferi cultivated in vitro was exposed to blood, suggesting that B. burgdorferi expresses BBL01 in the tick host preferentially, while mammal host conditions induce a suppression of the protein; protein suppression is confirmed by the fact that BBL01 antibody response was not detected in B. burgdorferi infected mice [Borrelia turicatae phage portal protein in human urine, if confirmed, might suggest that B. turicatae has a prophage system, and that the B. turicatae differential expression of phage derived proteins in the invertebrate and vertebrate host is different than B. burgdorferi’s.cterized . Prophagi genome . Bacterinduction . A circuproteins . Previouted mice . Our detBorrelia that can be targeted for future treatment strategies.Thus, proteomic analysis of urine from symptomatic patients, reveals a long list of functional molecular insights about the physiologic state of the pathogen at the time of specimen collection, and generates further insights into vulnerabilities of Trypanosoma cruzi [Mycobacterium tuberculosis [Toxoplasma gondii [Borrelia derived peptides and protein fragments detected in peripheral fluids have been proposed [Borrelia peptidoglycan in the synovial environment of patients suffering from Lyme arthritis was detected for several weeks after antibiotic treatment [Borrelia spirochetes can disrupt the architecture of lymph nodes, and impede the formation of stable germinal centers [Borrelia immunology: generation of anti-Borrelia neutralizing antibodies is MHC class II independent [Borrelia primed dendritic cells undergo cytolysis mediated by natural killer cells, thus releasing their antigenic content [Borrelia expressed MHC class II molecules and effector cytokines involved in lymphocyte activation. Culture studies demonstrated that primary mouse fibroblasts were able to internalize Borrelia [Borrelia antigens and peptides derived from immune cells is unknown and presumably exceedingly low. This supports the use of highly sensitive analytical technologies, and of pre-analytical sample processing steps aimed at concentrating low abundance analytes. A urinary Borrelia peptide identified in our clinical study was compatible with MHC II presentation thus supporting the involvement of immune cells in its shedding [Similarly, to other pathogens , two poproposed : a) spirproposed . Persistreatment . It has  centers . Animal ependent . It has  content . ExperimBorrelia . The pershedding .Borrelia peptides can be direct shedding by spirochetes present in different tissues . Proteiree form  and vesiree form ,50. Non-analytes  (Figure Borrelia derived peptides, similarly to cancer derived biomarkers, can enter blood circulation by passively penetrating blood vessel walls [Borrelia derived molecular analytes offers a definitive means of distinguishing whether or not persistent symptoms are associated with persistent presence of the pathogen itself (el walls . Blood cel walls . (Figureel walls . Thus urn itself .Sensitive and accurate direct tests can adjudicate active pathogen infection and contribute to solving long-standing controversies about the etiology of posttreatment persistent symptoms causing great suffering for Borrieliosis patients. High throughput technologies such as nextGen sequencing and proteomics are promising approaches for a direct test that is insensitive to strain variability. We have shown that proteomics can provide functional information that has the potential to contribute to our understanding of pathogen behavior in the human host and can complement information provided by nucleic acid amplification based techniques. High throughput highly sensitive technologies need to be validated longitudinally on controlled cohorts of well characterized Borreliosis patients, before and after antibiotic therapies.


Ricinus communis) oil that contains the 18-carbon HFA ricinoleic acid. Since castor seeds also produce the extremely toxic protein ricin, efforts are underway to engineer valuable HFAs into more suitable crops. However, with few exceptions, the bioengineering of unusual fatty acid production into model and crop plants to date has failed to achieve levels near those of native species. Oilseed plants accumulate triacylglycerol (TAG) up to 80% of seed weight: the TAG fatty acid composition largely determines its nutritional or industrial value. The inefficient flux of transgenic fatty acids through membrane lipids prior to incorporation into TAG has been characterized as a major bottleneck to oilseed engineering. An alternative strategy that bypasses this bottleneck is the remodeling of pre-existing TAG. Bhandari and Bates (pp. 799–815) report that such TAG remodeling mechanisms seem to be at work in developing Physaria fendleri seeds, that have been shown to accumulate TAG containing >60% lesquerolic acid, an unusual 20 carbon HFA that accumulates at only the sn-1 and sn-3 positions of TAG. Isotopic tracing of developing P. fendleri seed lipid metabolism identified that phosphatidylcholine-derived diacylglycerol is utilized to initially produce TAG with only one lesquerolic acid. Subsequently, a nonhydroxylated fatty acid is removed from TAG (transiently reproducing diacylgylcerol) and a second lesquerolic acid is incorporated. Thus, a dynamic TAG remodeling process involving anabolic and catabolic reactions controls the final TAG fatty acid composition. The authors’ reinterpretation of P. fendleri transcriptomic data identified potential genes involved in TAG remodeling that could provide a new approach for oilseed engineering by altering the fatty acid composition of oils after initial TAG synthesis.Hydroxy fatty acids (HFAs) are used in a wide variety of industrial products, including lubricants, biodegradable polyesters, and biofuels. The major source of HFAs is castor  are emerging as major post-transcriptional regulators of gene expression. Brenes-Álvarez et al. (pp. 787–798) now report the participation of an sRNA called nitrogen stress-inducible RNA 4 (NsiR4) in the post-transcriptional regulation expression of two genes involved in CO2 fixation via the Calvin cycle, namely glpX, which encodes sedoheptulose-1,7-bisphosphatase/fructose-1,6-bisphosphatase (SBPase), and pgk, which encodes phosphoglycerate kinase (PGK). The overexpression of NsiR4 in the cyanobacterium Nostoc resulted in a reduced amount of SBPase protein and reduced PGK activity, as well as reduced levels of both glpX and pgk mRNAs. NsiR4 is also more strongly expressed in heterocysts than in vegetative cells, which may contribute to the heterocyst-specific shutdown of Calvin cycle flux. Post-transcriptional regulation of two Calvin cycle enzymes by NsiR4, an N-regulated sRNA, represents an additional link between N control and CO2 assimilation.In response to nitrogen (N) deficiency, some filamentous cyanobacteria form a heterocyst, a specialized cell type devoted to the fixation of atmospheric Nβ-glucuronidase (GUS) transgene as the reporter, they found that LDH-delivered functional dsRNA efficiently induces RNAi of the GUS gene and reduces the GUS enzyme activity. These findings demonstrate that an LDH delivery platform with the size of up to 50 nm in diameter can facilitate delivery of functional exogenous dsRNA into intact plant cells.Transgenes expressing double-stranded RNA (dsRNA) have been particularly effective in conferring resistance to viruses and pests, and in manipulating metabolic pathways in plants. However, the efficacy of genetic engineering and virus-facilitated trait modification varies greatly between different plant species. In the past decade, many nanoparticles have been developed to mediate delivery of biomolecules into plants. These include single-walled carbon nanotubes that can deliver plasmids and siRNAs into leaves via infiltration. Nanoparticle delivery platforms have many advantages, such as low toxicity, high delivery efficiency, species independence, and subcellular targeting. Despite their potential, however, there have been few reports of nanoparticle-mediated delivery of functional biomolecules directly into intact plant cells. Layered double hydroxide (LDH) is a family of bio-compatible and degradable clay materials. Plate-like LDH nanoparticles consist of mixed divalent/trivalent metal hydroxide layers, which are bonded together with anions and water molecules between them. The layered structure and positively charged hydroxide layers make the LDH nanoparticle a suitable carrier to deliver negatively charged biomolecules such as DNA, RNA, and proteins. Yong et al. (pp. 886–899) show that LDH nanoparticles are taken up by developing pollen. Using Arabidopsis thaliana), more than 30 RALF peptides have been predicted. Nevertheless, to date, there is no comprehensive, family-wide functional study of RALF peptides. Abarca et al. (pp. 996–1010) have analyzed the phylogeny of the proposed multigenic RALF peptide family in the Arabidopsis ecotype Col-0, and tested a variety of physiological responses triggered by RALFs. They show that the majority of AtRALF peptides, when applied exogenously as synthetic peptides, induce seedling or root growth inhibition and modulate ROS production in Arabidopsis. These findings suggest that alkalinization and growth inhibition are, generally, coupled characteristics of RALF peptides. Finally, the authors provide evidence that for the majority of these peptides, their responses are dependent on the receptor kinase FERONIA, suggesting a pivotal role for this receptor in the perception of multiple RALF peptides.RAPID ALKALINIZATION FACTOR (RALF) peptides belong to a family of cysteine-rich plant peptide hormones that are involved in multiple physiological and developmental processes, ranging from pollen tube growth to the modulation of immune responses. They were originally discovered in a peptide hormone screen due to their ability to cause medium alkalinization of cell cultures. Later, RALF peptides with several conserved motifs were found to be ubiquitous in terrestrial plants. In Arabidopsis (Physcomitrella (Physcomitrium patens) mutants with altered respiration stemming from the inactivation of Complex I (CI) of the mitochondrial electron transport chain. Inactivation of CI caused a strong impairment of growth even in fully autotrophic conditions in tissues where all the cells are photosynthetically active. The impact of CI mutations on respiratory activity was assessed by measuring oxygen (O2) consumption rate. In CI mutants, O2 consumption in the dark was higher than in wild-type (WT). While this seems counterintuitive for mutants affected in a respiratory complex, this observation can be explained by the compensatory over-accumulation of Complexes II and II and alternative terminal oxidase. While CI mutants showed alterations in the stoichiometry of these respiratory complexes, the composition of the photosynthetic apparatus was not substantially affected. Photosynthetic functionality is nevertheless altered in CI mutants, and plants exposed to sub-saturating illumination show several differences. Both PSI and PSII showed a higher yield upon illumination in mutant plants, suggesting a lower saturation level in illuminated plants. CI mutants also show increased proton conductivity across the thylakoids membrane that, combined with an equivalent proton motive force, is consistent with a higher electron transport rate. The higher ATPase activity in the CI mutants causes a faster translocation of protons in the stroma and an increase in the lumenal pH, in turn, affecting photosynthesis regulatory mechanisms modulated by lumen acidification. These results demonstrate that alteration of respiratory activity directly impacts photosynthesis in P. patens and that metabolic interaction between organelles is essential in their ability to use light energy for growth.Respiration in photosynthetic organisms is essential for supporting energy demand in all plant tissues at night, in nonphotosynthetic tissues such as roots, and during developmental stages where photosynthesis is not active (e.g. seed germination). Increasing evidence, however, suggests that respiration is also important for sustaining optimal photosynthetic activity, and that there are strong functional links between the bioenergetics of chloroplasts and mitochondria. To further elucidate these links, Mellon et al. (pp. 931–946) isolated Zea mays) mutant  versus WT. These two genotypes were grown in two contrasting soil textures: sand and loam. Transpiration rate and leaf water potential were measured continuously for different soil moisture conditions using the root pressure chamber method. Based on their findings, the authors propose a soil–plant hydraulic model to explain the relationship between transpiration rate and leaf xylem water potential. They used this model to systematically compare the two genotypes in the two soil textures and to investigate whether, thanks to root hairs, the two genotypes would exhibit distinct root hydraulic properties. Contrary to previous observations with barley, they found no significant effect of root hairs on water uptake and soil–plant hydraulic conductance in both sandy and loamy soils.Although the role of root hairs in nutrient acquisition has been well documented, their contribution to water uptake remains controversial. Many of these studies have involved comparisons of hairless mutants to WTs, but the general conclusions drawn have often been contradictory. Cai et al. (pp. 858–872) hypothesize that the differences in the interactions between root hairs and soils of different textures and hydraulic properties may reconcile many of these seeming discrepancies. They hypothesize: (1) root hairs attenuate the drop in soil matric potential around the roots, by extending the surface of the roots extracting water and thus reducing the flow velocity at the root–soil interface. This effect is expected to be particularly marked in coarse-textured soils, such as sandy soils, whose hydraulic conductivity abruptly drops with decreasing soil matric potentials; (2) the effect of root hairs on the relation between transpiration rate and leaf water potential is more pronounced in sand than in loam, due to the pronounced drop in soil hydraulic conductivity of sand; (3) mutant plants develop longer and thicker roots to partly compensate for the absence of root hairs. To test these hypotheses, they compared the maize (


Background and Objectives: Retroperitoneal schwannoma is a very rare case of schwannoma which commonly occurs in the other part of the body. However, it is difficult to distinguish schwannoma from other tumors before pathological examination because they do not show specific characteristics on imaging study such as ultrasound, computed tomography (CT), and magnetic resonance image (MRI). Case summary: A 60-year-old male showed a retroperitoneal cystic tumor which is found incidentally during evaluation of coexisted bladder tumor. Neurogenic tumor was suspicious for the retroperitoneal tumor through pre-operative imaging study. Finally, a schwannoma was diagnosed by immunohistochemical examination after complete surgical excision laparoscopically. Conclusion: As imaging technology is developed, there may be more chances to differentiate schwannoma from other neoplasm. However, still surgical resection and histopathological examination is feasible for diagnosis of schwannoma. Schwannoma (neurilemmomas) are benign tumors that originate from nerve sheath cells along peripheral nerves . They geA 60-year-old male presented with a two-week history of intermittent gross hematuria. There was a 2.5 cm-sized papillary tumor at the left anterolateral wall of the urinary bladder on cystoscopy. A contrast-enhanced abdominal computed tomography (CT) scan incidentally revealed a 4.8 cm cystic mass with septa at the left pelvic wall. Lymphangioma or cystic changes of the neurogenic tumor were suspicious for a cystic tumor. Magnetic resonance image (MRI) was recommended for differential diagnosis of a necrotic metastatic lymph node, which was the least-likely suspected diagnosis. MRI revealed that the bladder tumor was likely muscle-invasive with Vesical Imaging-Reporting And Data System (VI-RADS) category 4, and the pelvic mass exhibited cystic changes, suggestive of a neurogenic tumor  [5]. TheFirst, TURBT was performed under general anesthesia with the patient in a lithotomy position. The bladder tumor was histologically confirmed as a high-grade papillary urothelial carcinoma, Ta. One month later, laparoscopic pelvic mass excision was completed under general anesthesia using four trocars with the patient placed in the supine position. A 12-mm incision port for the laparoscope plus three other working ports were placed. The left retroperitoneal space was exposed after an incision of the left abdomen peritoneum. The plane was developed by a combination of sharp and blunt maneuvers. The left ureter and left common iliac vessels adherent to the pelvic mass were identified and dissected carefully. Finally, the mass was gently separated from the pelvic wall and extracted into a laparoscopic specimen bag. The specimen was sent to the pathologic department for routine staining and sectioning . SuctionSchwannoma most commonly occur in the head and neck . AlthougCystic changes happen when the tumor overgrows its blood supply . Over haStill, surgical excision and immunohistochemical examination are necessary to reach a definitive diagnosis. Intense staining of the S-100 protein is used to confirm the diagnosis of benign nerve sheath tumors, including schwannoma. The recurrence rate is very low in patients with benign schwannoma, while malignant tumors are associated with a very poor prognosis and high recurrence rate . In thisWe reported a case of an incidentally discovered retroperitoneal schwannoma, which is very rare and difficult to diagnose preoperatively. As the use of radiological examinations becomes more common, we expect that there will be increasing cases of incidental schwannoma. However, as imaging studies are limited in their ability to achieve definitive diagnosis, complete surgical resection and regular follow up are necessary.


Epstein–Barr virus (EBV) is a human herpesvirus that is common among the global population, causing an enormous disease burden. EBV can directly cause infectious mononucleosis and is also associated with various malignancies and autoimmune diseases. In order to prevent primary infection and subsequent chronic disease, efforts have been made to develop a prophylactic vaccine against EBV in recent years, but there is still no vaccine in clinical use. The outbreak of the COVID-19 pandemic and the global cooperation in vaccine development against SARS-CoV-2 provide insights for next-generation antiviral vaccine design and opportunities for developing an effective prophylactic EBV vaccine. With improvements in antigen selection, vaccine platforms, formulation and evaluation systems, novel vaccines against EBV are expected to elicit dual protection against infection of both B lymphocytes and epithelial cells. This would provide sustainable immunity against EBV-associated malignancies, finally enabling the control of worldwide EBV infection and management of EBV-associated diseases. Epstein–Barr virus (EBV) is a double-stranded DNA virus that belongs to the gamma herpesvirus family. It causes endemic infection in over 95% of the worldwide population , and is The transmission of EBV within the population is mainly mediated by saliva, and the infection involves both B lymphocytes and epithelial cells . PrimarySimilar to other herpesviruses, EBV is an enveloped virus, comprising a membrane decorated with envelope glycoproteins , which are crucial for receptor recognition, attachment, and virus–host membrane fusion . As EBV The complex molecular machine of surface glycoproteins brings challenges for not only understanding the complete fusion mechanism of EBV, but also choosing appropriate antigens for vaccine development. Therefore, an ideal prophylactic vaccine against EBV should be able to elicit potent neutralizing antibodies against EBV infection in both B lymphocytes and epithelial cells, which requires careful selection of antigens during vaccine design.® as vaccine for reducing the risk of post-transplant lymphoproliferative disease (PTLD)  . The vace (PTLD) 2.Since the gH/gL complex plays a critical role in the infection of B cells and especially epithelial cells, there is increasing focus on the gH/gL complex as the antigen for new vaccine candidates. The recently identified anti-gH/gL dual-tropic neutralizing antibody AMMO1  further gp42 is a subunit of the gH/gL/gp42 heterotrimer on the EBV virion membrane. It was identified as the ligand for HLA class II molecules mostly participating in B cell infection and was recently found to hinder the infection of epithelial cells , 65, indEBV gB is the fusion protein on the viral surface mediating viral–host membrane fusion and recognizing NRP1 on epithelial cells. Most prophylactic antiviral vaccines target the viral fusion protein, such as influenza HA –68, HIV With the development of protein structure analysis, the fusion status of fusion proteins becomes increasingly important for elucidating the fusion mechanism and understanding the connection between conformational changes and the fusion process. Pre-fusion status is often regarded as the natural conformation on the viral membrane , 76 befoAfter primary infection, EBV undergoes a short period of replication in the oropharynx, after which further infection of B cells ensues, during which glycoproteins encoded by the EBV genome become eclipsed by certain lytic and latent genes, which drive the B cell transformation and latency as summarized by a review . TherefoHence, during EBV vaccine development, rational and careful antigen selection is necessary to ensure both robust and comprehensive immunity against EBV infection. There is still a lot of space for extensive study on immunization efficacy of single glycoproteins, especially gH/gL or gB. Additionally, combinatorial use of multiple antigens as vaccine candidates, including glycoprotein sets or glycoprotein-latency protein combinations, deserves further study for eliciting both sufficient neutralizing antibody titers and T cell responses.The outbreak of the COVID-19 pandemic has brought significant challenges for global vaccine development, prompting a continuous stream of innovative designs of candidate vaccines against SARS-CoV-2, and thus giving great impetus to next-generation vaccine development. The rapid application of the first clinically used mRNA vaccine developed by Moderna and BioNTech , 99 achiBecause EBV tends to establish a latent infection of host cells, a general approach to induce EBV replication and cell lysis requires complicated procedures and results in a low yield of live virus. Consequently, the development of attenuated virus or inactivated virus vaccines based on authentic EBV is challenging due to limited viral material. Thus, there are few reports on the use of inactivated or attenuated EBV as vaccine candidates. Alternatively, modification of EBV the genome for direct production of defective virions without genomic DNA could be a viable approach for EBV-derived vaccine development. EBV-derived virus-like particles (VLPs) are based on different EBV mutants with various deletions of sets of oncogenic genes or DNA packaging genes , producevia the classic MHC-I pathway and activation of antigen-specific cytotoxic CD8+ T cells   and founAdjuvants incorporated in components of the antigen for vaccine formulation can modulate the immune response. In addition to the original immunogenic profile of the selected antigen, a carefully selected adjuvant can broaden the use or enhance the efficacy for immunization. For vaccine platforms such as inactivated virus or protein-based subunit vaccines/VLPs, the loss of bioactivity greatly diminishes the immunogenicity of the antigen itself, which further requires powerful adjuvants for pre-stimulation of immune recognition, prolongation of antigen retention, as well as both humoral and cellular immunity enhancement –129.th century, adjuvants such as Freund’s adjuvant, lipid A, immune-stimulating complexes (ISCOMS), and aluminum hydroxide  encouraged the use of NHPs as challenge models for EBV vaccine evaluation . The facth century, the discoverer of EBV, Epstein et al. as well as Emini et al. used cotton-top tamarins and common marmosets  for gp350-based vaccine evaluation of both neutralizing antibody titers and challenge protection , which shares a high level of genomic sequence similarity with EBV , EBV canAs the most widely used animal model, mice play an important role in EBV vaccine evaluation. Most prophylactic EBV vaccine studies used mouse immunization to primarily assess the serum antibody titer and neutralizing antibody titer , 44, 46.Some studies also used rabbits as animal models for EBV vaccine evaluation , 156. ThTupaia belangeri subsp. chinensis) could also be a viable animal model for EBV vaccine evaluation. Following intravenous injection of virus, 8/10 tree shrews displayed symptoms of EBV infection including detectable expression of EBV-related genes and increase of anti-EBV antibodies. Despite positive results in early challenge, only a small portion of tree shrews showed EBER, LMP, and EBNA2-positive cells in spleen or mesenteric lymph node samples. The negative staining for EBV markers in the lungs and nasopharynx also indicated that epithelial cell infection might also be absent in the tree shrew animal model .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The handling Editor declared a past co-authorship with one of the authors MSZ.


A 50-year-old male presented for loss of consciousness. He was initially treated with intravenous epinephrine and fluids, and an electrocardiogram (ECG) displayed an ST-segment elevation in lead aVR with global ST-segment depressions. A subsequent left heart catheterization revealed that the middle segment of the left anterior descending artery (LAD) demonstrated severe stenosis during systole but would become patent during diastole, which was suggestive of myocardial bridging. After stopping the epinephrine and increasing the fluid infusion, the ECG changes rapidly resolved. The patient had later admitted to significant dehydration all day. Myocardial bridging is a congenital anomaly in which a coronary artery segment courses through the myocardium instead of the usual epicardial surface. Occasionally, myocardial bridging may present similarly to acute coronary syndrome in severe dehydration or hyperadrenergic states. The diagnosis can be made through coronary angiography, which reveals a dynamic vessel obstruction pattern corresponding with the cardiac cycle. Long-term effects may also include accelerated atherosclerosis. Treatment consists of reversing precipitating causes during acute presentations and decreasing the risk of coronary artery disease on a chronic basis. New-onset ST-segment changes on electrocardiogram (ECG) are usually a cause of concern, with coronary artery occlusion among the lead differential diagnoses. Myocardial bridging is a peculiar condition that may lead to such changes, yet the vessel obstruction is mostly dynamic and dependent on the cardiac cycle. Though usually asymptomatic, it can manifest in emergent cases such as acute coronary syndrome when precipitated by some factors and long-term atherosclerosis.A 50-year-old male with a past medical history of hypertension presented for loss of consciousness. He was hypotensive with a mean arterial pressure of 55 mmHg, tachycardic with a heart rate around 140 bpm, and minimally arousable on initial evaluation while complaining of ill-defined chest pain. Physical exam showed a regular tachycardic rhythm, normal heart sounds, decreased bilateral air entry, and absent jugular venous distention or peripheral edema. Treatment started in the emergency department with intravenous fluids and epinephrine. The electrocardiogram (ECG) displayed an ST-segment elevation in lead aVR, along with global ST-segment depressions, most notably in the inferolateral leads . Point oMyocardial bridging is a congenital anomaly in which a segment of the coronary arteries courses through the myocardium, thus rendering it “bridged” by the overlying cardiac tissue. While often benign, it may lead to emergent clinical presentations and long-term vascular disease . MyocardThe treatment of myocardial bridging involves both avoiding the acute limitation of blood flow and the long-term decrease in symptoms and atherosclerosis incidence. Patients should refrain from conditions that may induce an adrenergic drive and dehydration. Beta-blockers have been the cornerstone of therapy as they increase the diastolic phase and decrease inotropy, in addition to nondihydropyridine calcium-channel blockers as an adjunct therapy or when beta-blockers are not tolerated . As discThis case highlights the importance of identifying myocardial bridging as a potential cause of myocardial ischemia, especially in patients presenting with dehydration and hyperadrenergic states. Early recognition may guide proper treatment and avoid disease exacerbation.


TM is a popular biosynthetic semi-permeable skin substitute conventionally applied onto non-excised partial thickness burn wounds to facilitate healing. The use of BiobraneTM for definitive coverage after excision of partial-thickness thermal burns has not been reported. We highlighted our experience of immediate BiobraneTM application for definitive coverage of tangentially-excised partial thickness thermal burn wounds in four patients. This technique is safe and efficient, minimizes painful and costly dressing changes, avoids the complications associated with autologous skin grafting, and eliminates the unpredictability of burns wound conversion. We believe this method expands the indications for BiobraneTM usage, accelerates wound healing, and provides better aesthetic outcomes.Biobrane The cosmetic appearance of skin grafted sites is also frequently unsatisfactory.2 Introduced in 1979, BiobraneTM  is a highly-conformable synthetic acellular material comprising of a flexible inner trifilament nylon fabric mesh embedded with hydrophilic type I porcine collage and an outer semipermeable silicone membrane.3,4Early excision and autologous skin grafting is the gold standard treatment for burns.TM to the wound reduces pain and permits early joint mobilization. The silicone membrane functions as a protective epidermal barrier against infection and prevents wound desiccation, whilst permitting water vapour permeability to prevent fluid from collecting.3,4When placed on a clean partial-thickness burn, collagen on the inner surface preferentially binds to fibrin from the wound exudate. Adherence of BiobraneTM from the re-epithelised wound.3,4Over the next 10-14 days, proliferating epithelial cells from hair follicles within the deep dermis migrate across the wound surface, forming ever-enlarging epidermal islands that eventually become confluent, and in the process causing spontaneous detachment of BiobraneTM is changed, which reduces patient discomfort and duration of hospitalization. As BiobraneTM is transparent, tracking the progress of wound healing and monitoring for potential complications is straightforward.3,4 Numerous authors have shared their protocols and techniques to maximise successful use of BiobraneTM.4-6 Wound selection is paramount: an exudative wound bed is a prerequisite for adherence, hence BiobraneTM should not be applied to non-exudative deep dermal and full-thickness burns that require excision and skin grafting. Meticulous aseptic wound bed cleaning, as well as removal of unviable blisters and surface debris is also essential to prevent BiobraneTM infection and premature separation from the wound bed.5During this period, only the gauze dressings on top of the BiobraneTM should be applied within 24-48 hours after the burn injury, beyond which the risks of wound colonization leading to prosthesis infection and failure become significant.4-6 BiobraneTM is primarily utilised as a dressing for non-excised superficial and mid-dermal thermal burns, as well as for skin graft donor sites, to expedite re-epithelization. BiobraneTM has also been deployed for temporary coverage of excised deep dermal and full-thickness thermal burns to prepare the wound for autografting,7 and has reportedly been used for definitive coverage of partial-thickness alkaline burns with good results.8 The use of BiobraneTM for immediate definitive coverage of thermal burn wounds that have been tangentially excised has not been described in the literature. We reported our experience using this method on four patients with partial-thickness thermal burns. BiobraneTM was secured to the wound under slight tension with sterile adhesive tape  and surgical staples, and wrapped in gauze soaked with povidone-iodine . Written informed consent was obtained from each patient. After induction of general anaesthesia and aseptic cleansing, the area of burns to be excised received a subcutaneous injection of tumescent solution . A tourniquet was not utilized. Ultra-thin tangential excision was performed with an air-powered dermatome  at 4/1000 inch cutting thickness until uniform punctate bleeding was observed from the wound. Topical adrenaline solution-soaked laparotomy sponges were then applied for hemostasis. BiobraneCASE 1TM was then applied to all burns. The area of excised burns was observed to be healing well underneath the BiobraneTM dressings  partial-thickness thermal burns from a flash fire at an industrial site . The timressings  on post-ressings . CASE 2TM was then applied to these excised areas. Her wounds were observed to be healing well on POD 7  and left calf (4% TBSA) were found to be poorly blanching with multiple areas of fixed-staining . These aon POD 7 , and epion POD 7 . A summaThe study was performed in accordance with the principles of the Declaration of Helsinki. Written informed consents were obtained. Institutional review board/ethics committee approval was not required for retrospective case reports. Patients provided written informed consent for the publication and the use of their images. Written informed consent was obtained from the patient for publication of this case series and accompanying images. A copy of the written consents was available for review by the Editor-in-Chief of this journal on request.TM usage recommend that to be applied to newly-burnt, exudative, partial-thickness wounds that have been carefully prepared.4-6 This maximises adherence and reduces the risks of wound colonization and consequent prosthetic infection and failure that plagued many earlier studies.9,10 Our case series demonstrated that ultra-thin, tangential excision allowed conversion of an older (>48 hours) burn to a fresh burn which permitted successful application of BiobraneTM. Not only does this remove colonizing bacteria, the raw excised surface also produces renewed exudate, enhancing the adherence of BiobraneTM to the burn wound.5Contemporary guidelines on BiobraneTM is applied. If a part of the wound is poorly-blanching, only that particular area requires tangential excision, and BiobraneTM can then be applied to the entire wound (excised as well as non-excised). The latter extends the “trial-of-life” concept, where BiobraneTM is applied over burns of uncertain dermal depth in a wound that is hopefully still capable of spontaneous healing.5As illustrated above, this technique lends itself well to burns of homogenous or heterogenous depth. If capillary refill is uniformly reduced across the burn, the entire wound is tangentially shaved and Biobrane11,12 including our own13 have shown that BiobraneTM has reduced the need for autologous skin grafting in partial-thickness burns. By improving adherence of BiobraneTM to areas of deeper injury, this novel technique potentially decreases the need for autologous skin grafting  even further. Nonetheless, it is imperative that patients should be counselled pre-operatively that BiobraneTM may not adhere or “take” over the excised areas and that subsequent re-excision with autologous skin grafting may be necessary. Numerous groups,TM adherence after tangential excision. Laser Doppler imaging may be a useful adjunct in determining burn depth (and the suitability of this technique), if uncertainty persists after clinical assessment.14 Greenwood et al. have warned about the risk of applying BiobraneTM to an excised wound, with blood clots and subsequent fibrovascular ingrowth into the nylon scaffold making removal difficult.5. Caution should be exercised when applying this technique to deep dermal and full-thickness burns, in these wounds it is highly unlikely that the remnant dermis will be able to produce enough exudate to allow sufficient BiobraneTM was only secured onto the raw wound surface, once all visible bleeding had been arrested. We did not encounter any problems related to spontaneous detachment of BiobraneTM from the wound, once epithelization had occurred. It has been demonstrated that BiobraneTM is capable of controlling bacterial growth in wounds with an initial bacterial count of less than 105 per gram of tissue, and wounds with a bacterial count exceeding 105 per gram of tissue precludes BiobraneTM “take”.4,6,15We thus paid meticulous attentiontowards achieving hemostasis using a combination of adrenaline-containing tumescent infiltration, bipolar electrocautery and manual pressure. BiobraneTM is applied within 48 hours of injury,4-6 with a recent retrospective cohort study showing evidence of significantly faster re-epithelization and decreased need for skin grafting, if BiobraneTM is applied within 12 hours.12 Nonetheless, there is no conclusive evidence that its use is contraindicated in older burns.4,6 We successfully performed ultra-thin tangential excision with immediate BiobraneTM coverage on burns with a mean age of 105.5 hours  from the time of injury without any infectious complications. Nonetheless, we do not recommend that this technique is attempted on burn wounds that display overt clinical signs of infection. As burn wounds become progressively colonized by bacteria with time, most authors advise that BiobraneTM. This promotes greater adherence of BiobraneTM to the wound, and in so doing facilitates accelerated re-epithelisation.5,6 The correlation between burns healing time and subsequent development of hypertrophic scarring and contractures has been widely reported, with burns taking more than 21 days to heal at significantly greater risk of hypertrophic scarring and its sequelae .2This approach allows more definitive intervention on burns of equivocal healing potential, the excized surface exudes fibrin that promotes binding to collagen on the inner surface of Biobrane16 In our study, all excised wounds re-epithelised after an average of 9.75 days (range 7-13 days), and none had developed hypertrophic scarring at 12 months of follow-up. BiobraneTM was shown to be a versatile alternative to conventional dressings in the management of thermal burns. While its cost, benefit ratio has yet to be fully elucidated; BiobraneTM permits reduced frequency and discomfort of dressing changes, potentially reduces the extent of and the need for skin grafting, as well as decreases hospitalization time.5,6,17-19More recent work has shown that the rates of hypertrophic scar are multiplied by 1.138 for every additional day taken for the burns to heal (even within the 21 day period); therefore, every effort should be made to accelerate wound healing.TM should be limited to superficial to mid-dermal partial-thickness thermal burns within 48 hours of injury. Preparatory tangential excision expands the indications for BiobraneTM use, allowing earlier definitive treatment of equivocal burns, whilst simultaneously avoiding the complications of skin grafting and retaining the benefits of BiobraneTM that have made it so popular. Larger prospective case-controlled studies are required to further establish the average time to wound healing, cost-effectiveness, outcomes  and complications of this novel technique.However, current recommendations may restrict its usage. Although the small population and descriptive nature of this series make it impossible to perform any meaningful statistical analysis, these promising early results challenge the widely-accepted doctrine that application of BiobraneTM can achieve good functional and cosmetic results. Contrary to existing guidelines, this technique can be successfully employed on burns that are more than 48 hours from the time of injury. This accelerates re-epithelisation, minimizes the complications of delayed wound healing and may reduce the need for patients to undergo autologous skin grafting. Ultra-thin tangential excision of partial-thickness thermal burns with equivocal capacity for spontaneous healing followed by immediate definitive coverage with the biosynthetic dressing Biobrane


In a somatic mutation pan-cancer analysis, aggregated mutations of those predicted to be missense versus truncations were differentially associated with gene expressions of cancer drivers, and somatic truncation eQTLs were further identified as a new multi-omic classifier of oncogenes versus tumor-suppressor genes. AeQTL is easy to use and customize, allowing a broad application for discovering rare variants, including coding and noncoding variants, associated with gene expression. AeQTL is implemented in Python and the source code is freely available at https://github.com/Huang-lab/AeQTL under the MIT license.Concurrently available genomic and transcriptomic data from large cohorts provide opportunities to discover expression quantitative trait loci (eQTLs)—genetic variants associated with gene expression changes. However, the statistical power of detecting rare variant eQTLs is often limited and most existing eQTL tools are not compatible with sequence variant file formats. We have developed AeQTL (Aggregated eQTL), a software tool that performs eQTL analysis on variants aggregated according to user-specified regions and is designed to accommodate standard genomic files. AeQTL consistently yielded similar or higher powers for identifying rare variant eQTLs than single-variant tests. Using AeQTL, we discovered that aggregated rare germline truncations in  Samples with concurrent DNA- and RNA-sequencing (DNA-seq and RNA-seq) provide opportunities to discover expression quantitative trait loci (eQTLs), i.e. genetic variants associated with variations in gene expression3. Most existing eQTL tools focus on applying various statistical models to test for association between individual pairs of a variant and the associated gene expression6. However, for rare variants, the underlying power of the statistical testing is often limited and identifying eQTLs from rare variants remains a challenge7.Advances in sequencing technologies have enabled the generation of large-scale disease cohorts with concurrently available genomic and transcriptomic data11, yet similar strategies have rarely been implemented for identifying eQTLs. In addition, these tools are not readily compatible with standard variant call files resulted from sequencing data, including VCFs/MAFs and RNA-seq data from large cohorts.Multiple methodologies and tools using aggregation strategies to group and identify rare variants associated with disease status have been developed12. Here, we show AeQTL’s aggregation algorithm can increase the statistical power in order to discover rare variant eQTLs with a larger size of grouped carriers. Further, we demonstrate AeQTL’s capacity in identifying both germline variants and somatic mutations associated with gene expression changes, which can help prioritize disease susceptibility genes or cancer driver genes. In sum, AeQTL offers a much-needed versatile multi-omics tool to integrate DNA-seq and RNA-seq data.Here, we present AeQTL, a software tool that performs eQTL analysis on aggregated variants in specified genomic regions and is designed to accommodate standard file formats generated from sequencing data. Previous studies have found that rare germline variants are significantly enriched at both high and low extremes of gene expression in promoter regions2.AeQTL implements standard eQTL analysis with user-defined variant-aggregation and its workflow is shown in 2.1.trans-eQTL discovery mode. This user-constructed BED file allows flexibility in the design of eQTL analysis for testing both cis- and trans- eQTLs. We also provide a coding exomic region BED file on our Github page, which can be used for testing and exploratory purposes.The input region file (i.e. a BED file) is provided by the user to set up desired association tests between gene expressions and variants. Each line of the file contains a genomic region followed by one or more genes to be tested against. An association test will be set up between the expression level of each specified gene and aggregated variants in the genomic region. If no genes are specified, AeQTL by default will test each region against every gene’s expression in the expression file in a While all variants with matching samples in the expression file will be included in the tests, users can further restrict the aggregation by setting two optional thresholds: the number of mutated samples per region and the number of variants per region, in which case regions with samples or variants below the thresholds will be filtered out. Both thresholds are set to 1 by default.2.2.https://github.com/bxlab/bx-python). We used a standalone wrapper of the interval tree  for easier compilation. The interval tree is designed for fast intersect queries on one-dimensional intervals. Compared to other simple positional intersection methods, the interval tree has two major strengths: (1) it allows each interval to be annotated and the annotations will be preserved in queries; (2) the interval tree is implemented in Cython which is faster and more computationally efficient. AeQTL creates an interval tree for each chromosome. For each genomic region provided in the BED file, an interval is specified by the start and end positions, annotated by the region name, and added to the interval tree of its corresponding chromosome. Then, AeQTL finds the intervals that overlap with the given variant to extract its region name and aggregates variants of the same region. AeQTL accommodates different types of variants including single-nucleotide variants (SNVs), insertions, and deletions. After aggregation, AeQTL maps each region to samples and defines a regional mutation status by assigning a genotype “1” if a sample has any variants in this region and a genotype “0” otherwise.AeQTL aggregates variants by finding overlaps between variants and regions using the interval tree data structure, which is part of the bx-python package  and thus the intermediate files enable flexibility for users.AeQTL outputs mapped files with variant genotypes, expression, and covariates for each region, which can be readily routed into other aggregational statistical tests such as SKATp-values and coefficients of the intercept and all dependent variables, including regional genotype and covariates. To correct for multiple testing, AeQTL also reports adjusted p-values with false discovery rate (FDR) based on the Benjamini-Hochberg (BH) procedure.All the regression results are compiled in a summary file where AeQTL reports both 3.3.1.To demonstrate the aggregating effect on the statistical power of identifying rare variant eQTLs, we performed a simulation analysis using AeQTL. Based on VCF files and expression matrices of 10 rare variants  with a series of sample sizes, we ran AeQTL on both single variants and grouped variants specified by BED files . Gene exOverall, statistical analysis of aggregated variants consistently demonstrated comparable or higher powers than individual variants. When the sample size was small, the powers of grouped and single variants were similarly low for both effect sizes. As the sample size increased, the powers under all testing conditions increased as expected. However, the powers of grouped variants increased noticeably faster than those of single variants. When effect size = −20, the increased power fold change provided by the AeQTL aggregation method was the most substantial within the sample size interval of 600 to 2,000. The power of grouped variants reached 97% with sample size = 2,000, while single variants required three times the sample size to reach a similar power. When effect size = −10, the power of grouped variants reached 95% with sample size = 6,000 and single variants did not reach the same power until sample size = 15,000. At a sample size of 5,000, the powers of all testing conditions except for single variants with effect size = −10 were higher than 90% and were saturated (> 99%) when the sample size reached 8,000.3.2.13. We tested the hypothesis that rare truncations in cancer susceptibility genes are associated with their cis-expression in tumor samples. For the input BED file, we specified each of the genes on chromosome 17 as a region of interest and tested truncations in each gene region against the expression of its located gene. We used the level 3 TCGA RNA-seq gene expression data in RSEM14 from breast invasive carcinoma (BRCA) patients and incorporated six covariates: age, gender, ethnicity, tumor stage, as well as the top two components from the principal component (PC) analysis on population structure (accounting for > 80% of the top 20 PCs). Because low gene expression levels would likely present technical noises, we filtered out genes with median expressions lower than log(2)  on chromosome 17 of the TCGA PanCanAtlas cohortn log(2) . This gep-values on a QQ-plot (BRCA1 was significantly associated with aggregated rare truncations in the BRCA1 exomic region (P = 0.033) in the BRCA cohort, demonstrating that AeQTL could efficiently identify grouped genotype-expression association. In addition, SLC25A39 (P = 0.030) was among the top-ranked genes whose expressions were negatively associated with the aggregated rare truncations in their regions. We also carried out a sensitivity analysis of adjusted genotype p-values against region sizes, which suggested no significant correlation between the two, indicating the lack of false-discovery from large genes , the calcium channel gene CACNA1A from lung adenocarcinoma (LUAD), and TP53 from BRCA; the top-ranked gene-cancer pairs with missense mutations include JAK2 from stomach adenocarcinoma (STAD), TP53 from lung squamous cell carcinoma (LUSC), and FGFR3 from bladder urothelial carcinoma (BLCA).We interrogated a subset of 299 genes that were reported as likely driver genes by Bailey et al.To demonstrate the computational capacity of AeQTL, we expanded the analysis to the entire dataset, including 335,866 truncations and ~2 million missense mutations from 10,208 samples. AeQTL identified 1,179 gene-cancer pairs with truncations and 3,241 gene-cancer pairs with missense mutations significantly associated with their respective gene expressions (FDR < 0.05).16. The top-ranked somatic eQTL genes with truncations include OR8D1 in LUSC, SOX10 in head and neck squamous cell carcinoma (HNSC), and PSG7 in kidney renal clear cell carcinoma (KIRC). The top-ranked somatic eQTL genes with missense mutations include USP29 in cholangiocarcinoma (CHOL) and AMELX, CNTN5, and OR1L3 in lymphoid neoplasm diffuse large B-cell lymphoma (DLBC). Multiple recent reports highlight the functionality of the “long-tail driver genes” found with lesser mutations in multiple cancer types21. These somatic eQTL genes and their expression-associated mutations represent new candidates that warrant further investigations.For significant gene-cancer pairs with truncations, 156 overlapped with the likely driver genes. For significant gene-cancer pairs with missense mutations, 115 overlapped with the likely driver genes. Interestingly, we also identified many top-ranked genes that were not previously identified drivers by TCGA PanCanAtlas driver project3.4.25 that lack considerations of their downstream consequences. To understand whether eQTL patterns could capture the distinction between oncogenes and TSGs, we further investigated the significant genes classified as oncogene or TSG from Bailey et al.’s DNA mutation-based study16.Cancer driver genes, depending on their mutated cancer type and pathway context, can be subclassified into oncogenes and tumor suppressor genes (TSGs). But most existing methods to classify oncogenes and TSGs leveraged cohort-level mutation dataIn the likely-driver-gene subset analysis, the genotype coefficients of truncations showed a strong association with their respective predicted classifications of oncogenes or TSGs. Genes predicted to be oncogenes or possible oncogenes had larger positive genotype coefficients while genes predicted to be TSGs or possible TSGs had larger negative genotype coefficients , demonst3.5.8 and SKAT-O9, and performed the same multiple testing correction based on the BH procedure using FDR. For each gene-cancer pair, we analyzed the difference between the adjusted p-value from AeQTL and the adjusted p-values from SKAT and SKAT-O. The majority of the adjusted p-values from AeQTL were lower than the ones from SKAT and SKAT-O (median difference for truncations = −0.076 (SKAT) and −0.017 (SKAT-O), median difference for missense = −0.012 (SKAT) and −0.010 (SKAT-O), We used the intermediate mapped files from TCGA somatic likely driver subset to test two of the most popular variant-aggregation methods, SKATMost existing variant-aggregation methods are designed to conduct association tests on quantitative traits, most notably for SNP-array genotype data. While each gene expression value can be considered as a continuous trait for analyses using these methods, few of those readily accommodate sequencing data formats such as large VCFs/MAFs and expression matrices from cohorts. To address this challenge, AeQTL can complement the existing methods since it provides intermediate mapped files which can be routed into other aggregational statistical tests based on the users’ preference and hypothesis. We believe such user-friendly functionality would be essential to help the field adopt aggregated eQTL testing from sequencing data.4.AeQTL increases the power of eQTL detection by aggregating variants in a defined genomic region. We have applied AeQTL to both synthetic and real datasets. The synthetic dataset demonstrated that variant aggregation consistently yielded similar or higher powers for rare variant eQTL detection. For real datasets, we used rare germline truncations in breast cancer to showcase that AeQTL can efficiently identify significant associations between grouped variants and gene expressions. Furthermore, we applied AeQTL to somatic mutations in a pan-cancer dataset and identified top-ranked gene-cancer pairs that were significantly associated with either truncations or missense mutations in their respective gene regions.15. The application procedure is described in detail and included as an example on Github.To facilitate users’ adoption of AeQTL, we also provide input files to conduct analyses using MAF datasets, as used by TCGA PanCancer somatic mutation datacis-eQTL mode.AeQTL is easy to use and customize. Out of the three required input files , both variant and expression files can be directly taken by AeQTL without any complicated reformatting or pre-processing, while the user-constructed region file allows great flexibility for setting up association tests. Moreover, we provide the exome BED file used in our TCGA analyses on the Github page so that users can easily explore the tool in the The simplicity of AeQTL’s method design means that it can be broadly applied to datasets without imposing on them excessive assumptions or limitations. We have demonstrated AeQTL’s promising performance when applied to cancer datasets. However, with more genomic and transcriptomic data being collected and made available in other fields such as neurodegenerative diseases and psychiatric diseases, we believe AeQTL will contribute to multiple areas of study. Aside from research, another important application of AeQTL is in educational settings. From processing standard sequencing data formats, to building classic regression models, and to producing FDR-controlled outputs, AeQTL has a clear and simple workflow that can facilitate the learning process of eQTL analysis.There are a few aspects of the method that may be improved. First, a potential downside of having a simple method that suits more datasets is that the aggregated genotype of each region is not weighted. Having unweighted variants does not necessarily lead to worse performance, since the underlying mechanism is often unknown and having preset weights may actually confound the results. Nevertheless, we would like to offer more options for users in cases where there are known variations in the magnitudes of effect for certain variants. We plan to introduce more optional settings such as an annotated variant file with a scaling factor, either specified by the user or generated using other algorithms.25. Herein, we present truncation eQTL patterns revealed by AeQTL as a potential new method to distinguish oncogenes (elevated expression) from tumor suppressors (reduced expression). In TSGs, truncations including nonsense variants or frameshift variants may introduce early stop-codons that likely have led to nonsense-mediated decay (NMD), thus abolishing gene transcripts. In contrast, oncogene truncations show a higher frequency of inframe indels16, albeit the mechanisms through which they are associated with higher gene expression warrant further investigation.Traditional methods to classify oncogenes or tumor suppressors rely on algorithms considering only DNA-mutation patterns or functional curationWith increasingly available cohorts of matched genomic (e.g. whole-genome sequencing) and transcriptomic (e.g. RNA-seq) data, we expect that the robust and versatile AeQTL tool can be applied broadly for discovering rare coding and noncoding variants associated with gene expression.


Aged individuals are at higher risk for morbidity and mortality following acute stressors than similarly stressed young people. Evaluation of age-associated metabolic changes could lead to the identification of specific therapeutic targets to improve outcomes from acute stressors, such as infections, in the elderly. We thus compared the plasma metabolomes of both young and old mice following cecal ligation and puncture (CLP), an accepted model of acute infection and stress.  Young (9-17 wks) and aged (78-96 wks) male C57bl/6 mice were subjected to a retro-orbital bleed and two-week recovery prior to sham surgery  or CLP. Animals were sacrificed at 4 h, 8 h, or 12 h following intervention, and plasma was isolated from blood for subsequent analysis. Metabolomic analysis of samples were performed . χ2p = 0.0004), confirming enhanced frailty. Principal component analysis and partial-least squares discriminant analysis of 566 metabolites demonstrated distinct metabolomic shifts following sham surgery or CLP in both young and aged animals. Identification of metabolites of interest using a consensus statistical approach revealed that both the histidine/histamine pathway and the nicotinamide pathway have significant age-associated alterations following CLP.  Aged animals demonstrated greater intraprocedural mortality than young (30.2% vs. 17.4%,  The application of untargeted plasma metabolomics identified key pathways underpinning metabolomic responses to CLP in both young and aged animals. Ultimately, these data provide a robust foundation for future mechanistic studies that may assist in improving outcomes in frail patients in response to acute stressors such as infection, trauma, or surgery. The medical advances of the past century, including the identification of antibiotics and the discovery and implementation of vaccine programs, have led to a significant increase in life expectancy worldwide. In turn, the number of individuals aged >65 years is expected to surpass 1.5 billion by 2050 . Much ofAging itself is a progressive decline in functional capacity resulting from a combination of degenerative processes in cellular metabolism, with multiple consensus hallmark features –8. DNA rOne of the key limitations of the murine analyses to date is the lack of evaluation of physiologic insult in young vs. aged animals. Based on prior murine metabolomic studies, we hypothesized that aging would be associated with distinct metabolomic trajectories following physiologic stress. To address this, and to bridge the gap between murine modeling and the human cohort data, we performed plasma metabolomic profiling of both young and aged mice at various time points following a standard cecal ligation and puncture (CLP) or sham surgical intervention. These data provide a robust signature both of the baseline metabolomic differences between young and aged mice and several differences in metabolite responses to surgical stressors and sepsis. In particular, we identified significant signature differences in the nicotinamide metabolite axis as well as in histidine and carnosine amino acid biology, which provide a clear path forward for future investigation and potential intervention to medically treat some of the biological sequelae of aging in the acute care setting.via cardiac puncture for analysis. These timepoints were chosen based on data from biometric monitoring with implantable sensors following CLP, which underscored that initial physiologic changes take place within this time period following the initial CLP insult [Animal protocols were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. The experiments were performed in adherence to the US National Institutes of Health guidelines on the use of laboratory animals. C57BL/6 mice ages 11-18 weeks (young) or 86-96 weeks (aged) were utilized for the experimental protocol. For retro-orbital blood sample collection, intraperitoneal injection of a ketamine-xylosine mix was used for procedural sedation, and blood samples were harvested using heparinized capillary tubing. Animals were observed during immediate postprocedural recovery and then allowed to recover fully for a period of two weeks prior to randomization to either immediate sacrifice, sham surgical intervention consisting of a laparotomy and closure in layers, or cecal ligation and puncture done as previously described . AntibioP insult .μL prior to flash-freezing in liquid nitrogen followed by long-term storage at -80°C.Following collection, all blood samples were stored briefly on ice in heparinized 1.5 mL microcentrifuge tubes. Samples were spun at 500 g for 15 minutes to isolate the plasma fraction, which was distributed into multiple aliquots of 150-200 Circulating AST/ALT was measured using the Liver Panel chemistry set and associated Heska analysis platform . Serial dilutions were performed of plasma samples to ensure appropriate reads within the linear range of the detector, as specified by manufacturer's instructions.http://www.metabolon.com) using a multiplatform system encompassing tandem ultrahigh-performance liquid chromatography-mass spectrometry as well as tandem gas chromatography-mass spectrometry systems with different column ionization parameters. Peak calling, compound identification, and batch-effect normalization accounting for day-to-day interrun variability for individual metabolites were accomplished according to Metabolon's proprietary methodology and internal library of standards. A total of 834 metabolites were identifiable, of which 566 had specific KEGG or HMDB identifier labels.Plasma metabolomics was accomplished by Metabolon , an open-source suite of metabolomics analysis tools maintained by the Xia Lab at McGill University [Statistical analysis as described in the text was undertaken using MetaboAnalyst  or 86 and 96 weeks (aged), were utilized for all studies according to the experimental protocol described graphically in http://www.kegg.jp) accession numbers [http://www.metaboanalyst.ca [χ2, p = 0.77, α-linolenic acid metabolism), carbohydrate metabolism (pentose and glucuronate interconversions), cofactor and vitamin biology (nicotinamide and retinol metabolism), and amino acid regulatory pathways (histidine metabolism). Detailed information from this analysis is compiled in Figure Plasma samples isolated according to protocol were sent to Metabolon  for metabolite detection and annotation . A total of 834 discrete metabolites were identifiable across the dataset; 566 of which had identifiable Human Metabolome Database (HMDB) or Kyoto Encyclopedia of Genes and Genomes , but by 12 h, there was a notable colocalization with the retro-orbital bleed metabolome . In conttabolism . A detaitabolism .χ2, p = 0.06, Next, we examined the metabolomic effects of CLP or sham on the aged animal groups. In contrast to the young animals, sham intervention in the aged animals produced a differentiation from the retro-orbital bleed metabolome that persisted throughout the assayed duration . Similarα-linolenic acid metabolism and linoleic acid metabolism were identified in the young versus aged retro-orbital bleed group and the young CLP-sham comparison; however, on closer examination of these pathway structures, these were not informative and largely incompletely represented by the raw data (data not shown). Thus, detailed statistical analysis on both histidine metabolism and nicotinamide metabolism was performed.Having established key differences between young and aged animals at baseline, as well as critical pathways associated with the response to surgical stress in both young and aged animals, temporal trends within critical pathways across the entire dataset were evaluated. Tabulated outputs from MetaboAnalyst pathway analysis tools were compared, identifying overlapping metabolic pathways among the three comparator groups Figure . Nicotinhttp://www.kegg.jp) were represented in the dataset and are organized according to biochemical relationships in Using the raw data, the elements of the histidine metabolism axis over the time course of the experiment grouped by age and surgical intervention was graphed. A total of 11 metabolites from the annotated KEGG pathway  or surgical stress combined with infectious insult (CLP), underscoring the contribution of aging to metabolomic homeostasis. Furthermore, some metabolic pathways were found through statistical analysis to contribute more to the observed metabolomic alterations than others, highlighting important potential targets for the mitigation of age-associated adverse outcomes following surgical intervention.The experimental design was chosen to provide maximal insight into multiple different metabolic trajectories. This is reflected in the specific analyses reported here, including initial comparison of young vs. aged animals, evaluation of global metabolomic trajectories from baseline in both young and aged animals individually, and in the final evaluation of pathways of overlapping significance between the young and aged animals. Critically, for the evaluation of each of these specific pathways, we utilized a consensus-based methodology combining multiple statistical analysis tools for the identification of key metabolites. This approach was selected to mitigate biases in individual feature identification approaches, and to limit the possibility that any specific methodology  would overfit the data. In particular, the use of the PCA loading scores among the data selection criteria provided an important counterweight to the supervised methods, as PCA loadings are agnostic to group assignment and are only sensitive to the total variance of the system. This allowed for the generation of a metabolite feature list incorporating both the broad structure of the data as well as key group-defining metabolites. This approach generated a robust profile of the broad metabolomic differences for each of the distinct experimental comparisons.In the initial comparison, the baseline differences between young and aged animals from the retro-orbital bleed samples taken at the outset of the experimental protocol were evaluated. The data, summarized in Additionally, these data examined the temporal changes in the metabolome following surgical sepsis (CLP) or sham operation in both young and aged animals. These data provide significant insight into some key differences underpinning differential responses to surgical stress in aged vs. young animals. A key difference is the broad metabolomic landscape shifts that occur in both young and aged animals and how these differ over time. PCA-based analysis and 2-dimensional plotting demonstrated distinct temporal patterns for sham surgical intervention and CLP in both age cohorts. Notably, sham surgery demonstrated a metabolomic shift away from both the retro-orbital bleed controls and the 0 h sacrifice, with an apparent movement back to baseline by the 12-hour timepoint for both young and aged animals Figures . Interesβ-alanine and L-histidine, is an bioactive molecule with pleiotrophic function in different tissue compartments, including antioxidant scavenging, pH buffering, metal chelation, inhibition of advanced glycation end product formation, and inhibition of advanced lipoxidation end product formation  and overlapping pathways of interest were isolated across the three groups Figure . Within ander A. ). These ander A. –25. Howeander A. . In rat ander A. –29. Critander A. , 31 as wander A. , though The other key pathway that emerged from statistical evaluation of the metabolite data involved nicotinamide biology . Here, tThe work presented here, while providing intriguing evidence of circulating age-associated metabolic signatures in a murine CLP model, is not without limitations. The experimental design sought to develop a time course post-CLP that would capture a metabolic shift in the animals, based on telemetry data suggesting a biometric inflection point in the 5-10-hour range following CLP in young C57Bl/6 animals . The PCAIn summary, this manuscript presents a large-scale evaluation of the circulating plasma metabolome in both young and aged mice following CLP or sham surgical interventions. Several key metabolic pathways of interest were identified that differentiate between the young and aged response to surgical insult and subsequent infection providing significant foundational evidence for future study, including both mechanistic evaluation of the aged response to acute disease and potential therapeutic development.


To investigate the effectiveness of pre-procedural oral mefenamic acid compared with placebo in women undergoing Loop Electrosurgical Excision Procedure (LEEP) with intracervical lidocaine injection. A prospective double-blinded randomized control trial. Women undergoing LEEP for any indications were asked to participate in the study. The participants were randomly allocated into two groups. In group 1 , the participants were offered oral mefenamic acid (500 mg) for 30 minutes before procedures. In group 2 (placebo), the patients were given oral placebo  for 30 minutes before operation. All participants received immediate 10 mL of 2% lidocaine with 1:100,000 of epinephrine intracervical injection before undergoing the LEEP. All participants were excised in one piece of LEEP. No top-hat excision in this study. The patients graded their pain on a 10-cm visual analog scale (VAS) at different points during the procedure, including speculum insertion, at starting excision, and 30 minutes post excision. Primary outcomes revealed the difference of VAS during all steps of LEEP by generalized estimating equations procedure. Sixty participants (30 in mefenamic group and 30 in placebo group) participated in this study. The study did not find differences of VAS during all steps of LEEP and analgesic drug requirement at 30 minutes after LEEP procedure. All patients reported no immediate complications and no intervention-related adverse events were observed. Using pre-procedural oral mefenamic acid before LEEP procedure was not associated with pain reduction during all steps of excision. The Loop Electrosurgical Excision Procedure (LEEP) is widely used to treat high-grade cervical intraepithelial neoplasia (CIN2-3) to prevent invasive cervical diseases . These procedures are usually performed in the outpatient setting, during which intracervical submucosal injection of lidocaine is generally performed to reduce pain. The other modalities of anesthesia for pain control during LEEP have been proposed in previous studies such as lidocaine spray, lidocaine gel, and oral analgesic. However, mild to moderate pain from patients who underwent LEEP are currently reported in speculum insertion, excision and post excision periods.To reduce mild to moderate pain, Mefenamic acid, a Nonsteroidal anti-inflammatory drug (NSAIDs), is widely used mostly in postoperative pain control . The actions of Mefenamic acid are achieved by blocking the effect of COX enzymes and decreasing prostaglandin products, which results in reduction of pain and inflammation . Mefenamic acid is rapidly absorbed via oral administration and has half-life of action of two hours . Single dose of 500 mg oral mefenamic acid is commonly used before minor gynecologic procedure such as hysteroscope, fractional and curettage for intraoperative and postoperative pain control and complication avoidance . Due to insufficient data regarding whether adding pre-procedural oral analgesic medication can diminish pain in women undergoing the LEEP procedure, the aim of this study was to compare the effectiveness of pre-procedural additional mefenamic acid with the standard intracervical lidocaine injection for pain control in all steps of the procedure. Setting and populationThis double-blind, parallel-group, randomized controlled trial was conducted at Srinagarind Hospital between July 2019 and February 2020. Women who were diagnosed as abnormal cervical cancer and planned to undergo LEEP procedure at our center were asked to participate in this trial. As for inclusion criteria, the participants were to be 18-60 years of age and able to communicate well in Thai; meanwhile, those who had pregnancy, history of allergy to NSAID or lidocaine injection, peptic ulcer disease, coagulopathy, chronic kidney disease, and pelvic infection were excluded from the study. Participants were withdrawn from the analysis once found to have anaphylaxis signs and symptoms such as exacerbated respiratory symptoms  and mucocutaneous reactions  after receiving the pre-procedural oral medication. Ethical oversight was approved by the Khon Kaen University Ethics Committee for Human Research (HE611536). Written informed consent was obtained from all of the participants. This trial was performed and reported in compliance with the CONSORT statement  and was registered with the Thai Clinical Trial Register (TCTR20190307001). Sample size calculationThe mean ± SD of pain score (VAS) from all steps of LEEP, including speculum insertion, excision, and 30-minute post excision were the primary outcome. Sample size in this study was calculated for analysis of generalized estimating equations based on a study by Vanichtantikul et al., 2013). We hypothesized that a combination of pre-procedural oral mefenamic acid with intracervical lidocaine injection before LEEP (group1) would achieve a higher score of pain reduction in all stages of LEEP procedure compared to that of group receiving placebo (group2). Based on a power of 80%, a type I error of 0.05 and the sample size needed for testing our primary outcome was 60 women, 30 of whom were assigned in each group. Randomization and intervention The participants who met the inclusion criteria were randomly allocated to one of the comparison groups using computer generated block randomization of varying block size. The assigned adjuvant treatment was noted on cards, which were sealed in secure opaque envelopes. The envelopes were then numbered in sequence, kept and opened by an independent researcher in an office outside the hospital. All LEEP procedures were performed by five gynecologic oncologists. The surgeon, participants and investigator involved in this study were blinded to the intervention allocated. Before performing LEEP procedures, participants having been allocated to the intervention group (group1) were given oral mefenamic acid (500 mg) at 30 minutes before LEEP procedures. Meanwhile, the participants having been allocated to the control group (group2) received oral placebo at 30 minutes before LEEP procedure. Oral placebo was prepared with the appearance similar to that of Mefenamic acid. All participants received the same operative excision procedure with local anesthesia under 10 mL of 2% lidocaine with 1:100,000 of epinephrine intracervical injection. All participants were excised in one piece of LEEP. No top-hat excision in this study. To minimize the effects of other variables, oral administration of others analgesic drugs or injected analgesic drugs before excision were not allowed.Outcomes and measurements According to the primary outcomes, VAS was different in all steps of LEEP procedures between two groups. Secondary outcomes were additional analgesic required at 30 minutes after LEEP procedure and adverse events. Pain scores were assessed by 10-cm visual analogue scale from 0 (no pain) to 10 (a maximum level of experienced pain). An investigator asked all individual participants to point their pain score based on a standard 10-cm visual analogue scale. Pain score assessments were recorded before speculum examination, at starting excision and at 30 minutes after LEEP procedure. The participants were instructed to inform nurses or investigators if analgesic drug was required at 30 minutes after LEEP procedure and if single dose of 500 mg oral paracetamol was given. Safety assessments were then recorded, including adverse events and immediate complications within 30 minutes after LEEP.Statistical analyses Statistical analyses were performed using Stata 10 . Descriptive statistics were used to report participants’ baseline characteristics. Student t-test was used to compare continuous variables. In addition, the Chi-square or Fisher-exact test were employed to compare categorical variables. Differences between the comparison groups were measured, including mean difference (MD) or relative risk (RR) with their associated 95% confidence interval (CI). Statistical analysis was conducted using generalized estimating equations. P-value < 0.05 was considered to be clinically significant. All analyses were carried out based on the intention-to-treat principle.Of the study population, 66 women were assessed for eligibility, 60 of whom met the inclusion criteria and were randomly allocated to either the mefenamic group (n = 30) or the control group (N = 30). According to the When subgroup analysis was applied, the size of the loop diameter did not affect the pain score in both groups . MoreoveThe results of this study demonstrated that the administration of pre-procedural Mefenamic acid 30 minutes before LEEP procedure did not result in statistically significant relief pain score in women who underwent LEEP procedures compared with placebo. Moreover, no differences in analgesic drug required at 30 minutes after LEEP procedure were observed. Furthermore, pre-procedural Mefenamic acid before LEEP was considered safe as no intervention-related adverse events took place. Preinvasive cervical cancer especially high-grade lesion are usually treated with excision procedures; thus, most tertiary hospitals performed LEEP excision in the outpatient setting (colposcopy clinic). Moreover, several guidelines recommended the analgesic prior to excisional procedures. Indeed, multiple anesthetic modalities for pain control during LEEP are available such as lidocaine gel, lidocaine spray, intracervical lidocaine injection, paracervical block . Premedical with NSAID can be used adjuvant with anesthetic block for additional pain control  . A previous study reported that premedication with oral Mefenamic acid 500 mg significantly reduced pain in 30 and 60 minutes after hysteroscopy . However, using oral Mefenamic acid 500 mg did not show the significant difference of pain score in fractional and curettage compared with paracervical block . Two small RCTs was used to evaluate oral naproxen before carbodioxide laser vaporization for preinvasive cervical cancer. Al-Kurdi et al., 1985 reported that premedication oral NSAIDs (Naproxen sodium) used in carbondioxide laser treatment of uterine cervix has minimal effect on the pain and causes discomfort during laser treatment. The likely explanation for the failure of naproxen sodium to relieve the pain during laser application may be that transmitting nerves are stimulated directly during applying the laser. To illustrate, naproxen sodium is not a centrally acting analgesic and would not be expected to reduce this sort of pain. However, naproxen sodium appears to have some advantages over placebo in the period following treatment when a response to tissue damage takes place. Therefore, if being routinely given naproxen sodium before laser treatment, patients may be prevented from distress after treatment. Frega et al., (1994) evaluated pain score during carbon dioxide laser vaporization for cervical intraepithelial neoplasia (CIN) among 3 groups; naproxen, placebo and no treatment. It may be argued from the study that analgesia before laser surgery for CIN is not required. In the present study, we reported pre-procedural Mefenamic acid before LEEP and we also found that it was not necessary either to relieve pain in women undergoing LEEP. A Cochrane review conducted by Gajjar et al., (2016) revealed that there was no difference in pain score in women receiving oral analgesics compared with placebo or no treatment during colposcopy treatment and in the postoperative period . The authors suggested that the evidence to be of a low to moderate quality and required further high quality, adequately powered trials to provide the data necessary to estimate the efficacy of oral analgesics, the optimal route of administration and dose of local anesthetics. Similarly, our study confirms that pre-procedural oral Mefenamic acid before LEEP is unlikely to significantly reduce pain during LEEP excision and post LEEP at 30 minutes. Al-Kurdi et al., 1985 reported that patients suffered from some pain during laser treatment particularly from dysmenorrhea, dental drilling and parturition. However, our study indicated that the factors such as the size of loop diameter, parity, menopausal status, cervical cytological and final histological type are not associated with pain score during excision and post LEEP 30 minutes by subgroup analysis. Moreover, in this study, a total of 70% of participants were performed LEEP by using 1 cm of loop diameter if using larger loop, it possible to be showed the different result.The strength of this study is that it was a prospective double-blinded randomized control trial, in which we used placebo prepared in similar appearance as Mefenamic acid. Another strength of this current study was that no participants dropped out from the study. The LEEP excision was performed in the standard technique by the same level of operators. In addition, as for postoperative pain evaluation, we applied analgesic using pain scores in order to address the bias of individual experience pain. Furthermore, this study is the first trial that evaluated the difference of pain scores in each time points and both groups were evaluated using generalized estimating equations. In conclusion, using pre-procedural oral Mefenamic acid before LEEP procedure is unlikely required. Further larger investigation with different approach in higher dose of Mefenamic acid and other pre-procedural oral analgesic administration would be explored to optimize pain during LEEP procedures.