Understanding the precise mechanism of vaccine-induced protection and the immune correlates of protection against coronavirus disease 2019 (COVID-19) is crucially important for developing next-generation vaccines that confer durable and protective immunity against COVID-19. Similar factors are also important for other infectious diseases. Here, I briefly summarize the mechanism of action of the currently used COVID-19 mRNA vaccines from the viewpoint of the function of the lymphatic system. Coronavirus disease 2019 (COVID-19) emerged suddenly in December 2019 in Wuhan, China. It is caused by a novel single-stranded RNA virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) .via the respiratory route, specifically through airborne macro- and micro-droplets (or aerosols) emitted by virus carriers. Two types of cell surface proteins are involved in the SARS-CoV-2 infection of human cells. One is angiotensin-converting enzyme (ACE2), to which the viral spike (S) protein of SARS-CoV-2 binds. It is expressed in varying degrees by a wide range of tissue types  and a receptor-binding domain (RBD), and a membrane-proximal S2 subunit; the S1 subunit binds to the ACE2 on human cells, whereas the S2 subunit is responsible for the fusion of viral and cellular membranes . These sSeveral strategies have been adopted to increase the immunogenicity of the COVID-19 mRNA vaccines –16. FirsBecause of these modifications, the COVID-19 mRNA vaccines are highly immunogenic, inducing strong serum neutralizing antibody responses in most individuals, whereas infection with SARS-CoV-2 produces more variable neutralizing antibody responses ; in bothAnother remarkable feature of the COVID-19 mRNA vaccines is that they can be readily incorporated into the lymphatic system. This is mainly because these vaccines are encapsulated in lipid nanoparticles approximately 100 nm in diameter. Lipids are known to be preferentially transported to the lymphatic system , and par18F]FDG in human volunteers confirmed that prominent vaccine-associated LN swelling occurs selectively in the axillary and supraclavicular LNs of the ipsilateral side in relation to the vaccine injection site (The COVID-19 mRNA vaccines are administered intramuscularly (into the deltoid muscle in most cases), and muscles are rich in both blood vessels and lymphatics. Because these vaccines are preferentially transported into the lymphatics as described above, following their intramuscular injection, they are swiftly delivered to the lymph nodes that drain the muscle. Studies with experimental animals revealed that vaccine transport after intramuscular injection is restricted mainly to the local LNs that drain the injection site and that the vaccine is not readily disseminated systemically –25, unleion site . Anotherion site . These LIn the draining LNs, although most phagocytic cells can internalize the mRNA vaccine, macrophages within the subcapsular sinus and dendritic cells (DCs) within the interfollicular area are the main groups that abundantly take up specific antigens. However, it is mainly DCs that exhibit high levels of translation of the vaccine mRNA and the upregulation of key co-stimulatory molecules (CD80 and CD86) essential for efficient antigen presentation to naïve T cells . These Din situ generated from the vaccine mRNA is degraded in endosome-derived proteasomes, and some of the generated peptides are transported to the endoplasmic reticulum, where they are loaded onto class I major histocompatibility complexes (MHCs). Additionally, part of the S protein is produced and secreted as an exogenous protein by the cells that took up the mRNA. It is then taken up by professional antigen-presenting cells, such as DCs, in which the protein is processed and presented in class II MHCs. Peptide presentation by class II MHCs also occurs on the surface of DCs after their uptake of cell debris containing the vaccine-encoded protein. These peptide-loaded MHC class I and class II molecules on the surface of DCs subsequently present their antigens to antigen-specific CD4+ and CD8+ T cells, respectively  of disease in some instances . Indeed,in vitro . BecauseIn addition to the potential ADE issue, one major problem with the current COVID-19 mRNA vaccines, although they are highly effective at reducing the incidence and severity of SARS-CoV-2 infection, is the waning of their efficacy with increasing time since the second dose , 55. HowWhile inducing durable immunity against SARS-CoV-2 does not appear to be an easy task, there may be at least a couple of ways of making the COVID-19 vaccine response more durable. One is to enhance the innate immune mechanism through which mRNA vaccines stimulate protective antibody responses. The other is to enhance the adaptive immune mechanism, particularly to enhance T-cell memory cell responses. Regarding the former, using other types of lipid particles or adding an appropriate adjuvant to the mRNA vaccines is a possibility, but this approach carries the possibility of increasing the incidence of adverse reactions as well. Regarding the latter, attempting to stimulate T cells with not only the S protein but also the matrix (M) protein and nucleocapsid (N) protein is also a possibility . The incWhile the current COVID-19 mRNA vaccines are highly protective against the development of severe disease, they elicit limited immune responses in the respiratory tract . VaccineCOVID-19 vaccine development is moving forward at unprecedented speed, but several challenges remain. For example, the current COVID-19 mRNA vaccines are less effective at blocking infection by the newly arising SARS-CoV-2 variants, although protection against severe disease remains well preserved. Additionally, only relatively weak immune responses are induced in the mucosa of many vaccine recipients, and even when such responses are successfully induced, they tend to wane rapidly. Furthermore, the virus continues evolving ways to evade our immune response. Thus, there is an urgent need to develop pan-coronavirus vaccines that can target not only the current SARS-CoV-2 variants but also future variants. The development of mucosal vaccines that are delivered across mucosal barriers may also present a promising strategy to promote durable protection against SARS-CoV-2 at the mucosal level.The author confirms being the sole contributor of this work and has approved it for publication.https://jp.edanz.com/ac) for editing a draft of this manuscript.I thank Katie Oakley, PhD, from Edanz (The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Esophageal carcinoma (EC) is a common malignant cancer worldwide. Esophageal squamous cell carcinoma (ESCC), the main type of EC, is difficult to treat because of the widespread morbidity, high fatality rates, and low quality of life caused by postoperative complications and no specific molecular target. In this study, we screened genes to establish a prognostic model for ESCC. The transcriptome expression profiles of 81 ESCC tissues and 340 normal esophageal mucosal epithelium tissues were obtained from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) cohorts. The transcriptome expression datasets of 19 esophageal squamous carcinoma cell lines were downloaded from Cancer Cell Line Encyclopedia (CCLE). The R software Limma package was used to identify 6,231 differentially expressed genes and 647 differentially expressed immune-related genes between normal and ESCC tissues. Gene functional analysis was performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Weighted gene co-expression network analysis (WGCNA) was used to screen out 18 immune-related prognostic genes. We then established the prognostic and risk signature using these genes, and the patients were divided into low-risk and high-risk groups. Compared with high-risk group patients, the low-risk group patients had longer overall survival. M1 macrophages and resting dendritic cells were differentially distributed between the low-risk and high-risk groups and were related to patient survival. We also examined the functional immune cell and immune molecule levels in low-risk and high-risk group patients, with significant differences in the tumor microenvironment between the two groups. To further verify the accuracy of the prognostic risk model, we performed area under the ROC curve (AUC) analysis. The AUC value was 0.931 for the prognostic risk, which was better than the microsatellite instability (MSI) and Tumor Immune Dysfunction and Exclusion (TIDE) scores. In conclusion, we found 18 immune-related prognostic genes related to the occurrence of ESCC and established a prognostic model for predicting disease severity. Esophageal carcinoma (EC) is a common malignant cancer worldwide. In 2018, about half of all new or fatal global cases of EC originated in China. Esophageal squamous cell carcinoma (ESCC), the main type of EC, is more difficult to treat because it is frequently diagnosed at a late stage . At presFor EC patients, especially those with advanced disease, it is of practical significance to better distinguish patients who will potentially respond to immunotherapy. The immune-related gene prognostic index is a recently emerging algorithm that calculates the score of each patient based on the expression levels of related genes. It can be used to predict the patient’s immunotherapy response and prognostic risk index . In thishttps://portal.gdc.cancer.gov/). The 340 normal esophageal mucosal epithelium samples were obtained from GTEx cohorts . The transcriptome expression datasets of 19 esophageal squamous carcinoma cell lines  were downloaded from CCLE (http://www.broadinstitute.org/ccle/home). Using R software to integrate TCGA and GTEx data, 274 normal esophageal mucosa samples and 81 ESCC samples were selected for further analysis. The immune-related gene list was downloaded from the ImmPort dataset (https://immport.org/home).The transcriptome expression profiles of 81 ESCC tissues were downloaded from TCGA and the corresponding clinical information  and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis results are shown in circular maps . The GenThe WGCNA package in R was used for the cluster analysis of immune-related differentially expressed genes and screening of prognostic differentially expressed genes. Kaplan-Meier (KM) survival curve analysis was used to further verify the accuracy of the impact of these genes on prognosis .p-value < 0.05 was a selective condition, and 73 ESCC samples were obtained for further analysis  was used to calculate the predictive ability of the ESCC prognosis model and the COX regression analysis was used to calculate the risk coefficients of ESCC patients. The effect was compared with conventional indexes .2 at 37°C.Human ESCC cell lines  and normal human esophageal epithelial cells (HEEC) were provided by cardiovascular thoracic surgery department of Tianjin Medical University General Hospital. Cells were cultured with RPMI-1640 containing 10% Fetal Bovine Serum (FBS). All cells were maintained in a humidified chamber containing 5% CO−ΔΔCT method.Total RNA of cultured cells was isolated with RNAiso Plus (TaKaRa) and reverse-transcribed to cDNA with PrimeScript Strand cDNA Synthesis Kit (TaKaRa). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed with TB Green Premix Ex Taq II (TaKaRa). The primer sequences are shown in The flow chart of the study is shown in Next, we downloaded the list of 6,196 immune-related genes from the ImmPort dataset and screened for any differentially expressed genes among them. There were 647 differentially expressed immune-related genes, with 418 upregulated and 229 downregulated in ESCC tissues relative to normal tissues. The differentially expressed genes are shown in Immune reactions and viral infections may be the main factors that maintain the malignant state of ESCC. GO and KEGG analyses were performed on the differentially expressed immune-related genes, with the results showing that the first eight most significant GO terms enriched were all related to immune response. Additionally, three of the first eight KEGG terms enriched were related to immune response and three were related to viral infection. The enriched KEGG terms, B cell receptor signaling pathway, cytokine-cytokine receptor interaction, and chemokine signaling pathway, were consistent with the enriched GO terms, such as immune response-activating cell surface receptor signaling pathway, immune response-activating signal transduction, and regulation of immune effector process .RELB, HNRNPL, ITCH, ILF3, PSMC4, CBL, SKP2, DHX33, PRKDC, ZMYND11, and CCDC88A) and seven that were related to poor prognosis   , 4A–R.PSMD6, RABEP2, GRN, STC2, ITCH, ILF3, and PSMC4). The expression levels of PSMD6, RABEP2, GRN, and STC2 were related to a poor ESCC prognosis, while the expression levels of ITCH, ILF3, and PSMC4 were related to a good ESCC prognosis. According to the prognostic risk model, we divided the training and test groups into high-risk and low-risk subgroups. For the training group, patients in the low-risk group had better survival rates, with similar results in the test group  scores of the high-risk and low-risk groups to further analyze the difference of immune status between the two groups. The results showed that the TIDE scores were higher in the high-risk group than in the low-risk group . The dysWe analyzed the relative mRNA expression levels of seven prognostic genes in the normal human esophageal epithelial cell (HEEC) and two ESCC cell lines  by qRT-PCR. As shown in With the emergence of immune checkpoint inhibitor therapies, the treatment options for cancer patients have significantly increased. For ESCC patients, the results of the KEYNOTE-181 clinical trial study highlighted the outstanding achievements of immunotherapy . For patPSMD6, RABEP2, GRN, STC2, ITCH, ILF3, and PSMC4). PSMD6 encodes a member of the protease subunit 26S family that is part of a multicatalytic proteinase complex with a highly ordered structure composed of two complexes, a 20S core, and a 19S regulator. PSMD6 colocalizes with DNA damage foci and is involved in the ATP-dependent degradation of ubiquitinated proteins, and PSMC4 is involved in the non-ATPase subunits of the 19S regulator lid  that are mainly used to control the initiation of T cell-dependent immune responses . Restingin vivo and in vitro (As a plastic and pluripotent cell population, macrophages exhibit significant functional differences under the influence of different microenvironments in vitro . Accordiin vitro . M1 typein vitro . Our resThe TIDE score is an advanced method to predict the sensitivity of tumors to immune checkpoint treatment by using gene expression information. The TIDE score calculated by the software consists of two parts: dysfunction score and exclusion score. The calculation principle of the dysfunction score is that genes with immune disorders have a higher weight, and it can be obtained by multiplying by the expression level. The exclusion score is obtained by multiplying the weight of immune rejection genes by the expression level . The TIDMSI refers to the phenomenon that new microsatellite alleles appear in the tumor from a change to the microsatellite length caused by the insertion or deletion of a repeat unit . MSI hasHere, we found 18 immune-related prognostic genes related to the occurrence of ESCC and used several of them to establish a prognostic model for predicting disease severity.


C. elegans hermaphrodite germline, and how this redundancy may contribute to robustness. We focus on the various types of redundancy utilized to regulate this balance, as well as the approaches that have enabled these redundant mechanisms to be uncovered.The proper production of gametes over an extended portion of the life of an organism is essential for a high level of fitness. The balance between germline stem cell (GSC) proliferation  and differentiation (production of gametes) must be tightly regulated to ensure proper gamete production and overall fitness. Therefore, organisms have evolved robust regulatory systems to control this balance. Here we discuss the redundancy in the regulatory system that controls the proliferation vs. differentiation balance in the  C. elegans germline to ensure proper regulation of the balance between germline stem cell (GSC) proliferation and differentiation.Proper development of multicellular organisms requires the ability to adapt to both internal and external perturbations. The ability of an organism to adapt to such changes requires its biological processes to be robust, meaning they are able to continue to develop and function, even when perturbations are encountered . This roStem cells are necessary for proper development and tissue homeostasis; therefore, the processes regulating their behavior must be robust. Stem cells have the ability of maintaining themselves over much of the life of the animal, while also providing the cells that form the differentiated tissue . For steThe reproductive success of animals within a population is key to that population being maintained; therefore, the processes involved in ensuring proper gamete formation must be robust. This includes the balance between proliferation and differentiation of GSCs. If this balance is disrupted, or is unable to withstand various environmental insults, the reproductive success of the animal would be significantly reduced; too much self-renewal would result in germline tumors of proliferating cells and a reduction or elimination of differentiated gametes that are formed, while too much differentiation would result in the GSC population being depleted and a lack of sustained gamete formation.C. elegans germline has revealed a core genetic pathway that contains many redundant factors, and many additional inputs that appear to fine-tune the balance. This regulatory mechanism has been extensively reviewed elsewhere , which is a somatic cell that caps the distal end of the gonad arm and serves as the niche cell for the GSC stem cell population (sygl-1 (synthetic germline proliferation defective) and lst-1  genes (fem-3 mRNA binding factor) and other PUF (Pumilio and FBF) homologues (PROM−1 (skp1-cullin-F-box protein progression of meiosis-1) pathways, based on their founding members  genes  (Kershnet) genes , whose pmologues . This PUmologues . These p members .PROM−1 pathways to increase  of a loss of the redundant effectors, but also the mechanism by which each effector functions. With many of the examples of redundancy in regulating the proliferation vs differentiation balance in the C. elegans germline has proven to be a powerful system to uncover redundant factors and mechanisms due, in part, to the availability of sensitive genetic backgrounds and the ease with which subtle phenotypes can be observed. C. elegans are transparent, allowing observation of the germline in living animals [reviewed in  gain-of-function allele, perhaps the strongest identified glp-1 gain-of-function allele, and which also contain an additional wild-type copy of glp-1 on a free duplication, have completely tumorous germlines with no evidence of cells entering meiosis , all stem cells differentiate prematurely in early larval development, resulting in a total of approximately four to eight stem cells, which differentiate into 16–32 sperm  and glp-1(q175null) result in these extreme opposite phenotypes, there are many other alleles of glp-1, including weak gain-of-function alleles, and partial loss-of-function alleles, which result in milder phenotypes, especially at permissive temperatures (glp-1(oz264gf) or glp-1(ar202gf) at higher temperatures result in increased proliferation of the mitotic cells and a delay of entry into the differentiation pathway, resulting in a larger than wild-type progenitor zone , have stem cells entering the differentiation pathway more distally in the gonad arm, resulting in a smaller progenitor zone than wild-type  as potentially being involved in regulating the proliferation vs. differentiation balance whose involvement have been characterized by analyzing the Glp and tumorous phenotypes that result when combined with weak glp-1 loss and gain-of-function alleles [Reviewed in , function downstream of GLP-1/Notch signaling within the C. elegans germline   . The higof fem-3 . No singfbf RNAi , supportfbf-1 and fbf-2 identified an additional role for fbf in regulating the balance between proliferation and differentiation of GSCs  mutants as the progenitor zone did not become fully depleted until later in larval development, and depletion did not fully occur at higher temperatures , a catalytic subunit of the CCR4-NOT (carbon catabolite repressor factor 4—negative on TATA-less) deadenylation complex   . Localizality 1) . Therefoality 1) . Althougality 1) . These vality 1)  for discality 1) .fbf-1 and fbf-2 within the C. elegans germline suggested that these genes displayed molecular redundancy, with their highly conserved PUF domains allowing them to recognize and repress the same mRNA targets; however, more recent evidence of differing binding partners and localization, combined with their single mutant phenotypes, has uncovered that these two genes likely function using distinct mechanisms to repress target gene expression    works redundantly with fbfs to robustly promote GSC proliferation. PUF-11 was identified as a strong candidate as it displays similar RNA-binding specificity to FBF and interacts with LST-1 in a large scale yeast two-hybrid screen (Although the complete loss of the GSC pool in adult enotype) . In the /animal) . The inad screen . PUF-3, d screen .puf-11 and puf-3, have ∼90% nucleotide identity with each other, and 88% identity at the amino acid level  mutants , based on mRNA in situ data showing expression within the GSC pool  knockdown, resulted in a Glp phenotype during larval development  normally and result in over-proliferation of the mitotic cells, with most cells failing to enter meiosis . Its placement in this pathway is based on the finding that gld-2; nos-3 double mutants show synthetic tumors while gld-1; nos-3 double mutants have normal meiotic entry )  that functions in parallel to the GLD-1 and GLD-2 pathways to inhibit proliferation and/or promote meiotic entry. First, the synthetic tumorous germlines in ic cells . Second,p-1(gf)) . Finally mutants . Therefoic entry .PROM−1 was found to bind CYE-1, potentially targeting it for degradation  that functions redundantly with the GLD-1, GLD-2 and SCFPROM−1 pathways to promote meiotic entry and/or inhibit proliferation  E3 ubiquitin-ligase complex which includes the PROM-1 F-box protein. SCFradation . Loss ofpathways . Further mutants . Howeverferation .PROM−1 pathways display cellular process redundancy in their regulation of the proliferation vs differentiation decision in the C. elegans germline ) and prp-17 (Yeast PRP17 related splicing factor) were identified , cyn-4/mog-6 and prp-19  mutants , which encodes an ⍺-subunit of the 20S proteasome  was found to be one of these proliferation promoting proteins, and is targeted for degradation by the E3 ubiquitin ligase RFP-1 (ring finger protein) (glp-1(gf) background, suggesting that there could be multiple targets whose regulated degradation is necessary for the proliferation vs differentiation decision to be properly maintained  . Other fintained . As mentintained .glp-1 partial loss-of-function alleles, or enhance the tumorous phenotype of glp-1 gain-of-function alleles [reviewed in , which could impact over-proliferation and under-proliferation phenotypes. Additionally, the type and degree of redundancy may not be constant throughout development and ageing. For example, it is possible that some factors are redundant in early larval development as the progenitor zone is being established, but not in adulthood while it is being maintained.The genetic pathway regulating the proliferation vs. differentiation decision in the Presumably, even slight disruptions in the proliferation vs. differentiation balance would negatively impact long-term reproductive success. Therefore, there is likely significant evolutionary pressure ensuring that the system regulating the proliferation vs differentiation balance is robust, which could involve a significant level of redundancy. It is thought that redundancy can provide robustness to a system by decreasing the phenotypic consequence of mutations, or by broadening the flexibility of the system so that signals can still be properly controlled in fluctuating cellular environments, different genetic backgrounds, and environmental stresses such as changes in temperature and availability of food . TherefoC. elegans germline. In some cases, the initial discovery of redundant factors and their presence or lack of similarity suggested that they were an example of one class of redundancy. However, for some redundant factors, as understanding increased as to their modes of action and their potential targets, it actually became less clear as to which class of redundancy most accurately described their relationship. Furthermore, some redundant factors may exist in more than one class; for example, two redundant proteins with a high degree of amino acid sequence identity may function on a group of common targets, but may also have targets that are unique to each redundant factor. Conclusive classification of redundant factors requires complete understanding of the biochemical functions of the factors and a knowledge of all their targets. Obviously, we are not yet at that level of understanding with respect to the regulation of the proliferation vs differentiation decision in the C. elegans germline. However, perhaps our level of understanding of the biochemical functions and targets involved in this process is relatively high, which has revealed that the redundant relationships between factors are more complex than previously appreciated. Indeed, the relationships between redundant factors in other systems may also be found to be more complex than once thought as more is learned about the biochemical functions and targets of these redundant factors. However, what is clear is that many types of redundancy are utilized in regulating this proliferation vs. differentiation balance in the C. elegans germline, and that this redundancy likely contributes to its reproductive success and fitness.In this review we have discussed some of the different types of redundancy, highlighted with specific examples, that are utilized in regulating the proliferation vs differentiation decision in the


On March 11, 2020, the novel coronavirus disease (COVID-19) was declared a pandemic. Since the declaration, countries have implemented response measures to stop the spread of the virus, while multiple vaccines combatting the virus have also been developed. However, vaccine rollout and providing vaccine access has been very challenging in many African countries, including The Gambia. This article briefly assesses the efforts and challenges facing The Gambia´s COVID-19 vaccine rollout and implementation. The article also provides recommendations that policymakers and program implementers can use to address the low COVID-19 vaccination rate in The Gambia. It is based on a narrative review of existing literature on COVID-19 vaccination efforts and challenges in The Gambia. COVID-19 vaccination efforts in The Gambia: the first case of COVID-19 in The Gambia was confirmed on March 17, 2021, and as of February 28, 2022, the total number of COVID-19 cases reached nearly 12,000 with 365 total deaths from the disease [ disease . Since t disease . Howeverth February 2022.The Gambia initially proposed goals of vaccinating 40% of its population by December 2021 and implemented mobile vaccinations campaigns in the summer of 2021 which increased vaccination rates in that timeframe. Furthermore, house-to-house outreach campaigns between December 2021 and January 2022 improved the population reached by vaccination campaigns when compared to the fixed-site strategy used since March 2021 when vaccines initially reached The Gambia . In JanuChallenges of the COVID-19 vaccination rollout in The Gambia: a major challenge in The Gambia´s vaccination efforts against COVID-19 has been finding a balance between mobile outreach campaigns and fixed-site vaccination options. While outreach campaigns have increased demand transiently, maintaining a consistent level of vaccination rates has proven difficult for The Gambia. Furthermore, while projects such as the IOM campaigns targeting mobile at-risk groups present a step in the right direction, the continued low vaccination rate increases the risk for virus transmission and novel, more-transmissive variant emergence. In addition, although COVID-19 vaccinations are available in The Gambia, challenges such as misconceptions about and lack of trust in COVID-19 vaccines, lack of a robust electronic database to track vaccinated individuals, limited vaccination points and staffing shortages may also be significantly affecting the efficiency of the COVID-19 vaccine rollout in The Gambia [e Gambia . Of these Gambia .While the study did not detail The Gambia, it studied the neighboring Senegal, finding that 50% of surveyed Senegalese citizens agreed with the statement that the coronavirus threat was exaggerated, while 32% of Senegalese surveyed reported that they would not be willing to receive a COVID-19 vaccine. Furthermore, only 29% of Senegalese respondents strongly agreed that a COVID-19 vaccine was safe, with 23% of respondents strongly disagreeing that a COVID-19 vaccine was effective . While tFuture directions for COVID-19 vaccinations in The Gambia: a variety of steps are needed to increase the vaccination rates against COVID-19 in The Gambia. For instance, considering The Gambia has only received enough vaccines to protect a small subset of the population, increasing vaccine availability through more shipments and securing more vaccines through initiatives such as COVAX would aid in getting more Gambians vaccinated.Furthermore, increasing mobile outreach and house-to-house campaigns encouraging vaccination could reinvigorate the rate of COVID-19 vaccination in The Gambia. Following the lead of other African countries with more successful vaccine rollouts could also pay dividends. For example, Ghana has made use of drones to reach communities situated far from distribution centers, while Botswana made use of emergency operation centers to coordinate vaccine transport. Training vaccinators and ensuring that sufficient support staff is present for either fixed-site centers or mobile initiatives would also increase the ability to use available doses efficiently. Fighting vaccine misinformation may also play a pivotal role in increasing The Gambia´s COVID-19 vaccination rate. On this issue, The Gambia could turn to programs in other African countries and try to emulate them. In Ghana, for instance, a misinformation and rumor management task force has been established by the Ghanaian authorities to address false claims on COVID-19. In Senegal, the free call centers aim to provide fact-based information to citizens to alleviate hesitancy. To adequately track vaccinated individuals, a system is needed to digitize data and verify vaccination. As of now, increasing vaccine access and availability through mobile outreach seems to be a successful path to pursue ,8, whileConclusion: to mitigate the challenges of COVID-19 vaccine implementation and rollout in The Gambia, the development of a robust digital system to effectively track vaccinated persons is urgently needed. Additionally, increasing mobile outreach campaigns as well as door-to-door vaccination campaigns could benefit dwindling vaccination rates. Ensuring proper staffing and training for medical and allied health professionals on these campaigns will be crucial, as will finding a way to address vaccine hesitancy and mistrust. Lastly, The Gambia has shown an ability to use a high percentage of available doses, suggesting increasing dose availability may help vaccinate more Gambians.


This review brings attention to a crucial yet under-investigated subject which is vitamin–drug interactions. Fat-soluble vitamins such as vitamins A, D, E, and K have been proven to possess many beneficial effects in the treatment and prevention of COVID-19. Moreover, it has been reported that deficiencies of fat-soluble vitamins have been directly linked to COVID-19 infection-related morbidity and mortality, especially in high-risk populations such as cancer and autoimmune patients. However, many vitamin–drug interactions exist between most of the newly COVID-19 FDA-approved medications and fat-soluble vitamins. Hence, this mandates personalizing the COVID-19 treatment protocols, especially for patients who have any deficiency in any of these vital vitamins. Weighing the risk-to-benefit ratio of supplementing any of these fat-soluble vitamins with COVID-19 medications is considered crucial to maximize the therapeutic benefit and decrease the side effects of these drugs. COVID-19 is a recent pandemic that mandated the scientific society to provide effective evidence-based therapeutic approaches for the prevention and treatment for such a global threat, especially to those patients who hold a higher risk of infection and complications, such as patients with autoimmune diseases and cancer. Recent research has examined the role of various fat-soluble vitamins  in reducing the severity of COVID-19 infection. Studies showed that deficiency in fat-soluble vitamins abrogates the immune system, thus rendering individuals more susceptible to COVID-19 infection. Moreover, another line of evidence showed that supplementation of fat-soluble vitamins during the course of infection enhances the viral clearance episode by promoting an adequate immune response. However, more thorough research is needed to define the adequate use of vitamin supplements in cancer and autoimmune patients infected with COVID-19. Moreover, it is crucial to highlight the vitamin–drug interactions of the COVID-19 therapeutic modalities and fat-soluble vitamins. With an emphasis on cancer and autoimmune patients, the current review aims to clarify the role of fat-soluble vitamins in SARS-CoV-2 infection and to estimate the risk-to-benefit ratio of a fat-soluble supplement administered to patients taking FDA-approved COVID-19 medications such as antivirals, anti-inflammatory, receptor blockers, and monoclonal antibodies. It was announced that the global pandemic COVID-19 was caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In December 2019, Wuhan city in China reported the very first reported cases of this virus [The relationship between autoimmunity and cancer results from a shared immune system dysregulation, which subsequently raises the risk of infections and other consequences ,3,4. TheRecent research has looked at how several fat-soluble vitamins can reduce the overall severity of COVID-19 infection. The total risk of morbidity and mortality in SARS-CoV-2-infected individuals was found to be reduced as a result of strong evidence that vitamins A, D, E, and K hold a significant role in boosting the defensive mechanism and reducing the cytokine storm and other inflammatory reactions . It is aNonetheless, several studies revealed a link between vulnerability to infection and worse prognosis in COVID-19 patients with deficiencies in one or more of these fat-soluble vitamins . VariousThe authors aimed at exploring the effects of fat-soluble vitamins in cancer and autoimmune diseases and their link to COVID-19 infection, as well as the vitamin–drug interaction in COVID-19 infection. The authors screened the National Library of Medicine (PubMed). To search databases, the descriptors or keywords used were: “SARS-CoV-2”, “COVID-19”, “Vitamin A”, “Vitamin D”, “Vitamin E”, “Vitamin K”, “Cancer”, “Autoimmune Disease”, “Drug-Drug Interactions”, “Vitamin-Drug Interactions”, “Clinical Trials”, “Systemic Lupus Erythematosus”, “Multiple Sclerosis”, “Rheumatoid Arthritis”, and “Fat-soluble Vitamins”, to cover as many articles as possible in the literature. Relevant publications with detailed information were included includingresearch articles, review articles and book chapters were evaluated and summarized in order to fulfill the purpose of this review article. A summary of the search strategy from initial search, data retrieval, and screening for eligible studies to the final included studies is illustrated in Cancer patients are enlisted as a high-risk group in the population by the WHO, due to their susceptibility to complications due to their immune-compromised status . HoweverReduced T cell counts were considered a characteristic of late-stage cancer patients infected with the virus. Longer prothrombin time, elevated D-dimer and C-reactive protein levels also enhance the risk and mortality of cancer patients when encountering COVID-19 infection ,19. MoreAutoimmune diseases are another class of diseases that have been on the rise and still require deep investigations, due to the comorbidities and mortalities associated with this subclass of diseases ,22. RheuFurther studies emphasizing the link between COVID-19 and autoimmune diseases prognosis is much needed due to the complex heterogeneity of these diseases and the questionable effects of COVID-19 . MoreoveThere are significant global efforts underway to identify suitable therapeutic modalities to treat COVID-19 infection. One of the quick-acting strategies was drug repurposing, although it had the disadvantage of having poor selectivity and limited value within virus families. Examples of drugs that were repurposed in COVID-19 therapy are listed in The WHO launched many interventional clinical trials of several drugs including antivirals, anticoagulants, anti-inflammatory and some other adjuvant drugs. Remdesivir, an inhibitor of RNA-dependent RNA polymerase, was the first medication to receive FDA approval for use in COVID-19 treatment, especially in emergency circumstances. Later, numerous clinical trials showed that remdesivir inhibits SARS-CoV-2 both in vitro and in vivo . MoreoveThe pulmonary microvascular thrombosis caused by SARS-CoV-2 is thought to be the primary factor causing acute lung injury. Controversial studies exist concerning the use of heparin as an anticoagulant agent for thrombo-prophylaxis against COVID19-associated coagulopathy. This was further supported by a retrospective study, which found that heparin treatment reduced the mortality risk in COVID-19 patients . On the Some other approaches are under clinical investigation to measure their clinical benefit in COVID-19 patients including immunotherapy such as neutralizing monoclonal antibodies and intravenous immunoglobulins. Due to their capacity to regulate the immune response and cytokine storm linked to severe COVID-19, monoclonal antibodies have drawn significant attention in clinical research. One of the main cytokines linked to COVID-19 acute inflammation and the cytokine storm is the interleukin, IL-6.A decreased mortality rate in COVID-19 hospitalized patients was reported to be associated with the utilization of monoclonal antibody against IL-6 such as tocilizumab . TocilizFurthermore, monoclonal antibodies against the viral proteins also represent a great promise in COVID-19 treatment. For instance, monoclonal antibodies against the S protein of the virus, an essential protein in viral entry and initiation of host immune responses, block the interaction with ACE2 and consequently prevent viral entry . RecentlAnother immunotherapeutic approach is the use of intravenous immunoglobulin (IVIg) that was used to treat many inflammatory and autoimmune disorders, which includes immunoglobulin G (IgG) obtained from the plasma of healthy donors. Such therapeutic approach could be implemented for the treatment of COVID-19. Although the precise mechanism of how this approach would help to reduce the cytokine storm associated with COVID-19 remains unknown, studies showed that critically ill COVID-19 patients recovered when subjected to high-dose injection of IVIg . AdditioSeveral reputable institutions formed specific guidelines that are continuously updated aiming to reduce the negative outcomes and lethality of cancer during the pandemic, such as ESMO and the NCCN . ResuminFor stable cancer patients, delaying chemotherapy and surgical interventions is recommended, specifically due to the increased risk of nosocomial transmission through hospitals . For patSLE patients are generally more susceptible to viral infections and the complications associated with the diseases such as diabetes, hypertension, and obesity, which places them at a stressful position during the pandemic, where their hospitalization and mortality rates exceeded those of healthy individuals . This isImmunosuppressive drugs dedicated for SLE patients were not contraindicated during the pandemic, according to the American College for Rheumatology (ACR), as they did not appear to affect either the COVID-19 infection cycle or the symptoms . HoweverMS is another important autoimmune-related neurodegenerative disease with several clinical manifestations that unfortunately result in several varying disabilities in patients . The genTreatment of MS by disease-modifying treatments (DMTs) has been proven to be a double-edged weapon for patients during this pandemic . Amor etAn individualized approach should be tailored to each MS patients when being infected with COVID-19 to ensure that highest therapeutic output with least risk of adverse events. MS patients under immunosuppressive agents might have altered and compromised immune system rendering them good candidates to receive antivirals such as monoclonal antibodies against SARS-CoV-2 virus. Generally, RA has also been linked to risk of contracting infections and other accompanying disorders, yet the exact role of COVID-19 in aggravating RA is still debatable due to the common effects induced by both . HoweverACR, National Institute for Health and Care Excellence (NICE), and the Australian Rheumatology Association have provided in-depth analyses on the usage of anti-RA medications and endorsed the use of non-steroidal anti-inflammatory drugs (NSAIDs) in the chronic treatment of RA . In addiAntiviral therapies such as monoclonal antibodies against SARS-CoV-2 are considered the treatment of choice to RA patients as their immune system is compromised due to co-administration of immune suppressors. Among those monoclonal antibodies are Casirivimab and imdevimab . HoweverRecently nutrients have emerged as the panacea because of their efficacy to treat or prevent disease and exceedingly low risk levels ,93. ThusIt has long been known that vitamin A possess immunomodulatory roles and is essential for the appropriate performance of the immune system . For exaThe impact of vitamin D on COVID-19 has been examined in a number of reviews and clinical trials, where vitamin D supplements were reported to lower the risk of infection and could be taken in higher doses in COVID-19 infection . The maiVitamin K2 and the maintenance of serum 25-hydroxyvitamin D levels have been recently linked to death in COVID-19 patients, as reported by Goddek S., where there was an association of the vitamin levels with calcium deposition in bones, thus increasing its nutritional potential . A previA diminished activation of endothelial protein S and presence of other hepatic procoagulant factors and the matrix Gla protein (MGP), was linked to anti-thrombotic activity and vitamin K insufficiency . A novelSupplementation of Vitamin E along with vitamins C and D has been stated to be critical to the well-being of individuals as well as those infected with COVID-19 . This goOn the other hand, possible preventive intake of vitamin E was shown to provide beneficial effects on cancer patients due to the positive impacts associated with reduced tumorigenesis in animal models . MoreoveThe main goal of this review is to evaluate the risk-to-benefit ratio of vitamin supplementation to COVID-19 patients as part of their treatment plan. That is why it is essential to highlight any potential drug interactions between the approved COVID-19 treatment modalities and the four different fat-soluble vitamins.As mentioned earlier, vitamin A was identified to exhibit a potential therapeutic effect in COVID-19 patients, where it decreased the incidence of infection and shortened the duration of the disease . PreviouInterferon type 1A was a drug that has been postulated to benefit COVID-19 patients among many other viral infections. Vitamin A was known to carry a beneficial role through mounting the therapeutic efficacy of type 1 interferon . AnotherGiven that vitamin D may affect the metabolism of many medications that depend on CYP3A4 activation while the CYP3A4 gene contains a vitamin D response element, multiple studies into the vitamin D–drug interactions was deemed essential ,201. GivPreviously, the antimalarial drug hydroxychloroquine was used to treat autoimmune diseases such as SLE. Patients receiving hydroxychloroquine had significantly increased levels of vitamin D compared to those who are not taking it . A studyA high mortality rate in COVID-19 infection was significantly associated with the impaired coagulation process. The recent guidelines of COVID-19 treatment recommended that anticoagulants and antiplatelets should not be used in non-hospitalized patients . HoweverAlthough vitamin E is well known for its antioxidant properties, a study found a link between excessive vitamin E intake and an increased risk of gastrointestinal cancer, heart failure, and overall cause of death . MoreoveP-glycoprotein (P-gp), or multidrug resistance protein 1 (MDR1), is known to increase the efflux of some drugs limiting the intestinal absorption and bioavailability of these drugs. According to several in vitro studies, vitamin E at high concentrations may potentially enhance the activity of P-gp . LopinavNo data from in vivo studies have been published to support the fact that vitamin E might affect intestinal drug metabolism. In guinea pigs administered with vitamin E for six weeks, the expression of the CYP3A4 protein was found to be unaltered . LikewisIt is also worth mentioning that vitamin E is known to alter the metabolism of vitamin K as they are metabolized through the same pathway ,227. In Despite the immense efforts carried out by scientists for the discovery of new therapeutic modalities for treating COVID-19, and the evidence-based beneficial role of fat-soluble vitamins in preventing the cytokine storm associated with the severity of COVID-19, little is still known about the vitamin–drug interactions of fat-soluble vitamins and newly FDA-approved COVID-19 medications. Additionally, the pharmacodynamic and pharmacokinetic changes caused by these vitamins might alter the metabolism of COVID-19 therapeutic drugs. Such interactions could have negative consequences and hinder patients from taking the full therapeutic benefit out of the new modalities, particularly immunocompromised patients such as cancer and autoimmune patients. Thus, this review highlighted the critical vitamin–drug interactions between fat-soluble vitamins and the FDA-approved COVID-19 treatments and focused on personalized COVID-19 treatment protocols for cancer and autoimmune patients. This will help weigh the risks and benefits when tailoring personalized management protocols to such high-risk patients. Nevertheless, further research must be carried out to decipher the possible vitamin–drug interactions to maximize the benefits of these vitamins and prevent negative adverse events. Such information would be of great use by clinicians who will apply the concept of personalized medicine and implement personalized protocols and guidelines for preventing vitamin–drug interactions, particularly for high-risk patients such as autoimmune and cancer patients.https://clinicaltrials.gov/ct2/results?term=vitamins&cond=COVID-19 (accessed on 15 October 2022). Only one clinical trial (NCT04709744) focused on the impact of vitamin D supplementation on SLE patients during the COVID-19 pandemic. However, to date, no results have been posted or published from this clinical trial. Therefore, more clinical trials are needed to study the vitamin–drug interactions in order to draw a firm conclusion on the use of these vitamins during the course of COVID-19 infection. Additionally, studies are needed to study the drug–drug interactions between chronic medicines given to cancer/autoimmune patients and novel COVID-19 therapeutic modalities. This is in addition to exploring the prophylactic roles of these fat-soluble vitamins in COVID-19 infection, especially in immunocompromised patients. Furthermore, clinical studies should address the interactions between the forementioned vitamins in COVID-19-vaccinated individuals and specifically autoimmune and cancer patients.Currently, there are more than 200 clinical trials investigating the supplementation of vitamins in COVID-19 infection (


Current methods for the detection of brain malignancies often display low sensitivity and specificity. Noninvasive biomarkers can complement imaging techniques to improve the diagnosis of these tumors. The aim of this study was to identify circulating miRNAs in serum exosomes useful in all phases of the diagnostic and therapeutic path of patients with malignant brain lesions. Our data show a signature of exosomal miRNAs useful for the differential diagnosis of brain metastases and for monitoring tumor evolution over time.Circulating miRNAs are increasingly studied and proposed as tumor markers with the aim of investigating their role in monitoring the response to therapy as well as the natural evolution of primary or secondary brain tumors. This study aimed to evaluate the modulation of the expression of three miRNAs, miR-21, miR-222 and miR-124-3p, in the serum exosomes of patients with high-grade gliomas (HGGs) and brain metastases (BMs) to verify their usefulness in the differential diagnosis of brain masses; then, it focused on their variations following the surgical and/or radiosurgical treatment of the BMs. A total of 105 patients with BMs from primary lung or breast cancer, or melanoma underwent neurosurgery or radiosurgery treatment, and 91 patients with HGGs were enrolled, along with 30 healthy controls. A significant increase in miR-21 expression in serum exosomes was observed in both HGGs and BMs compared with healthy controls; on the other hand, miR-124-3p was significantly decreased in BMs, and it was increased in HGGs. After the surgical or radiosurgical treatment of patients with BMs, a significant reduction in miR-21 was noted with both types of treatments. This study identified a signature of exosomal miRNAs that could be useful as a noninvasive complementary analysis both in the differential diagnosis of BMs from glial tumors and in providing information on tumor evolution over time. Brain metastases (BMs) represent important causes of morbidity and mortality in cancer patients and are the most common intracranial tumors, occurring in approximately 10–30% of adult patients with cancer . The incSurvival seems to be primarily determined by the activity of extracerebral disease and systemic treatment options. The treatment of BMs continues to play a major role in preventing disease progression and in the deterioration of the neurological status and of the quality of life to limit the morbidity associated with systemic therapies .Treatment options for BMs include surgical resection with or without radiotherapy of the whole brain (WBRT), an independent method of WBRT, radiosurgery (SRS), SRS + WBRT and chemotherapy. Stereotactic radiosurgery is most frequently used in the treatment of solitary or multiple BMs, provided that the overall volume is less than 20 cc. The treatment’s effects on solitary BMs are comparable to those of surgical treatment . This meWhen a brain mass is detected in patients where extracranial malignancies are not diagnosed, conditions such as primary brain tumors, abscesses or granulomas are usually taken into consideration in differential diagnosis ,8. MRI iRecently, there is an increasing interest in liquid biopsy in the field of neuro-oncology. The search for a non-invasive test that can give information on the state of the disease, in primary or secondary brain tumors, is the challenge of recent years. Among these, the role of circulating biomarkers in the cerebrospinal fluid, such as circulating tumor DNA, microRNAs and metabolites , and bloExosomes, small extracellular vesicles detectable in the blood stream, are emerging as promising sources of diagnostic and prognostic biomarkers in many forms of cancer, as they contain different types of molecules selectively derived from the cell of origin, including small non-coding RNAs, the microRNAs (miRNAs) . Many ofWe previously showed that the expression of three miRNAs—miR-21, -222 and -124-3p—in serum exosomes from patients with high-malignancy-grade gliomas (HGGs) was significantly increased compared with patients with low-malignancy-grade gliomas (LGGs) or healthy subjects and it drastically decreased after the surgical removal of the tumor .The aim of this study was to demonstrate how circulating miRNAs in serum exosomes can be useful in all phases of the diagnostic–therapeutic path of patients with malignant brain lesions.To ascertain the potential role in the differential diagnosis of malignant brain masses, we compared the expression of circulating miR-21, -222 and -124-3p in patients with different primary and secondary lesions and in healthy controls. In order to evaluate whether these miRNAs can provide information about the response to treatment, we analyzed their expression levels in the immediate post-operative period and one month after GK treatment. We found that the reduced expression of miR-124-3p associated with increased levels of miR-21 in serum exosomes was useful for the differential diagnosis of BMs. Mir-21 expression was significantly reduced both after the surgical removal of the tumor and GK treatment. To the best of our knowledge, this is the first study that compares the different expression of three miRNAs in patients undergoing surgery and radiosurgery. We previously demonstrated that the pre-operative miR-21, miR-222 and miR-124-3p expression in exosomes derived from the serum of patients with HGGs was significantly higher than that in healthy controls . Moreovep < 0.0001). No differences were found in the miR-21 expression between patients with BMs and HGGs. The miR-222 expression in patients with BMs was not significantly different from that in healthy controls, whereas it was significantly lower than that in patients with HGGs, in agreement with our previous data [The data reported in ous data   and Good Clinical Practice guidelines. The approval for the publication of these data was obtained from Ethics Committee and from all the patients enrolled in the study via written informed consent. Demographic and clinical features are reported in Blood samples were collected before surgery or GK. In some patients with BMs, one week after surgery (23 patients) or one month after GK (23 patients) a second blood sample was obtained. After clotting, samples were centrifuged and stored at −80 °C.miR-21, miR-222 and miR-124-3p expression in serum exosomes was measured as described . BrieflyGK SRS was performed in 65 patients with BMs—mean age of 65.14 years (range: 43–83), 23 M and 42 F—with 106 lesions. A blood sample was obtained from each patient before treatment and a second sample 1 month after GK SRS.Technical details of the GK procedure have already been fully described in the literature ,32,33.Briefly, the procedure consisted of positioning a Leksell stereotactic frame  on the patient’s head under local anesthesia.Neuroradiological localization was routinely performed using stereotactic MRI with specific algorithms and sequences (1 mm isovoxel volumetric with double-dose gadolinium-enhanced images). SRS procedures were performed with model C 201-source Co60 Leksell Gamma Unit and, since June 2008, with Gamma Knife (GK) Perfexion (both obtained from Elekta Instruments). Three-dimensional treatment planning was developed using Leksell Gamma Plan . Mean and range dose-planning parameters were as follows: gross target volume , prescription dose , prescription isodose , maximum dose  and shot number . SRS was characterized by PD and MD intensity delivered in compliance with the brain stem, the optic nerve, the chiasm and the pituitary peduncle.Regarding the clinical and neuroimaging characteristics, 40 patients with BMs underwent neurosurgery by craniotomy, with the aid of an intraoperative microscope. Of these, 24 were female, and 16 were male, with an average age of 64 years.p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****). ROC curves were plotted using GraphPad Prism 9.0 software. Cut-off values were calculated by optimizing the sums of sensitivity and specificity (Youden’s Method) for each miRNA and for the miR-21/miR124-3p ratio.All the statistics were carried out with GraphPad Prism 9.0 as specified in the figure legends. Differences were considered statistically significant with The differential diagnosis of brain tumors remains one of the challenges for neuroradiology. Our study demonstrates that the increased expression of miR-21 and decreased miR-124-3p in serum exosomes discriminated BMs from HGGs. Decreased exosomal miR-21 expression after both the surgical removal and GK treatment of BMs indicated that this miRNA could complement neuroradiological imaging in the longitudinal follow-up of patients with BMs. To the best of our knowledge, this is the first report showing dynamic changes in exosomal miR-21 expression after the treatment of BMs with GK.


O6-Methylguanine-DNA-methyltransferase (MGMT) promoter methylation was shown in many studies to be an important predictive biomarker for temozolomide (TMZ) resistance and poor progression-free survival in glioblastoma multiforme (GBM) patients. However, identifying the MGMT methylation status using molecular techniques remains challenging due to technical limitations, such as the inability to obtain tumor specimens, high prices for detection, and the high complexity of intralesional heterogeneity. To overcome these difficulties, we aimed to test the feasibility of using a novel radiomics-based machine learning (ML) model to preoperatively and noninvasively predict the MGMT methylation status. In this study, radiomics features extracted from multimodal images of GBM patients with annotated MGMT methylation status were downloaded from The Cancer Imaging Archive (TCIA) public database for retrospective analysis. The radiomics features extracted from multimodal images from magnetic resonance imaging (MRI) had undergone a two-stage feature selection method, including an eXtreme Gradient Boosting (XGBoost) feature selection model followed by a genetic algorithm (GA)-based wrapper model for extracting the most meaningful radiomics features for predictive purposes. The cross-validation results suggested that the GA-based wrapper model achieved the high performance with a sensitivity of 0.894, specificity of 0.966, and accuracy of 0.925 for predicting the MGMT methylation status in GBM. Application of the extracted GBM radiomics features on a low-grade glioma (LGG) dataset also achieved a sensitivity 0.780, specificity 0.620, and accuracy 0.750, indicating the potential of the selected radiomics features to be applied more widely on both low- and high-grade gliomas. The performance indicated that our model may potentially confer significant improvements in prognosis and treatment responses in GBM patients. Despite some advances in standard multimodal treatment, including surgical resection followed by adjuvant chemoradiotherapy and adjuvant chemotherapy, the median survival of patients remains pretty low, at only 14–16 months3. GBM is considered a deadly disease with a poor prognosis due to its biological complexity, high frequency of chemotherapeutic resistance, and frequent recurrence after surgical treatment4.Glioblastoma multiforme (GBM), one of the most common brain tumors, accounts for approximately 45% of all malignant types of central nervous system tumors and is more likely to develop as primary (de novo) GBM5. This agent exerts its cytotoxic effects through methylating O6-methylguanine, which in turn causes DNA damage leading to cell death7. Thus, a major obstacle to the successful treatment of GBM is inherent and/or acquired chemoresistance to TMZ regulated by an enzyme called O6-methylguanine-DNA methyltransferase (MGMT), which is a highly evolutionarily conserved DNA repair enzyme that removes alkylated guanine residues at the DNA level, thereby antagonizing the effects of alkylating therapeutic agents9. Since methylation of CpG islands in the MGMT promoter region leads to suppression of MGMT transcription11, it could be a potential predictive biomarker for TMZ resistance and poor progression-free survival. It is thus important to identify the MGMT methylation status to have an accurate treatment strategy and improve success rates for GBM treatment.The standard chemotherapeutic agent for GBM treatment is temozolomide (TMZ), an alkylating drug that makes cells more sensitive to radiation12, while other techniques such as activity assays, immunohistochemistry, and methylation chip analysis have technical constraints13. In addition, the possibility of incomplete biopsy sampling, high prices for detection, and the high complexity of intralesional heterogeneity render these invasive techniques less useful in hospitals14.Although molecular techniques using surgical specimens are considered as reference standards for evaluating the MGMT methylation status, determining this epigenetic modification by a methylation-specific polymerase chain reaction often requires a large volume of tissue sample and a strict sample cryopreservation procedure15. Radiomics has been used to quantitatively extract and analyze noninvasive medical imaging features, including intensity distributions, textural heterogeneity patterns, spatial relationships, and many other characteristics17. In recent years, some radiological research has developed radiomics models for predicting survival rates16, distant metastasis18, and characterizations of molecular characteristics19. As the MGMT methylation status is considered an important prognostic biomarker for guiding GBM treatment decisions, several computational models were also developed to preoperatively predict the MGMT methylation status based on magnetic resonance imaging (MRI)22. In a recent study, Le et al. proposed a radiomics-based eXtreme Gradient Boosting (XGBoost) model that achieved relatively high performance for predicting the MGMT promoter methylation status, with an accuracy of 88.7% and an area under the receiver operating characteristics curve (AUC) of 0.89623.Recently, experts’ interest has shifted toward using non-invasive techniques such as radiomics to discover links between clinical symptoms and genetic characteristics23, we proposed a hybrid machine learning feature selection model in this paper to obtain the most informative radiomics feature set and meticulously evaluate its capability of accurately classifying MRI images into methylated and unmethylated ones. The proposed feature selection approach consisted of two steps: the XGBoost algorithm was first employed to extract features relevant to the MGMT methylation status; the selected feature set was then fed into a genetic algorithm (GA)-based wrapper model, in which the radiomics feature set with the best prediction power were selected under schemes similar to and inspired by natural selection in order to identify the “fittest” set of features for predicting MGMT methylation status. The performance evaluations of the GA-based approach also revealed that our GA-based hybrid model achieved a better performance for detecting the MGMT methylation status compared to other models, indicating the potential of the proposed hybrid approach in effectively predicting MGMT methylation status.Inspired by this previous work of Le et al.Our proposed method for building an effective model for predicting MGMT methylation status consists of the following steps. Upon downloading the pre-processed and segmented multimodal MRI (mMRI) features from the TCIA public database, a two-stage radiomics feature selection approach was conducted on the mMRI feature set to identify the most informative features for MGMT methylation status classification purpose. The procedure for conducting feature selection and evaluating the efficacy of the features was illustrated in Fig. 24 collections were downloaded from Bakas et al.25. Only data entries with MRI modalities of at least one of the following types were selected: T1-weighted pre-contrast (T1), T1-weighted post-contrast (T1-Gd), T2, and T2-FLAIR (fluid-attenuated inversion recovery). As a result, 53 GBM patients were included in this study. Totally 704 radiomics features were obtained from Bakas et al.25 and can be classified into seven categories. The categories include (1) first-order statistical features (intensity), (2) volumetric features26, (3) textural features describing the statistical relationship between image voxels 27, gray-level run length matrix (GLRLM)29, gray-level size zone matrix (GLSZM)30, neighboring gray-tone difference matrix (NGTDM)31, and wavelet-based features32, (4) histogram-based features33, (5) morphologic features34, (6) spatial features, and (7) glioma diffusion properties extracted from glioma growth models36.The pre-processed and segmented multimodal MRI features from The Cancer Genome Atlas (TCGA)-GBMFeature selection is an important task in eliminating noisy variables, keeping only features that are helpful in the classification tasks. In this study, we performed a two-stage radiomics feature selection using XGBoost and a genetic algorithm (GA) to discover the most appropriate subset of features that contributed to improving the MGMT methylation status prediction. The TCGA-GBM dataset with 704 radiomics features was used as the input. In the first feature selection stage, we used the XGBoost classification model  to preliminarily determine features that may be important for our model. The gain score was utilized in determining feature importance. The XGBoost-selected feature set was then fed into the next stage of feature selection using the GA. By subjecting the feature selection process to an evolutionary-based mutational model, the GA could optimize the radiomics feature subset for highly effective MGMT methylation status prediction. x GA-chromosomes for one population in each generation, in which the length of each GA-chromosome was the total number of XGBoost-selected features. The value of x was set to 100.Generation of the initial population: this stage is to create a set of solutions , and each solution is one “chromosome (termed GA-chromosome hereafter)” indicating the inclusion or exclusion of the radiomics features. Each potential feature has an equal probability to be included (1) or excluded (0) from each GA-chromosome, resulting in a vector of 0 and 1 bits . Initially, the algorithm randomly generatedFitness assessment: In this stage, the suitability (i.e. whether the combination of selected features results in good prediction) of each GA-chromosome in a population is evaluated based on the “fitness values” representing the ability to predict MGMT methylation statuses. The idea of a wrapper model, in which the fitness was defined as the accuracy of a machine learning model built on the selected feature set, was incorporated in determining fitness values. Several ML models, including support vector machine (SVM), random forest (RF), and XGBoost were evaluated to choose the most suitable model for the prediction purpose. More detailed description of the ML models will be explained in more detail in the Classification Algorithms subsection. Repeated five-fold cross validation was utilized for evaluating the model accuracy. In addition, elitism was applied in our GA to preserve the best feature set from generation to generation, in which two GA-chromosomes with the highest accuracy were copied into the next generation.i and ith GA-chromosome and the mean accuracy evaluated by the “fitness function” for the ith GA-chromosome. This step allows fit individuals to be selected with a higher probability while still giving some chances for good characteristics of less fit GA-chromosomes to be passed to the next generation.Selection of Parents: The individual genomes were selected for mating and crossover purposes as part of the GA algorithm, in which the probability that an individual genome was selected is proportional to its fitness value. The selection probability was calculated by the following formula:n and m chiasmata) in each pair of parent chromosomes were sampled within the GA-chromosome. A single-(SCs) or double-crossover (DCs) were then conducted at the chiasmata, interchanging chromosome content between the two selected parent chromosomes. The crossover rate was set to 0.8. This crossover procedure was applied repeatedly until x offspring were generated.Crossover: In each generation, selected parent solutions exchange their GA-chromosome segments to create new individuals. The “cross-over” points  or (0) values representing either the inclusion or exclusion of the randomly sampled features were then flipped.Mutation: To protect against stagnation or in-breeding and maintain the diversity of GA populations, mutations are introduced to each child chromosome of a population. Inspired by the state-of-the-art GA algorithm developed by Her et al.Population replacement: A new generation consisting of child chromosomes generated from the aforementioned steps along with the two “elite” GA-chromosomes with highest scores were placed into the next generation and replaced the initial population. This process was repeated 5000 times for a GA run.The detailed workflow of the GA is as follows. An example that depicts how GA works can be seen in Fig. https://scikit-learn.org/) was used for the model development. These three models were incorporated into the GA, and the model with the highest performance was chosen for further analysis. The predictive accuracy of the training dataset was evaluated using the five-fold stratified cross-validation method.ML techniques are becoming increasingly popular in radiomics studies for their ability to handle high-dimensional features and their robustness in capturing complex interactions among features themselves and between feature combinations for building effective prognostic/predictive models. In this study, supervised ML models including RF, XGBoost, and SVM were used to conduct the binary classification between MGMT methylated and unmethylated classes in GBM patients. The RF and XGBoost algorithms are ensemble learning techniques that collect individual outcome predictions from numerous weak learners and select the final model based on the votes. On the other hand, SVM can identify the most effective hyperplane for discriminating different targets and is able to transform a non-linearly distributed feature space into a high-dimensional feature space. The parameters for the models were: SVM with the radial-basis kernel, RF with 100 trees, and XGBoost classifier with default settings. The Python language and Scikit-learn package (https://scipy.org/)38. In addition, values of the receiver operating characteristic (ROC) curve and area under the ROC curve (AUROC or AUC) were also calculated to evaluate the overall performance. The comparison against other methods was made by extracting the model performances from the published studies.The classification performances of the models were evaluated using sensitivity, specificity, and accuracy. The comparison between the three ML models used in this study was made based on the mean of running cross-validation 20 times and evaluated using Kolmogorov–Smirnov test. Data were analyzed using Scipy package  multimodal MRI dataset, which was also pre-processed and segmented from The Cancer Genome Atlas TCGA-LGGp < 0.001; Kolmogorov–Smirnov test), leading to a more accurate prediction of the MGMT methylation status in GBM patients.The numbers of MGMT methylated and unmethylated samples in the TCGA-GBM dataset were 26 and 27, respectively. The two-stage feature selection approach comprised of XGBoost followed by a GA algorithm for selecting the most representative features. Three different ML models  were evaluated for their efficacy in the GA fitness wrapper model see “”. As sho23). As shown in Fig. Next, we compared the 22 GA-RF feature subset with feature sets selected by different methods, including all features 704 features), the subset of 25 features extracted by the XGBoost algorithm based on the gain score, and the set of radiomics features originated from the F-score feature selection analysis  dataset. We benchmarked the features extracted from the three GA-based machine learning algorithms from the GBM dataset on the LGG dataset. The XGBoost-F-score model developed by Le et al.45 vary between 0.67 and 0.925 in terms of accuracy. Even though Support Vector Machine40, L1-regularized neural networks45, and XGBoost algorithm23 yielded higher performances than others, the GA-RF method that we proposed in this study outperformed others, achieving the highest sensitivity, specificity, and accuracy. The result showed that the feature set identified using the GA-RF model may be better indicator in classifying MGMT methylation status.To measure the effectiveness of our proposed method in predicting the MGMT methylation status, we compared the performance of our model against various published classifiers. As shown in Table In recent years, applications of radiomics have garnered a lot of attention because of their potential to provide meaningful interpretative and predictive information for guiding treatment strategies. Moreover, along with the exponential growth of imaging data, different ML and deep learning (DL) techniques have been applied to elucidate correlations between clinical symptoms and genetic characteristics to achieve more accurate prognoses and treatment responses. In this study, a hybrid ML-based radiomics feature selection model was developed to identify optimal radiomics feature sets and predict the MGMT promoter methylation status. We used a set of 704 radiomics features previously extracted from the TCGA-GBM dataset to test the performances of three different ML models using five-fold cross validation. The GA-RF algorithm in general outperformed the GA-XGB and GA-SVM Table , showing45, least absolute shrinkage and selection operator (LASSO)44, F-scores23, or even using the Mann–Whitney U-test with Bonferroni correction to analyze the correlation between MGMT methylation statuses and quantitative imaging features, followed by the ROC curve analysis to choose the cutoff value for the presence of MGMT methylation43. To the best of our knowledge, most of the radiomics feature sets for classifying MGMT methylation statuses provided by other studies were merely based on one feature selection technique, and this is the first time the genetic algorithm-based hybrid feature selection approach has been applied for classifying MGMT methylation statuses in GBM. Therefore, this study aimed to test the feasibility of using the two-stage feature selection technique comprised of feature selection performed using the XGBoost algorithm followed by a GA wrapper model in picking radiomics feature subset that could effectively predict the MGMT methylation statuses. We observed that the adoption of the GA led to a radiomics feature set exhibiting accuracies higher than most of those reported in previous literature for the prediction of MGMT methylation statuses  extracted by undecimated 3D wavelet transformations. Wavelet transformation is a technique by which the 3D image data can be split into various frequency components along three axes. Fine and coarse textural information extracted from wavelet-decomposed images can reflect the tumor heterogeneity at multiple scales51. A few studies also reported that wavelet features can act as important radiomics biomarkers to predict tumor phenotypes, since they are believed to have strong connections with tumor biological behaviors53. Our results suggested that wavelet-transform features could also play a crucial role in predicting the MGMT methylation status in GBM. In other words, the proposed method possesses great potential in “hunting” informative features for this prediction purpose.By using the two-stage feature selection, our GA-RF algorithm generated an optimal subset of 22 radiomics features, including 17 textural features, three histogram-based features, one volume feature, and one intensity feature Fig. . Interes54. Therefore, texture- and histogram-based features could play critical roles to facilitate the GBM clinical diagnosis. Textural features reflecting spatial intensity correlations and distributions of voxels could help quantify the “multiregional variations” in blood flow, edema, necrosis, etc. For example, GLSZM_GLV (gray-level variance) reflects the intensity variance between homogeneous subregions within the enhancement area, while GLSZM_SZE  is the distribution of short homogeneous zones in an image. On the other hand, histogram-based features illustrate the frequency distribution of intensity values that occur in an image. More specifically, these features quantify the statistical characteristics of an image and therefore reflect intratumor heterogeneity. Taken together, a combination of texture- and histogram-based features may boost the ability of machine learning models in discriminating methylated and unmethylated GBM tumors. Furthermore, our GA-RF feature sets also implied that to achieve an accurate prediction of the methylation status, a range of texture- and histogram-based features may be needed.Herein, we attempted to interpret the benefits of using texture- and histogram-based features in predicting the MGMT methylation status in a fundamental manner. Besides some major challenges associated with poor GBM prognoses such as late diagnosis, diffuse infiltration and pseudo-palisading necrosis, inter- and intra-tumor heterogeneity and the dynamic plasticity of cells were considered important characteristics that may exacerbate the ineffectiveness of GBM treatment and lower survival rates55. As the number of dimensions and data volume increases exponentially, sparser real differences may be obscured by random measurement noise57. This means that to gain statistical significance, high-dimensional feature space often demands a large number of samples. Second, this circumstance could also result in ML model overfitting when dealing with high-dimensional, small-sized datasets. We overcame the potentially overfitting problem by adopting cross-validation to make sure that the test portion does not interfere with the training process. We also note that even though the sample size is not very large, the features selected by the XGBoost feature selection algorithm and the published F-score study23 share a certain number of common features, indicating our two-step feature selection approach may have indeed selected meaningful features from this dataset. We also foresee that the approach can be further evaluated with the release of more processed radiomics datasets in the near future.Although this proposed method has yielded promising results, there are still a few limitations. One of the limitations in radiomics studies is that imaging features extracted from a small number of patients often cause high dimensionality-related problems59, there are still slight differences between GBM and LGG in terms of their radiological appearance. In comparison with GBM, LGG tumors showed less blood–brain barrier disruption (resulting in less leak of contrast in the scan period) and little to no edema formation due to the slow growth rate of this kind of tumor, thus leading to low-density areas in MRI scans25. Reaching an accuracy of 0.75 on the MGMT methylation status prediction accuracy despite the fundamental differences between GBM and LGG indicated that some of the identified features may also be important in distinguishing the MGMT methylation statuses in LGG. We also tried to mix the GBM and LGG datasets into one mixed set and check whether we can identify common features among the two diseases. The application of the two-stage feature selection approach achieved 0.75 accuracy, 0.84 sensitivity, and 0.38 specificity. This showed that such mixing effort does not lead to better outcome, as seen in the much-reduced specificity. We plan to continue investigating the “common feature for very similar disease” issue in our future work.The application of the GBM radiomics features on the LGG dataset implies that the radiomics features extracted from one disease may be applicable to other very similar diseases. As shown in Results and Table To the best of our knowledge, this is the first study implementing an ML-incorporated GA model for predicting the MGMT methylation status. Results of this study showed that our proposed method could noninvasively predict the MGMT methylation status with a superior performance compared to existing methods. The model with the highest performance (GA-RF) was tested on an independent dataset, which demonstrated that the model may be generalized to similar diseases. Predicting the MGMT methylation status by this state-of-the-art model could benefit clinical decision-making by accommodating treatment strategies for patients with GBM even before surgery.


The COVID-19 pandemic has led to major economic disruptions. In March 2020, the UK implemented the Coronavirus Job Retention Scheme –known as furlough –to minimize the impact of job losses. So far, little is known on the mental health impact of this scheme on older workers, and on whether this varies by job characteristics. Exploiting longitudinal data from Wave 9 (2018/19) and two COVID-19 sub-studies  of the English Longitudinal Study of Ageing we use logistic and linear regression models to investigate associations between changes of employment and mental health during the pandemic. About 10% of respondents aged 52-67 were furloughed in the initial phase of the pandemic. Overall, employment disruption was associated with changes in mental health, although results suggest differences by pre-pandemic job characteristics .


The use of ChAdOx1 nCoV-19 (Astra Zeneca) vaccine has proven beneficial, but in a limited number of the general population, it was found to be associated with vaccine-induced immune thrombotic thrombocytopenia (VITT). However, there have been no reports of this complication occurring in a microsurgical free tissue transfer.A 49-year-old man developed an acute myocardial infarction 3 weeks after receiving his first dose of ChAdOx1 nCoV-19 in June 2021. Three months later, he presented with right third toe wet gangrene with extension into the plantar foot nine days after receiving his second dose of ChAdOx1 nCoV-19 vaccine.Based on recent exposure to vaccination, the timing of inoculation before the development of his symptoms, and serology tests , the patient was diagnosed with VITT.Fasciectomy and sequestrectomy were performed for wound bed preparation. Limb salvage was done using free vastus lateralis muscle flap and skin graft for reconstruction.The flap was complicated by persistent microthrombi leading to superficial necrosis without vascular pedicle compromise. Repeated debridement of the superficial necrosis was done. Three months after the development of VITT, no further new superficial necrosis was seen. A well-contoured flap was seen 5 months after the initial surgery.We believe this is the first case describing microthrombi in the free flap due to VITT after microsurgical reconstruction. Patients and surgeons should be advised of this possible risk when contemplating microsurgery once VITT has developed after ChAdOx1 nCoV-19 administration. Vaccine-induced immune thrombotic thrombocytopenia (VITT) was one of the more lethal complications reported. This syndrome was specifically related to the SARS-CoV2 adenoviral vector vaccine. It causes symptoms, such as headache, visual changes, persistent chest pain, or lower limb swelling according to different thrombotic events that have happened. Pavord et al in 2021, found that in 220 cases with VITT in the UK, the overall mortality reached 22%. The most common sites of thrombosis are the cavernous sinus, the pulmonary artery, and the deep veins in the lower limbs. Currently, there have been no reports of this complication occurring in a microsurgical free tissue transfer.The coronavirus disease 2019 (COVID-19) pandemic, due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, has caused significant morbidity and mortality globally. As of early May 2022, there have been >500 million confirmed cases of COVID-19, including over 6 million deaths, reported to the World Health Organization. In order to prevent SARS-CoV-2 infection, several types of vaccine were made available to be used after approval by the Emergency Use Authorization Act in various countries since the end of 2020. However, different types of adverse effects and complications caused by the vaccines were soon reported.3/µL, D-dimer was 4916 ng/mL and anti–platelet factor 4 (PF4) antibodies were 83.77 ng/mL . According to the International Society on Thrombosis and Hemostasis interim guidance, a definite diagnosis of VITT was made. Conservative treatment without oral anticoagulant, intravenous immunoglobulin, or therapeutic plasma exchange was done, as the patient did not show any signs of headache, chest tightness or dyspnea and he did not have a trend of decreasing platelet count and fibrinogen levels. Due to progressive necrosis of the forefoot, the forefoot amputation was done with the 5th toe spared, as he was adamant that he did not want it amputated as the toe looked fine to him. Due to a resultant large soft tissue defect with bone exposure, a free vastus lateralis muscle flap with split-thickness skin graft was performed for limb salvage. The flap survived well and he was discharged 1 month later. This patient was regularly followed up at our outpatient clinics for wound care. Unfortunately, right 5th toe gangrene and partial dehiscence at the lateral plantar side of the right foot were noted, so he was admitted again for surgical debridement and amputation of the 5th toe. This time he agreed to the amputation of the 5th toe. The flap had survived well but was complicated by persistent partial necrosis of the superficial portion of the flap without vascular pedicle compromise. A possible etiology causing this was vaccine-induced microthrombi  on June 17, 2021. Three weeks later, he complained of chest discomfort and the cardiac echo showed dilated left ventricle chamber, anteroseptal wall hypokinesis with apical dyskinesis, and impaired left ventricle systolic function. Cardiac catheterization was arranged and 3-vessel disease was found. Coronary artery bypass surgery was performed on July 14 and the patient was discharged in early August. He recovered uneventfully. He received his second dose of the ChAdOx1 nCoV-19  vaccine on October 16, 2021. Nine days later, he presented to the Emergency Room for right third toe wet gangrene with extension into the plantar foot  vaccine. After debridement, we chose a free muscle flap for reconstruction as we felt that providing a highly vascularized soft tissue with a low resistant bed would provide a better run-off for the lower limb vessels as well as act as a nutrient flap for the wound. In the second admission, the flap had survived well with no signs of an infective process. But partial necrosis of the superficial layer of the muscle flap was noted even though both the arterial and venous pedicle was patent. This was different from what we usually encounter for partial flap or total flap necrosis. Often the cause of flap necrosis is thrombosis of the pedicle, either the artery or the vein; or thrombosis of a branch of the vessel from the main pedicle leading to a portion of the flap that becomes necrotic. Infection is also a cause of partial flap necrosis. But in all of these situations, we do not encounter superficial necrosis of the flap in a random pattern. described autopsy cases after VITT, showing microthrombi in the capillaries and veins of the lungs, glomerular tufts, liver, vasa vasorum of the aorta, and brain. Althaus et al reported 3 cases where autopsy also revealed arterial and venous microthrombi in various organs. This could be the same mechanism leading to microthrombi in our free flap.Although the mechanism of VITT is still not certain, it can cause insidious thrombotic events in the microcirculation apart from thrombosis in the sites previously mentioned. Fanni et al The effectiveness of the AZ vaccine was satisfactory according to the pooled interim analysis published by Voysey et al, and the overall vaccine efficacy was 70.4%. However; there were several articles that reported a rare but lethal complication, VITT, related to the AZ vaccine. The incidence rate was estimated to be 0.87 cases per million doses.Astra Zeneca vaccine, or ChAdOx1 nCoV-19 vaccine, is categorized as a viral vector vaccine. The chosen viral vector is a chimpanzee adenovirus which is a replication-deficient virus. It was engineered to express the genetic sequence of the spike protein from SARS-CoV-2 in host cells which then triggers sequential active immunity. Bye et al have proposed the hypothesis by in vitro fundamental study. They found that aberrant glycosylation of the heavy chain of the anti-SARS-CoV-2 IgG increased galactosylation instead of fucosylation. This enhances the binding affinity to the FcγRIIa on the platelet. This in turn triggers increased von Willebrand factor production by signaling transmission. This is believed to be the cause of the pro-thrombotic effect. The incidence rate of VITT was estimated to be 0.87 cases per million doses.The hypothetical pathogenesis of VITT after ChAdOx1 nCoV-19 vaccination is believed to be related to FcγRIIa (CD32a) receptors. in a 65-case VITT cohort study, found that all the patients who showed positive anti-PF4 antibodies in the acute phase also showed that titers of antibodies were still detectable by enzyme immunoassay 6 months (84.2%) and 9 months (78.6%) after vaccination. At the same time, 3 out of the 26 patients still showed recurrent episodes of VITT in whom anti-PF4 antibodies persisted for at least 3 months. The relationship between persistently elevated anti-PF4 antibodies and the pathogenesis of VITT is still unclear. Since VITT is still a novel syndrome after COVID-19 vaccination, further research into the duration of the thrombotic event and the relationship with the titer of antibodies are still required.The duration of the thrombogenic effect of VITT is also unclear. In our patient, the phenomenon lasted for five months at which time, no further microthrombi was seen and final reconstruction with skin graft could be undertaken. Schönborn et alIn the COVID-19 pandemic era, several types of vaccines were available to be used rapidly and commonly in order to prevent the SARS-CoV-2 infection. VITT was one of the more lethal complications associated with the vaccines. We believe this is the first case describing microthrombi in the free flap due to VITT after microsurgical reconstruction. Patients and surgeons should be advised of this possible risk when contemplating microsurgery once VITT has developed after administration.Conceptualization: Chen-Ting Hung, Honda Hsu.Supervision: Honda Hsu.Validation: Chen-Ting Hung.Writing – original draft: Chen-Ting Hung.Writing – review & editing: Honda Hsu.