Despite the common practice of using multigene tests to predict recurrence, existing recommendations are inconsistent. Our aim was to formulate healthcare recommendations for the question The European Commission Initiative on Breast Cancer (ECIBC) Guidelines Development Group (GDG), a multidisciplinary guideline panel including experts and three patients, developed recommendations informed by systematic reviews of the evidence. Grading of Recommendations Assessment, Development and Evaluation (GRADE) Evidence to Decision frameworks were used. Four multigene tests were evaluated: the 21-gene recurrence score (21-RS), the 70-gene signature (70-GS), the PAM50 risk of recurrence score (PAM50-RORS), and the 12-gene molecular score (12-MS).Five studies  were included; no eligible studies on PAM50-RORS or 12-MS were identified and the GDG did not formulate recommendations for these tests.The ECIBC GDG suggests the use of the 21-RS for lymph node-negative women , recognising that benefits are probably larger in women at high risk of recurrence based on clinical characteristics. The ECIBC GDG suggests the use of the 70-GS for women at high clinical risk , and recommends not using 70-GS in women at low clinical risk . The same rationale would apply to women with HoR-positive, HER2-negative invasive breast cancer with 1–3 positive lymph nodes.6 Several prognostic factors, including clinical-pathological features such as age, tumour size, percentage of ER- and PR-positive cells as well as Ki67-index,9 predict the risk of recurrence and can help identify women who would benefit the most from chemotherapy. Although these factors have been shown to discriminate different prognostic groups, they showed no or minimal predictive value on the response to chemotherapy.10Breast cancer is the most frequently diagnosed cancer among women.6 the application of the 70-GS also takes into account clinical prognostic characteristics, while the 21-RS has been proposed to substitute clinical risk-based treatment decisions.In the last 15 years, different tests have been developed to stratify patients with early breast cancer into different risk of recurrence groups by analysing the activity of various genes. Although these multigene tests use diverse techniques  and diverse target gene combinations, they all focus on genes involved in cell proliferation. The tests provide recurrence risk profiles categorised in different ways. Some tests have been explicitly proposed to provide additional information to clinical-pathological features, as the 12-MS and the PAM50-RORS. Based on the results of the MINDACT trial,11 The Guidelines Development Group (GDG) of the ECIBC prioritised a clinical question on the use of multigene tests to guide the use of adjuvant chemotherapy in HoR-positive, HER2-negative and lymph node-negative or up to 3 lymph nodes-positive invasive breast cancer. Four multigene tests, used to stratify women with breast cancer into different groups according to recurrence risk,17 are included in the clinical question (Supplementary Table The European Commission Initiative on Breast Cancer (ECIBC) aims to provide evidence-based recommendations for screening and diagnosis of breast cancer.Should multigene tests be used in patients who have HoR-positive, HER-2 negative, lymph node-negative or up to 3 lymph nodes-positive invasive breast cancer to guide the use of adjuvant chemotherapy” was structured following the Population, Intervention, Comparison and Outcomes (PICO) format , EMBASE (May 2018) and CENTRAL (May 2018) databases were searched, using pre-defined algorithms, for both systematic reviews and individual studies  and cohort studies , either from prospective or retrospective analysis, of stored specimen samples were included as long as they applied any of the four tests as predictive markers for guiding the use of adjuvant chemotherapy  according to the test results or according to usual clinical practice. The predictive value is assessed by comparing the outcomes from the testing-based arm versus the non-testing arm; (b) Treatment interaction design: patients are divided into groups based on the marker status (i.e. high and low marker status). Then they are allocated to receive endocrine therapy or endocrine treatment plus chemotherapy. The predictive value is assessed by observing the relative efficacy of treatment differences between marker status and treatment assignments.A predictive marker identifies the differential benefit of a treatment based on the marker status. Thus, we included the following assessment approaches: (a) Studies that only reported prognosis data based on marker status , individual observational studies, abstracts or conference communications not published as full text articles, and articles published in a language other than English were excluded.With respect to economic evidence, cost-utility, cost-benefit, and cost-consequences, analyses were included if conducted within clinical trials, as well as observational and modelling studies, published in English during the last decade (Supplementary Table Two reviewers (CCA and KP) independently assessed risk of bias and extracted the following information from each study: first author, year of publication, country, study design, inclusion and exclusion criteria, number of patients, age, participants’ characteristics and prioritised outcomes.19 Cohort studies were assessed with the “Risk Of Bias In Non-randomised Studies - of Interventions-I” (ROBINS-I) tool.20 For economic evaluations, one reviewer (MP) screened the search results and used the NICE methodology checklist to assess applicability and methodological limitations.21 Studies with poor applicability and/or high risk of bias were excluded  were reported as presented in individual studies.22The certainty of evidence per outcome and overall certainty was rated using the GRADE approach. For each recommendation, the GDG received a Summary of Findings (SoF) table and a first draft of an evidence to decision framework (EtD).deterministic decision tree model without discounting was built by PGR with input from the rest of the GDG to estimate the downstream consequences of testing patients with the multigene tests versus different scenarios of usual care .Only women with high clinical risk would undergo multigene testing, and only those with high genomic risk would receive chemotherapy. Women with low clinical risk would not receive it.Two strategies for implementing the multigene test to guide the use of adjuvant chemotherapy were considered as interventions .6 at five years, and the effect is maintained at 10 years; (b) the advantage from adjuvant chemotherapy is equivalent to that observed by Paik and colleagues26 for all women at high genomic risk, independent of their clinical risk. Distributions of the clinical risk within the multigene risk strata are those reported in the TAILORx trial.23Women would receive adjuvant chemotherapy only if the clinical risk is high. The model assumes that women with low clinical and high genomic risk, as well as those with low clinical and low genomic risk, do not benefit from adjuvant chemotherapy. For sensitivity analyses, in women of low clinical and high genomic risk, two different assumptions were used to estimate the benefits: (a) The advantage of receiving adjuvant chemotherapy is equivalent to that observed in the MINDACT trialTwo scenarios were considered as usual care comparators (“C” of the PICO framework) , took primary responsibility for the review and completion of the first draft of the SoF tables and the EtD frameworks, conducted initially by the systematic review team. The frameworks were used in the meetings to help the complete GDG formulate the recommendations. Subsequently they were reviewed by a technical team from the Joint Research Centre, the PICO responsible unit and the systematic review team. Finally, the recommendations and frameworks were approved by the GDG.The process the ECIBC GDG used to formulate recommendations has been described in a dedicated article published elsewhere.23 two secondary analyses of stored tissue blocks collected from former parent clinical trials27 and one pooled analysis of observational studies12 from four previously reported validation studies, including unpublished data  of 1.31 (95% CI 0.46–3.78), 0.61 (95% CI 0.24–1.59) and 0.26 (95% CI 0.13–0.53) in low, intermediate and high risk groups, respectively  in the low genomic risk group  and a potential advantage in the high genomic risk  -related outcomes among women with an intermediate genomic risk group (11 to 26 risk score) allocated to either endocrine therapy alone or chemotherapy plus endocrine therapy. The as-treated results suggest little to no difference in the risk of recurrence with chemotherapy plus endocrine therapy for invasive DFS . For distant metastases, local recurrence and OS, similar results were observed . The results for mortality were consistent with the observed pattern of the risk of recurrence but the evidence was also very uncertain  , of distant metastases free survival  and of OS   and genetic testing strategies , the number of women who avoid chemotherapy by using the 21-RS would change from more than 600 to about 200. Survival outcomes did not change substantially  of using the test to guide chemotherapy to be large and the undesirable effects (i.e. increase in recurrence) trivial, with very low certainty of the evidence. The costs were considered large, though no cost-effectiveness study was included. A negative impact on equity was considered a potential concern Table .https://healthcare-quality.jrc.ec.europa.eu/sites/default/files/Guidelines/EtDs/Updated/ECIBC_GLs_EtD_21_gene_recurrence_score.pdf).For women with HoR-positive, HER2-negative, node-negative invasive breast cancer, the ECIBC GDG suggests the use of the 21-RS to guide the use of chemotherapy 25 may experience larger net desirable consequences and provide a more favourable cost-effectiveness profile .In light of the results from the MINDACT trial,https://healthcare-quality.jrc.ec.europa.eu/sites/default/files/Guidelines/EtDs/Updated/ECIBC_GLs_EtD_70_gene_testing_high_risk.pdf).For women with HoR-positive, HER2-negative, node-negative or up to 3 lymph nodes-positive invasive breast cancer at high clinical risk, the ECIBC GDG suggests using the 70-GS test to guide the use of chemotherapy. The judgments favoured the intervention in the high clinical risk population due to the moderate desirable effect, a balance that probably favours the use of 70-GS testing, and the large savings Table . The recThe ECIBC GDG suggests the use of 21-RS in lymph node-negative women, recognising that benefits are probably larger in women at high clinical risk and suggests the use of the 70-GS only for women at high clinical risk.53 Previously, we described some limitations of our guidelines.54 The weakness of the deterministic decision tree model used is that it is, to some extent, a simplistic approach and some assumptions are questionable . Furthermore, we did not actually quantify the side effects of chemotherapy, considering that avoiding any unnecessary chemotherapy was a desirable effect.The strength of the recommendations includes the ECIBC’s adherence to the requirements for trustworthy development of guidelines.55 The panel decided to evaluate the evidence for the four commercially available multigene tests included in the ECBIC question, and for the IHC4 + C test.57 The NICE guidelines did not follow the GRADE methodology and also had a different goal, i.e. deciding which tests should be funded by the UK National Health Service. The NICE 21-RS recommendation is similar to the ECIBC recommendation, a conditional recommendation limited to those patients in which the risk of distant recurrence is intermediate, using a validated tool such as PREDICT58 or the Nottingham Prognostic Index  also provided recommendations on the use of multigene tests.The implications of our recommendations are context dependent. The criteria used for making decisions on the provision or not of adjuvant treatment differ between countries. Therefore, the cost-benefit profile of introducing one of the multigene tests might also vary across countries. Decreasing costs for the tests would support a more widespread use. For these considerations, the GDG decided not to establish a threshold of recurrence risk to recommend genomic risk assessment, or a threshold for adding adjuvant chemotherapy, since these thresholds are context specific.In conclusion, the ECIBC GDG recommendations for or against the use of 21-RS and 70-RS are justified based on the judgments made. The transparency of our approach allows understanding the rationale for making different recommendations for the two tests and risk groups.63Data protection issues may be of relevance for the 21-RS because processing of samples is centralised in one US lab and requires shipping samples abroad. Furthermore, transparent information on test results is not available and reproducibility has been questioned.https://healthcare-quality.jrc.ec.europa.eu/discover-ecibc/methodologies/guidelines-updating).The GDG recommends research on exploring in what subgroups the use of 21-RS would have larger anticipated benefits as well as carrying out longer follow-up studies for 70-GS. The recommendations will be updated according to the ECIBC monitoring strategy in place (64Furthermore, the GDG is exploring the possibility of evaluating biomarkers that may assist decision making regarding the administration of adjuvant chemotherapy on the basis of their ability to identify women with a sufficiently low risk of relapse that would allow them to be spared from chemotherapy. In contrast to the presented evidence evaluation, this would enable evidence-based and transparent recommendations based on prognostic cohort studies, randomised or not, that predict the recurrence risk of different subgroups. We are currently working on a healthcare question on the significance of Ki67 using this strategy. In a next step, such an approach might also make it possible to evaluate multigene tests in the assessment for which we found no usable evidence in the predictive search strategy used here, but for which data on the prognostic value are available.Supplementary material


Compared with the general population, people living with dementia have been unequivocally affected by the COVID-19 pandemic. However, there is a paucity of knowledge on the COVID-19 impact on people with dementia and their care partners. The objective of this scoping review was to synthesize the existing literature on the COVID-19 experiences of people with dementia and their care partners.Following Arksey and O’Malley’s scoping review framework, we searched five electronic databases  and an online search engine (Google Scholar). Inclusion criteria consisted of English-language articles focusing on the COVID-19 experiences of people with dementia and their care partners.Twenty-one articles met our inclusion criteria: six letters to the editor, seven commentaries, and eight original research studies. In the literature, five main themes were identified: (i) care partner fatigue and burnout; (ii) lack of access to services and supports; (iii) worsening neuropsychiatric symptoms and cognitive function; (iv) coping with COVID-19; and (v) the need for more evidence-informed research. Factors such as living alone, having advanced dementia, and the length of confinement were found to exacerbate the impact of COVID-19.Urgent action is needed to support people living with dementia and their care partners in the pandemic. With little access to supports and services, people with dementia and their care partners are currently at a point of crisis. Collaboration and more evidence-informed research are critical to reducing mortality and supporting people with dementia during the pandemic. The current coronavirus disease (COVID-19 from the SARS-CoV-2 virus) is causing global morbidity and mortality, with a disproportionate burden on people living with dementia. Recent data show that dementia is the most common comorbidity in COVID-19-related deaths  original research, commentaries/editorials, or letters to the editor; (ii) written in the English language; (iii) full-text, peer-reviewed journal articles; (iv) published between January 13 and September 15, 2020; and (v) focused on the COVID-19 experiences of people with dementia  and/or their family care partners. Exclusion criteria consisted of studies that were (i) published in languages other than English; (ii) not published between January 13 and September 15, 2020; and (iii) not focused on the review’s objective but rather clinical outcomes in terms of the incidence and mortality of dementia comorbidities in COVID-19 patients.This search identified 420 articles for possible inclusion in the review. Relevant search findings were imported into the bibliographic management software RefWorks, followed by a systematic de-duplication. After 206 duplicates were removed, the titles and abstracts of 214 articles were screened for relevancy independently by two reviewers. A total of 183 articles were excluded as they did not meet the inclusion criteria. Any discrepancies or uncertainty related to the data inclusion were discussed between the two reviewers. The full texts of the remaining 31 articles were reviewed, and 10 articles were excluded based on the full-text assessment. The final number of articles included in the scoping review was 21. Figure n = 1), Canada (n = 2), China (n = 2), France (n = 2), Ireland (n = 1), Italy (n = 2), New Zealand (n = 1), Spain (n = 2), Portugal (n = 1), India (n = 1), the UK (n = 2), and the United States (n = 4).From the 21 articles identified in the review, 6 were letters to the editor, 7 were commentaries, and 8 were original research. The articles were from Argentina ((i)Care partner fatigue and burnoutIn the literature on COVID-19 experiences of people with dementia and their care partners, five main themes were identified: (i) care partner fatigue and burnout; (ii) lack of access to services and supports; (iii) worsening neuropsychiatric symptoms and cognitive function; (iv) coping with COVID-19; and (v) the need for more evidence-informed research.Studies indicate that COVID-19 restrictions and imposed measures of confinement have added significant strain to the pre-existing workload of family care partners of people with dementia Lack of access to services and supportsGreenberg and colleagues Worsening neuropsychiatric symptoms and cognitive functionThe confinement from COVID-19 and physical distancing measures also contributed to social support challenges for people with dementia. Boutoleau-Bretonniere et al.  assert t(iv)Coping with COVID-19Twelve of the twenty-one articles addressed the impact of COVID-19 confinement on people with dementia’s neuropsychiatric symptoms and cognitive function Need for evidence-informed researchDespite the numerous articles documenting the COVID-19 challenges, only four articles discussed the coping strategies used by people with dementia and their care partners. A study on rural family care partners identified different coping strategies, including gardening, having alone time, being outdoors, making protective masks for care aides, spending time on their cell phones, and playing computer games , family care partners for people with advanced dementia were identified as being particularly vulnerable to experiencing care partner burnout from COVID-19  for people with dementia and their care partners. While technological interventions are not new, home-based interventions are relatively newly accessible to care partners. Accordingly, more research is needed to examine the implementation , scale-up , and evaluation of home-based interventions for people with dementia and their care partners.In developing COVID-19 policies to support persons with dementia, there is a critical need for collaboration and more evidence-informed research involving people with dementia and their care partners. Although several articles  have been written on COVID-19 and dementia, relatively few research studies have been conducted with people living with dementia. With personal expertise and lived experience of COVID-19, people with dementia can offer valuable knowledge and insight to identify COVID-19 challenges and mitigation strategies in the pandemic. Thus, partnerships and collaborative research are essential to developing effective COVID-19 policies to support people with dementia and their care partners.This review was limited to English-language, peer-reviewed literature published between January and September 2020. While a rigorous search method was performed, it is possible that relevant literature was excluded in terms of language and timeframe. For example, studies in different languages may provide a more comprehensive understanding of the COVID-19 experiences on people with dementia. Also, this review’s timeframe only captures the literature from the first wave of the COVID-19 pandemic. A follow-up review is required to compare and analyze literature focusing on the second wave of the pandemic.Another limitation of this review is that we did not search preprint repositories but focused specifically on peer-reviewed journal articles from electronic databases. As such, it is possible that some relevant articles may have been missed. Given the rapidly evolving COVID-19 situation, future reviews may consider searching preprint servers as a means to finding timely research. However, reviews focusing on preprint servers should be conducted with caution as preprints have not undergone peer review, and a recent article suggests a high retraction rate among COVID-19 articles and preprints (Yeo-Teh and Tang In addition, there are some limitations related to the generalizability of our study’s findings in terms of gender and residence. In the literature, only two studies briefly addressed gender. For example, women were more often identified as family care partners (Cohen et al. There is a growing need for more research to address the impact of COVID-19 on people with dementia and their care partners. Compared with the general population, people with dementia are a vulnerable population with risks not only from COVID-19 but also from social isolation and the physical distancing methods used to mitigate the pandemic’s spread. Confinement measures combined with the lack of access to health and support services have severely impacted the lives of people with dementia and their care partners (Savla et al. In developing COVID-19 policies to support people with dementia, there is a critical need for collaboration and more evidence-informed research involving people with dementia and their care partners. With personal expertise and lived experience of COVID-19, people with dementia can offer valuable knowledge and insight to address COVID-19 challenges and mitigation strategies. Collaboration and more evidence-informed research are critical to reducing mortality and supporting people with dementia during the pandemic.


Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in ~4.8 million deaths worldwide as of this writing. Almost all conceivable aspects of SARS-CoV-2 have been explored since the virus began spreading in the human population. Despite numerous proposals, it is still unclear how and when the virus gained the ability to efficiently bind to and infect human cells. In an effort to understand the evolution of receptor binding domain (RBD) of the spike protein of SARS-CoV-2, and specifically, how the ability of RBD to bind to angiotensin-converting enzyme 2 receptor (ACE2) of humans evolved in coronaviruses, we have applied an alignment-free technique to infer functional relatedness among betacoronaviruses. This technique, concurrently being optimized for identifying novel prions, was adapted to gain new insights into coronavirus evolution, specifically in the context of the ongoing COVID-19 pandemic. Novel methods for predicting the capacity for coronaviruses, in general, to infect human cells are urgently needed.proposed method utilizes physicochemical properties of amino acids to develop fully dynamic waveform representations of proteins that encode both the amino acid content and the context of amino acids. These waveforms are then subjected to dynamic time warping (DTW) and distance evaluation to develop a distance metric that is relatively less sensitive to variation in sequence length and primary amino acid composition.Using our proposed method, we show that in contrast to alignment-based maximum likelihood (ML) and neighbor-joining (NJ) phylogenetic analyses, all bat betacoronavirus spike protein RBDs known to bind to the ACE2 receptor are found within a single physicochemical cluster. Further, other RBDs within that cluster are from pangolin coronaviruses, two of which have already been shown to bind to ACE2 while the others are suspected, yet unverified ACE2 binding domains. This finding is important because both severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 use the host ACE2 receptor for cell entry. Surveillance for coronaviruses belonging to this cluster could potentially guide efforts to stifle or curtail potential and/or early zoonotic outbreaks with their associated deaths and financial devastation.Robust methods for predicting human ACE2 receptor binding by the spike protein of coronaviruses are needed for the early detection of zoonotic coronaviruses and biosurveillance to prevent future outbreaks. Here we present a new waveform-based approach that utilizes the physicochemical properties of amino acids to determine the propensity of betacoronaviruses to infect humans. Comparison with the established phylogenetic methods demonstrates the usefulness of this new approach in the biosurveillance of coronaviruses. Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus [Alphacoronavirus and Betacoronavirus are known to primarily infect mammals including humans [Coronaviruses are a diverse group of RNA viruses of the family Coronaviridae. There are currently four recognized genera within this family, namely, onavirus . Of thesg humans , 2. Therg humans .Betacoronavirus SARS-CoV that in November 2002 began circulating with human-to-human transmission in China [Betacoronavirus MERS-CoV that in 2012 made the leap to humans in the middle-east with human-to-human transmission limited primarily to healthcare settings [Betacoronavirus SARS-CoV-2 that began circulating in the human population of Guangdong Province, China in late 2019 [In the past 20 years, three of the seven known human coronaviruses have emerged, presumptively through zoonoses, as deadly human pathogens. These include in China , Betacorsettings , 13, andate 2019 . Of gravate 2019 , 16. Of ate 2019 . TherefoThe question of the origin of any organism, protein, or gene is a question of both homology and mapping of that homology. Current phylogenetic analyses mostly involve comparison and quantification of similarity among biomolecular entities, mainly at the nucleotide or protein primary sequence level. Arguably the most objective approach to classify genetic entities is using quantifiable traits even when that quantification is boolean in nature . This isWhen sequence identity as determined by primary sequence alignment is low and yet functional conservation is obvious, homology determination becomes difficult, that is, many such cases fall into the twilight zone of <30% identity among homologs where ambiguity rules . Of evenIn the early years of phylogenetics, perhaps when biomolecular sequences were not abundant, homology was predominately determined using traits expressed at the macro-morphological scale. This practice continues even today, albeit in a limited scope and most notably in cases where molecular data are not available or collection is not practical such as in the analysis of fossils  and fishRather than inferring the substitution of amino acids based on probabilistic models derived from the alignments of well-understood protein sequences, which underpin most scoring matrices commonly used for sequence alignment, the inference by the method presented herein is based solely on the assumption of conservation of structure/function within orthologous protein domains. Such structural conservation likely varies spatially within a protein and therefore our alignment-free method, not constrained by genomic segment contiguity relied upon by alignment-based methods, is able to account for such conservation at the structural/functional level and thus could resolve the twilight zone homology, where the alignment-based methods relying on genomic segment contiguity and probabilistic matrices become progressively less reliable.The physicochemical properties that we chose to mathematically represent the underlying structure of the RBDs of coronaviruses were HP and molecular weight (MW). While HP is well established as the premier factor with regards to the formation of stable protein–protein interaction , MW as aWe therefore build upon the use of HP by Lolkema and Slotboom  by incluPrimary amino acid sequences can be converted into vector representations according to the physicochemical properties of each individual residue. In doing so it becomes possible to visualize polypeptide sequences as waveforms . These are similar in concept to time series data except that periodicity is based on a serial representation of residues rather than observations at sampling times. The techniques employed are, therefore, conceptually similar to those used in the analysis of time series data such as speech recognition profiles.®Core™i5-7200u processor with 8GB of RAM. Each RBD sequence was converted into a vector according to the algorithm described below. The algorithm uses the values for residue MW and HP to compute residual value V  and originally published by Monera et al. [A complete list of the sequences used in our analyses can be found in  value V . The vala et al. .(1)V=; and H is the residue HP index.Where V for the residue of interest and the extrinsic value was obtained as the mean of the V value for the residue of interest and V value for the left flanking residue .The values in  residue . The vec residue . The vecRBD polypeptide sequences were analyzed by performing direct Euclidean and cosine comparisons using the ape, dtw, proxy and TreeTools libraries in R . Three-phttp://www.atgc-montpellier.fr/phyml/ (Accessed 02 November 2021), with automatic substitution model selection (default) and 1000 bootstrap steps [In addition to the waveform-based, alignment-free approach, we reconstructed phylogenetic trees for the amino acid sequences using alignment-based ML and NJ protocols. Amino acid sequences were used in our alignment-based analysis because the results from such analyses are directly comparable to our DTW-based approach which uses the physicochemical properties of those amino acids to derive distance and topological relationships. For the alignment-based analyses, multiple sequence alignments were performed using ClustalW version 2.1 as implemented in Jalview 2.11.1.3 with a gap initiation penalty of 10, a gap extension penalty of 0.2, and a Gonnet series scoring matrix (default) , 45. ML ap steps . NJ treeap steps .Three methods of determining the congruence among dendrograms were used. These included visual examination of tanglegrams, evaluation of the number of nodes with the exact same list of terminal taxa (aka common nodes) and calculation of the Robinson–Foulds Metric . TanglegX-axis warping. In other words, differences indicated by peaks in the plots of these waveforms may not be directly comparable across multiple waveforms unlike multiple alignment-based methods.To compare the waveform representation of RBD sequences from different coronaviruses including SARS-CoV-2, we used the Warping Correspondence function in Mathematica version 11.3.0.0  with a EThe MW and HP of amino acids were used to encode the RBDs of the betacoronavirus spike proteins. Those encoded sequences were subjected to direct comparisons using Euclidean and cosine distance and also DTW comparison using Euclidean distance. The resulting distance matrices were then subjected to hierarchical clustering analysis using both NJ and UPGMA methodologies and compared to the ML and NJ trees produced using a multiple alignment of the same primary sequences. As can be observed in While alignment-based ML and NJ are both commonly used in the analysis of protein sequences, the congruency between the dendrograms produced using these methods was low as indicated by tanglegram comparison of those dendrograms in DTW and strict Euclidean/cosine distance-based methods employed herein resulted in little ambiguity within and among clusters and were in general agreement, regarding cluster composition, with the alignment-based ML analysis. However, there is a major exception among ACE2-binding RBDs where the alignment-based ML method results in two separate clusters, a finding that has been reported by others as well , 55–58; The alignment-based NJ tree shows a less cohesive grouping of ACE2-binding RBDs than does our physicochemical clustering method and also exhibits numerous polytomies . AdditioCloser examination of congruency as indicated by the proportion of nodes with the exact same taxa (Common Nodes) in In addition to the cluster composition, overall tree topological congruency was evaluated using the Robinson–Foulds Metric. The greatest level of congruency was observed between the single- and three-peak DTW analyses and the alignment-based ML analysis (58%) as shown in Due to multiple polytomies in the tree produced using an alignment-based NJ methodology , the topThe death toll from SARS-CoV-2 has been growing steadily since the beginning of the COVID-19 pandemic in early 2020 . RecentlFour of the pangolin coronavirus RBDs used in our analyses have not been investigated with regards to ACE2 binding. However, two pangolin coronaviruses that have been previously investigated and confirmed to bind ACE2 (GXP2V and MP789) are found within our unified cluster , 21 raisWhile it is the most pronounced benefit, the ability to identify newly sequenced ACE2-binding RBDs is not the only value in our waveform-based approach. Some studies have focused on the divergent origin of SARS-CoV-2 coupled with recombination , 61. OthAnother way to approach the question of convergent evolution is by asking if there is excessive conservation of function given the observed level of phylogenetic diversification. Such functional conservation can be maintained semi-independent of primary sequence due to overlap in the physicochemical profiles of amino acids. Lolkema and Slotboom noted this physicochemical conservation and therefore made their previously mentioned statement to the effect that the 3D structure of proteins and thus their function is very tolerant of changes in their primary sequence . Herein,The true evolutionary history of the ACE2-binding betacoronavirus RBDs may have experienced convergent evolution, recombination or both. However, the most pressing question is whether or not a newly emerged betacoronavirus has the ability to bind to ACE2. Addressing this brings us closer to targeting the betacoronaviruses with the propensity to evolve to bind to human ACE2. The fact that our waveform-based approach results in a single cluster of ACE2-binding betacoronavirus RBDs when a large number of betacoronavirus RBDs were analyzed demonstrates the utility of the method in the identification of potential betacoronavirus zoonoses before they become an actuality in the human population. This is emphasized by the fact that prior to the identification of SARS-CoV-2, it would not have been assumed that a new RBD sequence belonging to the clade that we now know contains SARS-CoV-2  would beEMPH online.Conflict of interest: None declared.eoab032_Supplementary_DataClick here for additional data file.


For the first time, the mRNA technology was utilized to produce a vaccine against COVID-19 after the unprecedented pandemic equally affected every part of the world. Pfizer-BioNTech (BNT162b2) mRNA vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was granted emergency use authorization (EUA) by Food and Drug Administration (FDA) in December 2020. EUA has been widely discussed in the medical literature and the general public. The safety of the BNT162b2 vaccine has been investigated in short-term trials with data available for three months. We present a case of a 96-year-old female with a past surgical history of cholecystectomy who presented with acute onset severe abdominal pain a few days after getting the first dose of Pfizer-BioNTech COVID-19 vaccine. She was diagnosed with acute pancreatitis with a lipase level of 4036 U/L. Extensive history and investigations were unable to find any etiology. The patient was conservatively managed and discharged home without any complications. There has been some data available in medical literature showing an association between acute pancreatitis and COVID-19 infection. Trial data of Pfizer COVID-19 also shows one case of acute pancreatitis in the treatment group. There have also been individual cases of unexplained acute pancreatitis shared by medical professionals on online forums. Our main goal to write this case is to make medical literature aware of possible emerging side effects of the COVID-19 vaccine, one of such side effects being self-resolving uncomplicated acute pancreatitis. For the first time, the mRNA technology was utilized to produce a vaccine against COVID-19 after the unprecedented pandemic equally affected every part of the world. Pfizer-BioNTech BNT162b2) mRNA vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was granted emergency use authorization (EUA) by Food and Drug Administration (FDA) in December 2020. EUA has been widely discussed in the medical fraternity and the general public. Long-term side effects of mRNA vaccines could not be established at the time of EUA due to the limited availability of trial data [b2 mRNA vThe patient is a 96-year-old Caucasian female who presented to the emergency department with complaints of progressively worsening epigastric pain for two days. The pain was sharp, 10/10 in intensity, and radiating to the right lower chest. She had some nausea but no vomiting, diarrhea, or fever. The patient also denied any recent sick contacts, diet changes, or use of new medical preparations or herbal products. Her past medical history includes diastolic congestive heart failure, hypertension, and hypothyroidism. Past surgical history was significant for cholecystectomy and appendectomy. Her home medications included amlodipine, aspirin, bumetanide, carvedilol, levothyroxine, and lisinopril, which has been her regimen for the past three years without any recent changes. The patient denied any use of alcohol in the last 10 years. Notably, the patient got the first dose of the BNT162b2 COVID-19 vaccine a few days before coming to the hospital.On physical examination, the patient had epigastric abdominal tenderness, but otherwise, the exam and vital signs were within normal limits. Initial lab work including complete blood count (CBC), comprehensive metabolic panel (CMP), urinalysis, lipid panel, thyroid-stimulating hormone (TSH), troponin levels, and electrocardiogram (EKG) were unremarkable. Her lipase level was significantly elevated at 4036 U/L. Computed tomography (CT) scan of the abdomen did not show any acute pathological findings. The patient was diagnosed with acute pancreatitis and monitored overnight with conservative treatment. Remarkably, her pain was already improving on admission and nearly resolved overnight. Lipase levels trended down to 205 U/L. Her diet was gradually advanced to regular. The patient's symptoms resolved, and she was discharged home in stable condition. The brief duration of symptoms and the timing of COVID-19 vaccination administration led to the increased likelihood of the vaccine as a plausible culprit for her acute bout of pancreatitis.Acute pancreatitis, defined as inflammation of the pancreas leading to autodigestive injury to the pancreatic parenchyma, can develop due to multiple causes. Some of the most common factors include gallstones, hypertriglyceridemia, alcohol, and smoking. Toxins or drugs, autoimmune, idiopathic, and procedure-related infections are few other less frequent causes .AutoimmuSince the beginning of the COVID-19 pandemic, the greatest hope has been developing a vaccine effective against SARS-CoV-2. Now we have several options available, which have proven to be highly effective and safe enough to be used under EUA. Long-term side effects were not established at the time of EUA due to the unavailability of long-term data. Only three months of trial data were used for EUA. It is, however, essential to remember the potential side effects that these vaccines may have. One of these side effects can be acute pancreatitis. The safety of the BNT162b2 vaccine has been investigated in short-term trials with data available for three months, and local reactions were observed predominantly in patients who received the vaccine when compared to the placebo group [Although it is difficult to make conclusions about the likelihood of the vaccine being the etiologic factor of pancreatitis, it is essential to continue monitoring for possible under-reported side effects until we have extensive long-term data available in post-marketing surveillance for long-term and rare side effects. Because the vaccine is new, it is reasonable to assume that we have not yet seen many of the potential side effects that may occur in patients who have received it. Our main goal to write this case is to make medical literature aware of the possible emerging side effects of the COVID-19 vaccine, one of such side effects self-resolving uncomplicated acute pancreatitis.Our patient developed acute pancreatitis several days after receiving the BNT162b2 vaccine and had no other risk factors for the condition. We present this case to make medical literature aware of the possibility that the vaccine may have some side effects that are not yet reported or taken into consideration. The incredible benefits of the vaccine are apparent and not disputed in this article. However, some patients may likely be at risk of developing some unestablished and high-risk side effects, and being aware of these risks will aid in weighing the risks versus benefits when deciding who should be vaccinated.


The use of more potent medicine for local chemotherapy of retinoblastoma in order to minimize local and systemic adverse effects is essential. The main goal of this investigation was to assess the biodistribution of thiolated and methylated chitosan-carboxymethyl dextran nanoparticles (CMD-TCs-NPs and CMD-TMC-NPs) following intravitreal (IVT) injection into rat eyes with retinoblastoma.An ionic gelation method was used to fabricate Cy5-labelled CMD-TCs-NPs and CMD-TMC-NPs. The NPs were characterized. Cellular internalization of Cy5-labelled NPs was investigated using confocal microscopy and the absorption of labeled NPs was quantified by flow cytometry in human retinoblastoma (Y79) cells. In addition, the Cy5-labeled distribution of nanoparticles in the posterior segment of the eye was histologically imaged by confocal microscopy after IVT injection of NPs into the eyes of rats with retinoblastoma.CMD-TCs-NPs and CMD-TMC-NPs showed a mean diameter of 34 The ionic gelation technique was efficient in synthesizing a biocompatible polymeric nanosystem for drug delivery into the posterior segment of the eye. The current study demonstrated increased ocular bioavailability of CMD-TCs-NPs relative to CMD-TMC-NPs in retinoblastoma induced rat eyes. The presence of efflux transporters and the pigmented structure of the choroid are the major limiting factors affecting therapeutic molecule penetration from the choroid to the retina and subsequently into the vitreous.,3 Sclera mainly limits the delivery of lipophilic drugs. The effect of the molecular radius however is greater than that of lipophilicity, which affects the scleral permeability of the drug. Vitreous, which consists mostly of 99% water, also contains only a few solid components, such as collagen and glycosaminoglycans. The vitreous poses a substantial barrier to injectable therapeutic molecules, especially to the diffusion of suspended solids or combinations of high molecular weight.The blood–retinal barrier prevents large molecules from passing into the retina from the blood and choriocapillaris.,7,8 With recent developments in nanocarriers, polymeric carriers are being employed in facilitating drug delivery to the eye to improve the drug's bioavailability.,10,11,12 Natural polysaccharides are attractive for the formulation of ocular medications because they are nontoxic, economical, available, generally biodegradable and biocompatible, and usually amenable to chemical modification to fabricate new derivatives.,16,17,18 Chemical modifications have been recently used to fabricate derivatives with improved properties in terms of mucoadhesion, increased ocular bioavailability, and drug solubilization.,21,22,23Intravitreal (IVT) injection is the most popular method for delivering drugs into the posterior portion of the eye. It delivers the needed therapeutic concentration of the drug to the posterior segment with minimal but considerable hazards. When nanoparticles made of human serum albumin (HSA), hyaluronic acid, or a combination of the two are injected intravitreally, they can reach the retina. Polyethylene imine nanoparticles with positive surface charge cannot spread through the vitreous when intravitreally injected and are therefore not beneficial for IVT route. Nanoparticles fabricated from glycosylated chitosan (200–500 nm) can reach the retina when intravitreally injected but are not able to penetrate inner limiting membrane.The bioavailability of NPs in the retina can be enhanced using this technique. It is proven that PEG-coated polystyrene NPs with neutral surface charge up to the size of 750 nm could freely diffuse through bovine vitreous to reach the retina. Diffusion coefficients in nanoparticles were found to be greater at 100–500 nm rather than at 750 nm. Carboxylic groups coating was used to fabricate negatively charged beads, which were able to readily diffuse through the vitreous. Negatively charged nanoparticles, on the other hand, are more impacted by size than neutrally charged NPs, as a negative-500nm-particle was unable to efficiently disperse through vitreous fluid.To the best of our knowledge, no study has been performed to investigate the bioavailability of Cy5 fluorescent dye oligonucleotide labeled thiolated and methylated chitosan nanoparticles following IVT injection in the eyes of rats with retinoblastoma. Therefore, in this investigation, we characterized the effects of the surface charge of thiolated and methylated chitosan NPs on the diffusion and tissue distribution after a single IVT injection into the retinoblastoma bearing rat eyes.Medium-molecular-weight chitosan (Cs) with a degree of deacetylation of about 89% was purchased from Primex . N-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), carboxymethyl dextran (CMD) sodium salt , Ellman's reagent, RPMI-1640 tissue culture medium, fetal bovine serum (FBS), and dialysis tubing `1 were purchased from Sigma-Aldrich . N-Methyl-2-pyrrolidone (NMP), sodium chloride, hydrochloric acid, and sodium hydroxide (NaOH) were all purchased from Merck . The human retinoblastoma cell line (Y79). All chemicals were of analytical grade. The degree of quaternization (% DQ) was distinguished using 1H NMR spectrum of TMC which was prepared by a 600 MHz spectrometer . The %DQ was estimated by the following formula:TMC was synthesized according to the method reported by Sieval et al.DQ = [[(CH3)3 / [H] where DQ is the level of quaternization; [(CH3)3] is the integral of chemical shift of the hydrogens of N+(CH3)3 groups at 3.4 ppm; and [H] is the integral of H-1 peaks between 4.7 and 5.7 ppm. In the first step, 100 milligrams (mgr) of synthesized TMC was dissolved in 5 ml of distilled water (DI) and then 200 mgr of cysteine was added and then mixed until dissolved. In the second stage, EDC and NHS were added. The mixture was then incubated for 3 hr in the dark under continuous stirring at room temperature and the pH was balanced to 5. Afterward, the solution was dialyzed  using 1 mM HCl for three days at 4ºC. Eventually, the solution was lyophilized to obtain a powdery substance (TMC-cys) and stored at 4ºC. The amount of free thiol groups attached on the TMC backbone was determined by photometry with Ellman's reagent. The thioglycolic acid standards curve was used to determine the quantitative amount of thiol groups. FT-IR spectra of TCs were prepared with an FTIR spectrophotometer .The method of synthesis was according to Margit et al. Carboxymethyl dextran (CMD) was used as the cross-linking agent. Nanoparticles were prepared by adding CMD solutions to TCs or TMC solutions. Then, an instant vortex stirring was executed and samples were incubated at room temperature for 2 hr.The nanoparticles were fabricated by a simple coacervation technique.The particle size of the nanoparticles was distinguished by applying dynamic light scattering on a Malvern Zetasizer Nano-ZS . A Zetasizer Nano series  was performed to determine the surface charge of the NPs. Field emission scanning electron microscopy  and transmission electron microscopy  were used to study the morphology of nanoparticles. For this purpose, the Y79 cells were cultured in 6-wells at the density of 2 5 cells per well. When the cells reached confluence, the cells were then incubated with Cy5-labeled TMC-CMD-NPs and TMC-cys-CMD-NPs suspension to track their uptake in Y79 cells. As a result of this procedure, the nanoparticles were well-dispersed in the culture medium at concentrations of 100 μg/ml. Nanoparticle dispersions were incubated at 37ºC in a 5% COQualitative cellular uptake of Cy5-loaded NPs was investigated with a confocal laser scanning microscope .4 cells/well. After 24 hr of incubation, the cells were treated with Cy5-labeled NPs at 37ºC for 2 hr. After the incubation, cells were washed with PBS and analyzed for intracellular fluorescence of Cy5-labeled NPs using BD FACS Calibur flow cytometer .The cellular internalization of Cy5-labeled CMD-TMC-NPs and CMD-TCs-NPs were reconfirmed and compared by flow cytometric analysis in the Y79 cells. To execute, the cells were cultured in a 6-well plate at a density of 250 6 Y79 cells were intravitreally injected to the rat eyes. After retinoblastoma tumor formation, Cy5-labeled TMC-CMD-NPs and TMC-cys-CMD-NPs (100 µg/ml) was intravitreally injected. The control eyes received IVT normal saline as same concentration. All the animals were euthanized 24 hr after the IVT injection of Cy5-labeled nanoparticles and enucleation was performed on them. Afterward, tissues were cut into 5-µm thick layers using a microtome for investigation of qualitative ocular uptake and biodistribution of Cy5-labeled NPs, which was done with a confocal laser scanning microscope .For this study, 10 Wistar albino rats  were used. All rats were treated in accordance with the ARVO  Declaration on the Procedure of Animals in Ophthalmic and Vision Research, approved by the University of Medical Sciences of Tehran. Surgeries were performed by the same surgeon (FG). The rats were immunosuppressed with daily injections of Cyclosporin A (CsA) . Approximately 1 1H NMR spectrum of TMC is shown in Figure 1. In the 1H NMR spectrum of TMC, the signals at 3.3 to 3.8 ppm were attributed to the methyl group at the N,N,N-trimethylated site ([H3]–[H6]). FTIR spectroscopy is an efficient tool for the investigation of the physicochemical attributes of polysaccharide. In this study, the syntheses of TMC and TCs were corroborated by the FTIR spectra illustrated in Figure 2. The TMC-cys conjugate was synthesized by the development of amide bonds between the amino group of methylated chitosan and carboxylic acid group of cysteine. Meanwhile, for TMC the peak at 1470 cm1 corresponded to the characteristic absorption of N–CH3. The peak at around 1250 cm1 in the spectra of compound was accredited to the C–SH stretching band. Also, the spectra of thiolated-chitosan displayed two powerful characteristic absorptions at 1641 cm1 and 2500 cm1 which were attributed to the C = O double bonds of the amido group and stretching vibration of –SH, respectively [Figure 2].,38 Furthermore, the degree of substitution of thiols using Ellman's protocol was determined as 11%. In addition, CMD-TCs-NPs and CMD-TCs-NPs had diameters of 34  The size distributions of the CMD-TCs-NPs and CMD-TCs-NPs were 0.27 The The cellular uptake of Cy5-labeled CMD-TCs-NPs and CMD-TMC-NPs by Y79 cells was visualized using a confocal microscope after 2 hr of exposure [Figure 4]. Meanwhile, the nuclei of the Y79 cells were stained by DAPI (blue fluorescence) in order to ascertain the location of internalized NPs. A direct indicator of uptake enhancement by the Y79 cells could be increasing the number of uptakes of NPs, which was documented by the increase in the intensity of the red color as seen in Figure 4. In the Cy5-labeled NPs groups, the red signal that appeared, was mostly located in cytoplasm. Contrastingly, a stronger red signal was discovered to be distributed inside the cells treated with CMD-TCs-NPs. Compared with CMD-TMC-NPs, more bioadhesive CMD-TCs-NPs were better adsorbed by cell membrane, resulting in improved endocytosis of Y79 cells and efficacious cellular uptake [Figure 4].A rapid method for the determination of the absorption of nanoparticles in Y79 cells using flow cytometry has been used in this research. The cellular uptake of Cy5-labeled CMD-TCs-NPs and CMD-TMC-NPs by Y79 cells was further investigated by flow cytometry analysis. The fluorescence intensity of cell emission determined by flow cytometry can be a good marker of the amount of NPs internalized by Y79 cells. As shown in Figure 5, the peak of the fluorescence intensity shifted to a higher level when the CMD-TCs-NPs were used, suggesting the promoted Cy5-labeled CMD-TCs-NPs internalization by Y79.Within the first 24 hr after the IVT injection, the eyes were enucleated and severed into 5 μm thick sections. The NPs distribution was investigated by taking confocal images after the IVT injection of the Cy5-labeled NPs [Figure 6]. The two chitosan compositions showed various diffusion rates in the vitreous. After the injection, only Cy5-labeled CMD-TCs-NPs freely disseminated all over the vitreous cavity; 24 hr after the IVT injection, confocal microscopy demonstrated that the Cy5-labeled CMD-TCs-NPs had accumulated throughout the different retinal layers [Figure 6]. Also, it showed that cationic CMD-TCs-NPs with Zeta potentials +11 ,41Chemotherapy by nanoparticles has been an effective approach in ophthalmic research in overcoming poor intraocular bioavailability of drugs due to the presence of anatomical barriers and it has also contributed toward improving therapeutic efficiency. The main purpose in the engineering of nano-carriers in this investigation was to develop a promising vehicle via biopolymers to transport drugs to the posterior part of the eye. Chitosan is a polymer that has been discovered by researchers for the application of ophthalmic drug delivery systems. Mucoadhesive chitosan formulations were also considered as an effective strategy in overcoming the rapid elimination of topical ophthalmic drugs. The solubility of TMC-NPs may also be decreased as a consequence of a high degree of methylation (DQ%), which results in a high level of O-methylation. The beneficial approach of combining TMC and cys to fabricate TMC-cys conjugate in preparing desirable derivatives was used in this study to improve the solubility of fabricated NPs and minimize the formation of agglomerates.,44 Conjugation of polymers with the thiol group is the most common method used in the manufacture of mucoadhesive delivery systems. Endocytosis is the dominant mechanism in the adsorption of nanoparticles with a size of  By labeling NPs with Cy5, a qualitative assessment of their number can be obtained by evaluating the intensity of the staining seen through the confocal microscope to compare the cell uptake of the NPs. An increase in the red color intensity in the staining in the NPs-treated group, as compared to the control, could be due to better cellular uptake of NPs by Y79 cells. Therefore, the intensity of the red color that occurs after the labelling can be considered as a direct criterion for assessing the cellular uptake of NPs. As shown in Figure 4, to prove the presence of NPs in the cytoplasm, the cell nucleus was stained with DAPI. Flow cytometry was also utilized in order to provide a qualitative evaluation of the difference in cellular uptake between the two formulations of NPs. As can be seen in Figure 5, the cellular uptake of thiolated chitosan NPs by Y79 cells was better, which could be due to greater bioadhesion of TCs (TMC-cys) nanoparticles owing to the TMC combination with the thiol group.Due to its solubility in acidic solutions (pH = 6), the efficacy of chitosan can be reduced at the site of action. Hence, a chemical alteration of chitosan was employed to fabricate a water-soluble derivative of Cs. In this study, the NPs were fabricated using hydrophilic biopolymers such as TMC, TMC-cys (TCs) and CMD to design efficient and safe drug delivery systems for the posterior segment of the eye. Pitkänen et al showed that the major obstacle to nonviral gene delivery systems is the vitreous. Peeters et al. also stated that only PEGylated particles ,51In this research, TCs adhesion properties were founded by electrostatic interactions with cysteine. When the NPs are intravitreally injected, they must be able to cross the vitreous barrier to reach their destination. The vitreous body is a polyanionic gel-like mass which is made up of collagen fibers and glycosaminoglycan. Therefore, in this study, in order to achieve maximum bioavailability, IVT injection of nanoparticles has been used. CMD-TMC and CMD-TCs nanoparticles with identical sizes and diverse surface charges were employed to examine the connection between their diffusion rates and their composition. Confocal imaging was used to track real-time diffusion in the vitreous cavity of the injected Cy5-labelled nanoparticles. By comparing the images of the CMD-TMC-NPs, CMD-TCS-NPs, and the control groups, the Cy5 signal of labeled NPs was determined, and the variations of fluorescence intensity in the confocal images illustrated the distribution of Cy5-labelled NPs [Figure 6]. Two biocopolymers (TMC and TCs) could self-assemble into NPs with identical sizes and various surface charges. TCs could self-assemble into 34 nm NPs and with +11mV surface charges, while TMC NPs assembled into 42 nm NPs with a +29 mV zeta potential. The difference in the surface charges between TMC and TCs NPs may result in different diffusion rates of NPs after the IVT injection. In the two polymer confocal images of Figure 6, TCs with the 11 The drug bioavailability is dependent on the route of drug administration into the eye. Effectiveness of systematically administrated drug vehicles for ocular posterior segment drug delivery is limited by different factors including the wide drug distribution to the off-target sites and the presence of the blood–retinal barrier.Through comparing the fluorescence changes in the eye injected with TCs or TMC, the results illustrated in Figure 6 indicate that the appropriate surface charges on the particle surface (i.e., +11 In conclusion, an effort to facilitate the application of more potent medicine for the local chemotherapy of retinoblastoma to ensure minimization of local and systemic adverse effects, we embarked on the current study. One challenge that is evident in delivering the relevant medicine to the posterior of the eye is the ability of the medicine to break through the vitreous cavity. Nanoparticles have been determined to be a viable alternative in targeting specific areas for medication due to their ability to adsorb and diffuse medication in specific areas locally. Our study has revealed that appropriate surface charges (preferably +11 This research was funded by Tehran University of Medical Sciences, Tehran, Iran.The authors report no conflicts of interest in this work.


The COVID-19 vaccination has been the subject of unprecedented misinformation, false news, and public concerns. This study presents a unique analysis comprising persons who were not vaccinated and became ill. It investigates reasons for not vaccinating and evaluates how the personal experience of COVID-19 affected further attitudes and decisions related to health. The study included 730 consecutive unvaccinated patients hospitalized in 12 centers in Poland during the autumn 2021 pandemic wave. The most frequent reason behind the refusal to receive the vaccine was concern over the adverse effects, disbelief that the vaccine was sufficiently tested, and one’s conviction that COVID-19 will not affect a patient. Online information, friends, spouse, children/grandchildren, and other family members were most often the source of discouragement from vaccination. Most individuals regretted their decision not to receive a vaccine (66.0%), declared to promote COVID-19 vaccination after discharge (64.0%), and to receive a COVID-19 vaccine in the time recommended for convalescents (69.5%). Individuals expressing no regrets of vaccine refusal more frequently revealed conspiracy beliefs. The study shows that personal experience with severe COVID-19 can influence the perception of vaccination, but approximately one-third of unvaccinated hospitalized patients still appear to express vaccine hesitancy.  Vaccination campaigns represent one of the essential methods to fight the COVID-19 pandemic. The priority of vaccination is to decrease the clinical severity, need for hospitalization, and risk of death. The prevention of SARS-CoV-2 infection, whether symptomatic or asymptomatic, is considered as a secondary goal, yet it is achieved to some extent despite the emergence of novel, more transmissible variants adapted better to evade humoral immunity . Since uThere are many determinants of COVID-19 vaccine acceptance, including gender, trust in medical, scientific experts, and public health authorities, worry about severe disease, and family COVID-19 experience ,16. The The present study aimed to analyze the reasons for the lack of vaccination in Polish patients hospitalized with COVID-19 during the pandemic between November and December 2021. The sources of discouragement from vaccination were also identified. Moreover, the study assessed whether severe disease experience could potentially change the future perception of the COVID-19 vaccines in unvaccinated patients.We conducted a real-world multicenter observational study including consecutive unvaccinated adult patients with COVID-19 hospitalized between 1 November 2021 and 15 December 2021 in 12 infectious diseases departments in Poland. During this period, there were no shortages in COVID-19 vaccine availability. The vaccine coverage in the entire population exceeded 50% in mid-September 2021 and increased only by 4.7% until the end of this study, indicating a high vaccine hesitancy in Poland. Data were retrieved retrospectively from the medical documentation. Patient consent was waived due to the retrospective design of the study. The criteria for inclusion included being unvaccinated and being able to answer questions from a medical interview consciously.https://www.gov.pl/web/koronawirus/rada-medyczna, accessed on 1 March 2022). The information on the attitude to vaccinations after hospitalization was collected from patients who survived on the day of discharge as a part of a routine hospital procedure. In addition, data on the most advanced condition of the patient during hospitalization were collected from the medical records. The demographic data included age, gender, level of education, place of residence , occupation status , and whether an individual was a healthcare worker. Patients were asked about their reasons for vaccine refusal and whether this decision was influenced by other persons, institutions, or opinions found in media or the internet. Furthermore, each patient was asked whether they (i) regret the decision not to receive a vaccine, (ii) intend to promote vaccination when discharged from the hospital, and (iii) intend to receive COVID-19 vaccine when discharged from the hospital. The initial information was obtained as a part of a routine medical and epidemiological interview collected upon admission of a patient with COVID-19 to the hospital according to the standpoint 24 of the Medical Council at the Prime Minister of Poland of 15 September 2021  was used for data analysis. Because age did not meet the assumption of Gaussian distribution, a non-parametric Mann–Whitney U test was applied to test its effect. The differences in frequencies of given answers were assessed with Pearson’s χData from 730 consecutive unvaccinated patients hospitalized due to COVID-19 in Poland were retrospectively collected and analyzed. The demographic breakdown of the studied group was summarized in Studied patients declared various reasons for refusal to receive the COVID-19 vaccine. The most frequent included: concern about the adverse effects of the vaccine (44.2%), belief that the vaccine has not been sufficiently tested (24.8%), patients’ belief that they have too many comorbidities to receive a vaccine (20.4%), and one’s conviction that COVID-19 will not affect them (18.5%) A. ApproxOnly one patient admitted to being a part of the anti-vaccine organization. Healthcare workers  discouraged only 4.5% of patients. Effective discouragement by more than one source was reported by 28.9% of patients. p > 0.05 in all cases, Pearson’s χ2 test); age also was not a differentiating factor . Patients regretting this decision the least often had tertiary education (58.6%) followed by primary (65.6%), secondary (67.4%), and vocational (73.1%) . Only 54.5% of healthcare workers expressed such regret. Patients who did not require oxygen therapy or mechanical ventilation revealed the lowest frequency of regretting the refusal of the COVID-19 vaccine (The majority of patients expressed regretting their decision not to receive a vaccine (66.0%), 14.7% did not regret it, while 18.3% were unsure about their feelings. No difference in this regard was noted between men and women, those inhabiting urban and rural areas, and professionally active and inactive , doubt in vaccine efficacy , disbelief in the existence of SARS-CoV-2 , and refusal to receive foreign substances to the organism . No significant differences in frequency of responses were found for other reasons presented in Patients not regretting the vaccine refusal were more frequently indicating the following reasons: disbelief that vaccine was sufficiently tested , similarly to age . Individuals regretting their decision to refuse the COVID-19 vaccine were more frequently willing to promote the vaccinations  and to receive COVID-19 vaccine .Most of the studied patients (64.0%) declared to promote the COVID-19 vaccination when discharged from the hospital  and to receive their COVID-19 vaccine (69.5%) in the time recommended for convalescents . Gender, place of residence, and occupation status did not differentiate attitude toward these two aspects . Among healthcare workers, only 59.1% declared to promote COVID-19 vaccination, while 72.7% expressed a will to receive the COVID-19 vaccine. Patients with tertiary education were the least often willing to accept a COVID-19 vaccine after hospital discharge (59.6%) compared to those with vocational (71.2%), secondary (72.5%), and primary (75.0%) education levels  were also less frequently willing to receive the COVID-19 vaccine when compared to those on low- and high-oxygen supplementation as well as mechanical ventilation. Individuals requiring mechanical ventilation most often regretted the decision not to receive a vaccine, declared to promote COVID-19 vaccination after being discharged from the hospital and to receive COVID-19 vaccine as recommended. Significant differences between patients not requiring oxygen supplementation and those using oxygen therapy and mechanical ventilation were found, regarding regrets in their decision to refuse the COVID-19 vaccine, intent to promote the vaccine, and intent to receive vaccination after discharge, while between patients requiring oxygen therapy and those using mechanical ventilation a statistically significant difference was documented only for a declaration to receive a vaccine as recommended for convalescents .The present analysis presents the perspective on COVID-19 vaccines among unvaccinated patients hospitalized due to COVID-19 during the autumn 2021 pandemic wave in Poland, during which vaccination was freely available to everyone willing to receive it. Although there are numerous studies available assessing the level of COVID-19 vaccine acceptance and analyzing the reasons for their refusal in different populations and demographical groups ,19, the No universal profile of unvaccinated individuals was found, while various reasons behind the vaccine refusal were reported. Unsurprisingly, the concerns over adverse effects were the most frequent motive behind vaccine refusal (44%)—this finding is in line with many other investigations conducted in various populations ,21,22. SAs many as 52% of studied patients admitted that their decisions were influenced by others’ opinions or organizations, indicating information from the internet as the most common source. The anti-vaccine content published online, especially on social media platforms, has been a prominent issue throughout the pandemic . The mytA small percentage of patients (4.5%) in the analyzed group indicated healthcare workers as the source of information based on which the decision not to vaccinate was made. The share of this professional group in the overall analyzed population of unvaccinated hospitalized patients was also small (3.2%). Similar to other countries, Polish healthcare workers were prioritized in access to vaccination . They reThe added value of the present research was the assessment of the role of personal experience in shaping the perception and decisions related to health. Most hospitalized patients regretted their decision to refuse the COVID-19 vaccine, declared to support COVID-19 vaccination after being discharged, and expressed a will to receive their COVID-19 vaccine in the time recommended for convalescents. Importantly, severe COVID-19 experience did not convince approximately one-third of studied patients to receive vaccination. As evidenced, those who did not regret their refusal of the COVID-19 vaccine were more likely to support their decision with claims related to conspiracy theories, such as disbelief in the existence of SARS-CoV-2. This highlights the importance of misinformation on COVID-19 vaccines on some individuals who are emotionally attached to their beliefs and not prone to abandon them despite contradictory personal experiences. The pre-pandemic evidence shows that this may mainly concern those with anxiety traits, perceiving that society is under threat and that the situation lacks control ,39,40. AThe study has some limitations. Social attitudes may influence patients’ responses, therefore it cannot be entirely ruled out that some of them provided the answers expected by the medical doctor on whom their health and life depended. Moreover, the willingness of patients to receive vaccination after hospitalization was not verified later, and it is possible that some of the patients who declared a change in perception of COVID-19 vaccination ultimately did not receive the vaccine. The study’s strength is the collection of data from a real-world, diverse population representative of routine practice. Moreover, the data originated from a specific, unique group of unvaccinated patients hospitalized with COVID-19, not from the general population. In addition, the study comprised patients with various degrees of COVID-19 severity, including seriously ill individuals requiring intensive care treatment and mechanical ventilation in the further course of the disease. Our study found that despite wide access to vaccines some patients may still decide to risk their health and lives by not vaccinating. Suffering from severe COVID-19 appeared to change most unvaccinated patients’ attitudes toward vaccinations. However, one-third of unvaccinated hospitalized patients still expressed vaccine hesitancy. Its highest levels were seen in individuals who refused the COVID-19 vaccine based on conspiracy theories and false information. Unvaccinated individuals who suffered less severe disease and did not require oxygen supplementation were also less convinced of the benefits of COVID-19 vaccines. The study adds to the evidence that convincing some individuals to receive COVID-19 vaccination may be a challenging task, particularly in groups exposed to misinformation and conspiracy theories.


Clinical trials for COVID-19 vaccines initially excluded pregnant women due to safety concerns, and when the vaccines were authorized for emergency use, they were not recommended for this population. However, observational studies discovered that pregnant women infected with COVID-19 have higher risks of negative pregnancy and delivery outcomes compared to non-pregnant women, raising the question of the risks–benefits of administering COVID-19 vaccines to pregnant women. By mid-2021, there was general consensus on the relative safety of COVID-19 vaccination during pregnancy; therefore, it is critical to investigate the safety issues related to these vaccines, considering the increasing acceptance among pregnant women. To address these concerns, we developed a research project to study the short-term effects and outcomes of COVID-19 vaccination during the first trimester of pregnancy. Our research followed an observational retrospective design for 12 months from the beginning of the vaccination campaign, and included 124 cases of spontaneous abortions and 927 ongoing pregnancies. The odds of spontaneous abortion were non-significant for both versions of the mRNA vaccine . Overall, our data indicated that the risk of spontaneous abortion after mRNA COVID-19 immunization during the first trimester of pregnancy is commensurate with the predicted risk in non-vaccinated pregnant women. These findings contribute to the growing body of information regarding the safety of mRNA COVID-19 vaccination during pregnancy. Vaccine development in the European Union (EU) begins with preclinical in vitro and in vivo testing in the laboratory, followed by clinical testing in phase 1 conducted on healthy volunteers . In esseDuring pregnancy, many dynamic changes occur to prevent adverse immune reactions to the fetus and to protect both the mother and fetus from infections, but it is still unclear how these immune responses contribute to changes in infection risk ,6,7,8. IPregnant women represent a population that was not included in the initial clinical trials for COVID-19 vaccine authorization due to safety concerns about the new mRNA vaccines ,19. HoweThe COVID-19 vaccines, particularly the mRNA vaccines, such as BNT162b2 Pfizer and mRNA-1273 Moderna, were authorized by the Food and Drug Administration (FDA) in late 2020 , and a w® Pfizer/BioNTech BNT162b2  or the Spikevax® Moderna mRNA-1273  during their first trimester of pregnancy in comparison with unvaccinated pregnant women, between January 2020 and January 2022. Data for unvaccinated pregnant women were retrieved from the beginning of the COVID-19 pandemic in January 2020, while data for vaccinated pregnant women were collected from the beginning of the COVID-19 vaccination campaign in January 2021. All patients/pregnancies included in the study were followed in the outpatient setting of the Obstetrics and Gynecology Clinic of the Timisoara Municipal Emergency Hospital. The research protocol was approved by the Ethics Committee of the “Victor Babes” University of Medicine and Pharmacy in Timisoara, Romania, and by the Ethics Committee of the Timisoara Municipal Hospital.Our research followed an observational retrospective design to observe adult pregnant women vaccinated with the ComirnatyAll patients evaluated in the outpatient clinic of our university-affiliated hospital are required to sign an informed consent form that allows data retrieval for research purposes. Data collection consisted of a database interrogation and as search for the patient’s personal paper records by trained medical personnel. We compared the probabilities of getting a COVID-19 vaccination 4 weeks before spontaneous abortion to the odds of receiving one in the 4 weeks preceding index dates for continuing pregnancies. Data from a total of 3094 pregnant women were included in the final analysis.2/overweight = ≥25 kg/m2), parity status , vaccine type (Pfizer BNT162b2 or Moderna mRNA-1273), number of doses , existent chronic conditions affecting the mother , which were represented as a dichotomous variable (yes or no), previous miscarriage (yes or no), smoking status (smoking/not smoking), karyotype analysis , assisted reproductive techniques (yes or no), abnormal uterine or cervical anatomy (yes or no), status regarding SARS-CoV-2 infection (yes or no), number of COVID-19 vaccine doses , and other vaccines administered during the first trimester .The inclusion criteria comprised all pregnancies in mothers older than 18 years, that were evaluated from the start of their first trimester during the study period in our clinic. Only pregnant women vaccinated with the Pfizer BNT162b2 or Moderna mRNA-1273 were included. Patients who did not provide consent were excluded from this research. An initial evaluation considered the differences between vaccinated and unvaccinated mothers, while the second evaluation was stratified by the pregnancy outcome in vaccinated mothers only. Both spontaneous abortions and continued pregnancies were classified according to gestational age , maternal age , weight as determined by body mass index  . However, in the first group, there were significantly more infections with SARS-CoV-2 before pregnancy .During the study period, we identified a total of 3094 pregnancies with a first trimester presentation in the outpatient clinic, of which 927 had received at least one dose of mRNA vaccine. There were 124 (13.4%) cases of spontaneous abortions among those who received the COVID-19 vaccine, and 271 (12.5%) spontaneous abortions identified in pregnant women who were not vaccinated against SARS-CoV-2 infection (= 0.506) . The avep-value < 0.001). Similar findings were observed in regard to weight; 48.4% of patients with spontaneous abortions were overweight, compared with 35.1% in the ongoing pregnancies . Other significant differences between the study groups were observed in the proportion of chronic conditions, previous miscarriages, number of smokers, abnormal uterine or cervical anatomy, and utilization of assisted reproductive techniques in the population with spontaneous abortions, where all the aforementioned variables had significantly higher proportions , while just 39.0% of the ongoing pregnancies were women over 35  recommended that developmental and reproductive toxicology studies (DART) be conducted prior to enrolling pregnant women or individuals who are not actively avoiding pregnancy in clinical trials. The developmental and reproductive toxicology studies are meant to determine the impact of new medications or vaccinations on the whole reproductive range in animals. On 4 December 2020, Moderna, whose vaccine is based on mRNA, submitted these data to the FDA . Pfizer Although our study is important and it has a sizeable cohort of patients and the existence of a control group of unvaccinated pregnant women for statistical analysis, several limitations, including the homogeneity of the participants in terms of ethnic origin, background and occupation, and the retrospectively-collected data, could bias the selection, classification and results. Another possible limitation worth noting is the lack of information regarding the outcome of pregnancies in terms of congenital malformations and stillbirths in the cohort of patients who received the COVID-19 vaccine in the first trimester. We also believe that future studies should report symptoms that occurred after vaccination in the first trimester, and other short-term complications that were not quantified in our research.This study determined that the risk of spontaneous abortion after mRNA COVID-19 immunization during the first trimester of pregnancy is similar to the risk in non-vaccinated pregnant women. These findings contribute to the growing body of information regarding the safety of mRNA COVID-19 vaccination during pregnancy.


Pancreatic cancer is the fourth leading cause of cancer death in the United States and over 90% of the patients suffer from pancreatic ductal adenocarcinoma (PDAC). PDAC is the most lethal gastrointestinal malignancies and only 10% of the people survive more than 5 years, therefore, novel diagnostic, prognostic, and therapeutic strategies are an immediate necessity. Studies have demonstrated microRNAs in bodily fluids that are bound with membranes (exosomes) can act as stable biomarkers both for disease development and metastasis. The diagnostic, prognostic, as well as therapeutic roles of exosomal microRNAs in pancreatic cancer have been discussed in this review.Pancreatic cancer is the fourth leading cause of cancer death among men and women in the United States, and pancreatic ductal adenocarcinoma (PDAC) accounts for more than 90% of pancreatic cancer cases. PDAC is one of the most lethal gastrointestinal malignancies with an overall five-year survival rate of ~10%. Developing effective therapeutic strategies against pancreatic cancer is a great challenge. Novel diagnostic, prognostic, and therapeutic strategies are an immediate necessity to increase the survival of pancreatic cancer patients. So far, studies have demonstrated microRNAs (miRNAs) as sensitive biomarkers because of their significant correlation with disease development and metastasis. The miRNAs have been shown to be more stable inside membrane-bound vesicles in the extracellular environment called exosomes. Varieties of miRNAs are released into the body fluids via exosomes depending on the normal physiological or pathological conditions of the body. In this review, we discuss the recent findings on the diagnostic, prognostic, and therapeutic roles of exosomal miRNAs in pancreatic cancer. KRAS. In PDAC patients, the mutational activation of the proto-oncogene KRAS occurs in 85–90% of cases [KRASG12D, KRASG12V, KRASG12R, and KRASG12C mutations in PDAC are 39.2%, 32.5%, 17%, and 1.7%, respectively [KRASG12C mutations are only observed 1.7% of PDAC patients, its protein product has been extensively studied over the past few years and targeted with covalent inhibitors such as AMG510, MRTX849 [CDKN2A [TP53 [DPC4/SMAD4 [CDKN2A, TP53, and DPC4/SMAD4 is over 95%, up to 75%, and about 55%, respectively [The pancreas is a complicated dual-functional organ that links the digestive system with the endocrine system . Due to of cases ,11. The  [CDKN2A , TP53 [12A [TP53 , and DPCC4/SMAD4 . The ratectively .Surgical removal of the tumor mass is considered as the only curative treatment of PDAC ,18, but Recent findings demonstrated a critical role of the tumor microenvironment in the development and progression of PDAC ,34,35. OExosomes are membrane-bound extracellular vesicles (EVs) containing biological materials and play an important role in communication among cells . The ultThe term “exosomes” was proposed by Johnstone et al. in 1987 . EarlierAll exosomes are extracellular vesicles, but not all extracellular vesicles are exosomes. Once outside the cell membrane, their origin is difficult to determine ,70. TherThe communication between cells in the tumor microenvironment carried out by exosomal miRNAs is performed via complicated signaling events ,78,79,80In 2005, exosomal regulation of migration and cytotoxicity of natural killer (NK) cells was shown in a Colo357 PDAC cell line model for the first time . SubsequThe miRNAs are small, noncoding RNAs of ~19–24 nucleotide length that act on mRNA for silencing or degradation and post-transcriptional regulation. They bind completely or partially to 3′-UTR, the untranslated regions of mRNA, via the base-pairing principle ,107,108.Highly elevated levels of exosomal miR-17-5p and miR-21 were detected in serum samples of pancreatic cancer patients for the first time in 2013 . Since tThere are many promising circulating miRNA biomarkers that have been proposed over the last few years for pancreatic cancer. However, due to the heterogeneous nature of the circulating miRNAs, it has not yet been possible to establish any miRNA-based detection method for the clinic either for early diagnosis or for the detection of pancreatic cancer ,137. TheBlood serum is widely used for the isolation of exosomes from cancer patients. As mentioned above, serum exosomal miR-17-5p and miR-21 were the first indication of a differential overexpression of miRNAs in pancreatic cancer as compared to non-malignant pancreatic diseases as well as healthy controls . SometimPlasma exosomal miRNAs serve as non-invasive biomarkers for various types of cancer . Like inTo identify novel diagnostic biomarkers for pancreatic cancer, the most relevant biological fluid, the pancreatic juice, was utilized in a study for the isolation of exosomal miRNAs . The stuA clinical trial (NCT04636788) was initiated at the end of the year 2020 to identify potential exosomal miRNA biomarkers for early detection of pancreatic cancer among other small RNAs in combination with EUS-FNA tissues. The trial is still recruiting patients and healthy controls with a target of 102 participants. A total of 12 mL venous blood will be collected from participants and the study will assess miRNAs and other small RNAs by next-generation sequencing. Primary outcome measures include the sensitivity and specificity, and secondary outcome measures include the survival of patients.Though several exosomal miRNA biomarkers showed promise for the diagnosis of pancreatic cancer, no universal exosomal miRNA signatures have been recognized so far. This is due to the lack of reproducibility of the results obtained in different studies and associated with exosome isolation methods, utilized models, and miRNA analysis tools . TherefoThe exosomal miRNA-mediated cell-to-cell signaling in the tumor microenvironment plays a significant role in the progression of cancer ,166. In In an in vitro study, an elevated level of miR-23b-3p was detected in the exosome derived from pancreatic cancer cells. Further investigation with the overexpression of miR-23b-3p confirmed the involvement of this miRNA in cell growth, migration, and invasion . A similPancreatic cancer cells were shown to release exosomes enriched with miR-301a under hypoxic conditions. Mechanistically, miR-301a was found to contribute to the polarization of M2 macrophages via PTEN/PI3K signaling and the metastasis of pancreatic cancer cells  Figure . In the In contrast to a cancer-promoting role of exosomal miRNAs, a recent pancreatic cancer study found that exosomes derived from bone marrow mesenchymal stem cell (BMSC) contain elevated levels of miR-126-3p, which subsequently downregulate ADAM9 and suppress cancer progression . The BMSThe abundance of certain exosomal miRNAs can be utilized to assess therapy response. For example, plasma miR-221 levels are elevated in pancreatic cancer patients after just three weeks of lapatinib and capecitabine treatment. Further evaluation of data showed a relation to drug resistance . ExosomeIn addition to the cancer suppressive and progressive roles, generalized immunosuppression also can be accomplished by exosomal miRNAs in pancreatic cancer via the regulation of MHC-II molecule expression. Pancreatic cancer cells can transfer miR-212-3p to dendritic cells by releasing miR-212-3p-enriched exosomes. Consequently, it can suppress the expression of regulatory factor X-associated protein, leading to a downregulation of MHC-II molecules ,154.Recent studies have constructed the foundation of exosomal miRNA biomarker research for the assessment of proliferation, migration, and invasion in pancreatic cancer. However, the exchange mechanism of exosomes between cancer cells and stromal cells and how they regulate recipient cells remains to be defined. Sufficient longitudinal studies are essential for the proper identification of exosomal miRNA as prognostic biomarkers in PDAC.In 2020, engineered exosomes loaded with a cargo of choice have been utilized as a drug delivery system ,173. TheA growing body of research findings suggests that exosomes can be used for treating pancreatic cancer in the clinic for disease stabilization or reduction of aggressiveness . The useSeveral studies convincingly suggest that exosomal miRNAs are linked to pancreatic cancer therapy resistance. For example, standard therapeutic choice gemcitabine is usually administered for the treatment of pancreatic cancer. However, upon treatment it causes cancer-associated fibroblasts (CAF) to release exosomal miR-146a and miR-106b to modulate the transcription process ,161. MecIn a study, gemcitabine-resistant pancreatic cancer stem cells were developed to study exosomal miRNA’s role in resistance. The study provided evidence that exosomal miR-210 can induce horizontal transfer of drug-resistant abilities against gemcitabine from resistant to sensitive cells . Though KRAS, TP53, and CDKN2A [KRAS mutations in pancreatic cancer [KRAS-G12D mutated mesenchymal stem cells and loaded with a specific siRNA that targets KRAS-G12D. This proof-of-principle study provided evidence that the expression of mutant KRAS-G12D can be downregulated through exosome-mediated knockdown of the mutant gene in a mouse model for PDAC [G12DKRAS mutation (NCT03608631) [The initiation of pancreatic cancer is a multi-step process that starts with the mutations of certain genes, such as d CDKN2A . More thc cancer  that arefor PDAC . The anifor PDAC . These e3608631) . After sThus far, the use of engineered exosomes in pancreatic cancer is still in its infancy. In the year 2014, investigations tested intravenous delivery of miRNA or siRNA-loaded exosomes to inhibit the aberrant signaling of PDAC cells. The study suggests transportation of miRNA or siRNA to the target cells would occur without degradation as they are protected inside lipid bilayered exosomes from natural ribonuclease present in the blood . There aPancreatic cancer is a highly aggressive and lethal malignancy mostly due to its late-stage presentation. This malignancy is difficult to diagnose, monitor, and treat, hence the development of novel diagnostic and prognostic biomarkers and better therapeutic strategies are urgently needed. Several groundbreaking discoveries over the past decade on cancer-associated exosomes demonstrated an association between exosomal miRNA and the development, progression, and therapy-resistance of pancreatic cancer. Despite the reproducibility problem, several exosomal miRNAs, alone or in combination, showed superior potential as non-invasive diagnostic and prognostic biomarkers for pancreatic cancer. The International Society for Extracellular Vesicles has not declared precise standards for the isolation and processing of exosomes yet , but forThe area of exosome biogenesis and the mechanisms of exosome sorting and release in pancreatic cancer and other malignancies need to be explored further to gain a better understanding of the cellular processes that drive cancer progression and metastases that are being mediated by miRNAs and other cargos . Members of the endosomal sorting complex required for transport 1 (ESCRT-1) are crucial for the intracellular routing of a subset of cargo proteins that are destined for inclusion into exosomes. One of those ESCRT-1 factors is the tumor susceptibility gene 101 (TSG101), which was identified within extracellular vesicles almost 20 years ago and is now being employed as one of the markers for exosome isolation ,193. WhiIn future studies, the molecular and cellular processes by which miRNAs as exosomal cargoes exert their effects on the growth and invasive properties of cancer cells should also be examined under hypoxic experimental conditions. As discussed earlier, some cancer-relevant miRNAs are exclusively upregulated in hypoxic cancer cells. While most studies are being performed under normoxic conditions, the experimental results and may not be readily applicable to in vivo cancer models or humans. Another under-studied area is the feedback loop of exosomal-miRNA-based regulation besides unidirectional signaling, which also requires further attention. So far exosome-based treatment is limited to in vitro and in vivo experiments for pancreatic cancer. Besides preclinical, clinical investigations are essential to identify universal exosomal miRNA signatures for the diagnosis, prognosis, and treatment of pancreatic cancer. In summary, extensive research on exosomal miRNAs in pancreatic cancer is needed to overcome current obstacles and guide current progress ahead toward a novel biomarker discovery and a new therapeutic avenue.