Whereas hypertension is an established cardiovascular risk factor in the general population, the contribution of increased blood pressure (BP) to the huge burden of cardiovascular morbidity and mortality in patients receiving dialysis continues to be debated. In a large part, this controversy is attributable to particular difficulties in the accurate diagnosis of hypertension. The reverse epidemiology of hypertension in dialysis patients is based on evidence from large cohort studies showing that routine predialysis or postdialysis BP measurements exhibit a U-shaped or J-shaped association with cardiovascular or all-cause mortality. However, substantial evidence supports the notion that home or ambulatory BP measurements are superior to dialysis-unit BP recordings in diagnosing hypertension, in detecting evidence of target-organ damage and in prognosticating the all-cause death risk. In the first part of this article, we explore the accuracy of different methods of BP measurement in diagnosing hypertension among patients on dialysis. In the second part, we describe how the epidemiology of hypertension is modified when the assessment of BP is based on dialysis-unit versus home or ambulatory recordings. Hypertension among patients with end-stage kidney disease (ESKD) receiving either long-term hemodialysis or peritoneal dialysis (PD) is highly prevalent and often remains poorly controlled ,2. Two mIn the first part of this article, we explore the accuracy of various BP monitoring techniques in diagnosing hypertension among patients on dialysis. In the second part, we describe how the epidemiology of hypertension is modified when different methods are used in the assessment of BP.A literature search of the MedLine/PubMed database was performed from inception until September 2022 with the aim to identify studies that explored the diagnostic accuracy and predictive value of different BP monitoring techniques in ESKD patients on dialysis. Search terms used were “hypertension”, “hemodialysis”, “peritoneal dialysis”, “end-stage kidney disease”, “home BP monitoring” and “ambulatory BP monitoring”. Reference lists of prior reviews in this area were also searched for additional studies and information. All types of studies, such as diagnostic-test studies, cross-sectional analyses, prospective observational studies, randomized controlled trials and meta-analyses, were eligible for this article if they reported relevant data with respect to the diagnosis and epidemiology of hypertension. Studies that were conducted in the general hypertensive population or in patients with earlier stages of kidney disease were excluded.Peridialytic BP measurements are the recordings obtained by the dialysis-unit personnel at the beginning and at the end of each hemodialysis session ,2. TheseOne approach to improve the diagnostic performance of peridialytic BP measurements is the concomitant assessment of intradialytic BP. These measurements are obtained during the hemodialysis procedure, typically every 30 min, using an automatic cuff attached to the hemodialysis machine ,2. In daSince the calculation of an average is time-consuming and impractical, consideration of the median intradialytic BP from the measurements obtained during a single mid-week hemodialysis session also appears to be an appropriate method to screen for hypertension at the bedside. A mid-week median cut-off systolic BP of 140 mmHg provides 80% sensitivity and 80% specificity for the diagnosis of systolic hypertension. Similarly, a mid-week median intradialytic diastolic BP of 80 mmHg offers 75% sensitivity and 75% specificity in the detection of diastolic hypertension . FurtherHome BP monitoring (HBPM) is an established technique that is recommended by international guidelines for the assessment and management of hypertension in the general population or in patients with predialysis chronic kidney disease . HoweverThe technique of HBPM has been more widely adopted in the assessment of hypertension in patients receiving long-term PD, possibly because PD is a home-based modality for renal replacement therapy. Among 81 stable PD patients, a recent study showed that self-measured home BP for 1 week had at least similar or even better accuracy than standardized dialysis-unit BP in diagnosing hypertension, as confirmed by the reference-standard method of 24-hour ABPM . The areApart from its superior diagnostic performance, additional advantages of the technique of HBPM exist and merit attention. In DRIP, home BP was more sensitive than peridialytic BP in detecting the reduction in 44-hour ambulatory BP provoked by the probing of dry weight over 8 weeks of follow-up . ContrarSubstantial evidence also supports the notion that HBPM is a useful tool to guide therapeutic decisions and improve the management of hypertension . The feaThe chronobiology of BP should be taken into consideration in the design of a sufficient scheme for home BP measurements. Home systolic BP is characterized by a steady increase at a rate of 0.40 ± 0.25 mmHg/hour over the interdialytic interval. This rising pattern reaches a plateau after approximately 48 h . TherefoInterdialytic ABPM represents the “reference-standard” technique for diagnosing hypertension ,2. This Studies exploring the epidemiology of hypertension in patients on dialysis have used different definitions and provided considerably variable results with respect to the prevalence and control of BP ,39,40. TA more accurate description of the epidemiology of hypertension was provided by two larger studies that incorporated the reference-standard method of ABPM ,43. The Some earlier studies that were based on conventional dialysis-unit BP recordings suggested that hypertension control in patients receiving long-term PD is superior compared to those on hemodialysis. For example, among 1202 patients enrolled in the 1995 Peritoneal Dialysis Core Indicators Study, only 29% had an office systolic BP > 150 mmHg and 18% had a diastolic BP > 90 mmHg . HoweverThe risk association of BP with cardiovascular morbidity and all-cause mortality among patients on dialysis represents an area of controversy . In the Recent studies have shown that peridialytic BP should be interpreted within the context of hypervolemia and not as an isolated risk factor. In an analysis from the international Monitoring Dialysis Outcome Initiative (MONDO) database, the volume status of 8883 hemodialysis patients was assessed using bioimpedance spectroscopy . All-cauTwo separate prospective observational studies showed that the BP measurement technique is another factor that modifies the prognostic association of BP with all-cause mortality. The first study enrolled 150 hemodialysis patients who underwent a baseline assessment of BP with four different techniques: self-measured home BP for 1 week, 44 h ABPM and routine and standardized peridialytic BP measurements averaged over six consecutive hemodialysis sessions . During The second study included a larger sample size of 326 hemodialysis patients who underwent a similar assessment of BP at baseline. The duration of follow-up was longer also (median follow-up: 32 months) . In multThe notion that ambulatory BP provides superior prognostic information relative to office BP is further supported by a prospective cohort study of 108 stable PD patients . At baseThe superior prognostic significance of home or ambulatory measurements over dialysis-unit BP cannot be attributed to the higher number of BP recordings that these techniques provide. Observational studies showed that both home and ambulatory BP retain their ability to prognosticate the all-cause death risk, even when the average of a small number of randomly selected BP recordings is used as a risk predictor ,52,53,54The reverse epidemiology of hypertension demonstrated by observational data is not accordant with the results of two earlier meta-analyses of randomized controlled trials showing that deliberate lowering of BP with the use of antihypertensive therapy offers cardiovascular protection in patients on dialysis. The first meta-analysis included eight trials involving a total of 1679 dialysis patients. Relative to placebo or no therapy, the use of antihypertensive medications was associated with 29% reduced risk for cardiovascular events (risk ratio (RR): 0.71; 95% CI: 0.55–0.92), 29% lower risk for cardiovascular death  and 20% lower risk for all-cause death  . The secIn conclusion, although the present work provides mainly a qualitative evaluation of diagnostic accuracy and predictive value of different BP monitoring techniques in the dialysis population, a growing body of evidence supports that home and ambulatory BP measurements are superior to routine and standardized dialysis-unit BP recordings in diagnosing hypertension, in assessing the presence of target-organ damage and in prognosticating the risk of all-cause mortality. Undoubtedly, 44 h ABPM is the “gold-standard” technique that provides the highest accuracy and precision, but some weaknesses  limit the adoption of this method in daily clinical practice. A more feasible approach to improve the assessment and long-term management of hypertension is the broader use of HBPM. Large randomized controlled trials are now needed to demonstrate the superiority of home-BP-guided over predialysis-BP-guided antihypertensive therapy in causing regression of end-organ damage and in improving “hard” cardiovascular outcomes in this high-risk patient population.


To identify operational taxonomy units (OTUs) signaling disease onset in an observational study, a powerful strategy was selecting participants by matched sets and profiling temporal metagenomes, followed by trajectory analysis. Existing trajectory analyses modeled individual OTU or microbial community without adjusting for the within-community correlation and matched-set-specific latent factors.We proposed a joint model with matching and regularization (JMR) to detect OTU-specific trajectory predictive of host disease status. The between- and within-matched-sets heterogeneity in OTU relative abundance and disease risk were modeled by nested random effects. The inherent negative correlation in microbiota composition was adjusted by incorporating and regularizing the top-correlated taxa as longitudinal covariate, pre-selected by Bray-Curtis distance and elastic net regression. We designed a simulation pipeline to generate true biomarkers for disease onset and the pseudo biomarkers caused by compositionality. We demonstrated that JMR effectively controlled the false discovery and pseudo biomarkers in a simulation study generating temporal high-dimensional metagenomic counts with random intercept or slope. Application of the competing methods in the simulated data and the TEDDY cohort showed that JMR outperformed the other methods and identified important taxa in infants’ fecal samples with dynamics preceding host disease status.https://github.com/qianli10000/mtradeR.Our method JMR is a robust framework that models taxon-specific trajectory and host disease status for matched participants without transformation of relative abundance, improving the power of detecting disease-associated microbial features in certain scenarios. JMR is available in R package mtradeR at The online version contains supplementary material available at 10.1186/s12864-022-08890-1. Gut microbiota profiled by 16s rRNA gene sequencing or metagenomic  sequencing has been frequently used in observational studies of environmental exposures, immune biomarkers, and disease onset . One of Longitudinal profiling is a powerful strategy for the microbiome studies that aim to identify differential microbial trajectories between exposure groups or phenotypes , 10 or dOne concern about using generalized linear mixed model to test the association between 16S rRNA or metagenomic trajectory and disease onset is that the covariates in this model may contribute to disease risk. For example, the HLA haplogenotypes and early use of probiotics may affect infants’ gut microbiota and should be included as covariates. These factors were also found associated with islet autoimmunity among children enrolled in TEDDY . One usuMany of the existing methods for microbiome data are built on the transformed relative abundance, such as centered log-ratio or inter-quartile log-ratio. In our new method, transformation of compositional data is not considered, since transformation strategy may have profound impact on analysis result and interpretation . The comThe latent technical noise in microbiome was removed by converting raw counts to relative abundance, and Zero-Inflated Beta density  was adopFor simplicity, the aim of present research is to link the matched longitudinal microbiome samples to hosts’ matched disease risk and incorporate the unknown dependence between taxa in an univariate trajectory framework, without modeling the compositionality. Briefly, we develop a Joint model with Matching and Regularization (JMR) to detect taxon-specific compositional trajectory associated with disease onset, adjusting for the linear correlation with other taxa and matched-set-specific latent noises. According to the characteristics of disease risk and infant-age gut microbiota in the TEDDY cohort, we designed a simulation pipeline similar to , generatTEDDY is an observational prospective study of children at increased genetic risk of type 1 diabetes (T1D) conducted in six clinical centers in the U.S. and Europe . A total of 8,676 children were enrolled from birth and followed every 3 months for blood sample collection and islet autoantibody measurement up to 4 years of age, then every 3-6 months based on autoantibody status until the age of 15 years or diabetes onset . A primaDisease outcome for the matched participants are simulated by the procedure below. The observed relative abundance per taxon were simulated by different scenarios. We first generated raw counts for a single OTU by Beta-Binomial distribution to assess the robustness and power of our method JMR without covariate taxa. We also designed a shifting procedure to mimic inherent negative correlation in the true composition of microbiota and generated the temporal high-dimensional raw counts table to evaluate the performance of compared methods.sWe defined matched sets and subjects as ‘high-risk’ and ‘low-risk’ to generate the temporal OTU counts prior to disease onset. Subjects are matched at 1:1 ratio. For participant t in a matched set s was generated by j in set s at time point t was simulated by We first simulated the observed counts of a single OTU by Beta-Binomial  distribuWe also generated a high-dimensional counts table with entclass1pt{minimaFor a subject labeled as ‘high-risk’, we increased Data generation process for this scenario is similar to Scenario B1, except that the shift  with the following methods: a) a joint model with regularization but without matching indicator and correlated taxa (JR-NC); b) the ZIBR model with a Wald statistic jointly testing OTU-specific abundance or presence using either a single random effect (ZIBR-S) or nested random effects (ZIBR-N); c) LMM with arcsin-square-root transformation using either a single random effect (LMM-S) or nested random effects (LMM-N). For LMM and ZIBR methods, we used R package gamlss and set the sample age, genotype, disease status, and genotype-disease interaction term as the fixed effect covariates. It’s worth to note that the nested random effects used in LMM and ZIBR are independent of host disease risk, different from those in JMR.Acinetobacter sp. NIPH 236, Brachyspira murdochii, Streptococcus phage YMC-2011, Erysipelatoclostridium ramosum, Ruminococcus gnavus. Then we generated We randomly selected 6 OTUs with different relative abundance from TEDDY data and estimated the baseline parameters for each. These OTUs are In scenarios B1, B2 and C, we generated 10 replicates for each OTU table to assess the performance of competing methods. We evaluated the performance of each method at different size of set-level random effect  B1 Fig.  showed tity Fig.  and the ity Fig. . Resultso C Fig. .Fig. 3FPFor each raw counts table in scenarios B and C, more than half of simulated OTUs have the observed zero-inflation probability  during infancy among IA cases, with density shown in Fig. B.fragilis as part of the normal microbiota in human colon was found more abundant among IA cases compared to their matched controls  associated with IA onset exclusively detected by both JMR and JMR-NC at mentclass2pt{minimh cohort .Fig. 8DiFaecalibacterium prausnitzii and Escherichia coli visualized in Fig. F.prausnitzii, as one of the most abundant and important commensal bacteria of human gut microbiota that produces butyrate and short-chain fatty acids from the fermentation of dietary fiber, increased faster in IA cases after 6-month of age. This rapid change and abnormally higher level of F.prausnitzii prior to IA seroconversion may be a result of the sudden change of dietary pattern during infancy.There are two more abundant species E.coli, which was found as an amyloid-producing bacteria with temperal dynamics heralding IA onset in a subset analysis in DIABIMMUNE cohort [E.coli in TEDDY smoothly decreased from 4-month to 9-month for both cases and controls  by parallel computing. We also provided simulation functions StatSim and TaxaSim to generate (binary) disease status and temporal high-dimensional metagenomic counts of matched sets. The runtime of each method for different sample size and different number of OTUs were compared on an 8-core computer, with mean and standard deviation shown in Table We developed a joint model with nested random effects to test the association between taxa and disease risk, and adjusted for the correlated taxa screened by a pre-selection procedure in abundance and prevalence, individually. We implemented our method in an R package mtradeR  with illustration examples at The simulation of single OTU demonstrated the performance of each method at different relative abundance levels, implying that LMM with either single or nested random effect is still a robust method. The simulation of high-dimensional OTU tables also illustrated LMM’s overall performance in the test of intercept, but the unstableness of LMM is a concern in real data analysis. JMR yielded lower false or pseudo positive rate in the simulated datasets and higher detection power in slope analysis by adjusting for the top-correlated taxa. The pre-selection of top-correlated taxa in JMR was performed in relative abundance and presence, individually, being consistent with the two-part model strategy. According to the simulation study, a disadvantage of JMR is the limited power at small sample size and the dependence on tuning parameter. The prescreening procedure in JMR may occasionally select a true biomarker as covariate taxon, which is possibly confounding with the subject-level random effect. Hence, the adjustment of related taxa in JMR reduced the detection power compared to JMR-NC, although this strategy controlled the pseudo biomarker rate. Adding nodes in the GH approximation may improve the power of JMR, but more nodes will also lead to additional computation costs. Hence, future work should focus on improvement of JMR in both detection power and computation efficiency. Furthermore, the simulation results in Fig. Another limitation of our method is the potential bias in scaling parameter , the right-censoring of disease outcome should be addressed prior to the usage of JMR, such as multiple imputation. There are other important topics to be considered in the modeling of longitudinal microbiome data. One potential direction is high dimensional modeling framework, such as tensor singular value decomposition . A promiThe proposed framework JMR successfully controlled the false or pseudo biomarkers in taxon-specific trajectory analysis with improved detection power by incorporating the matching of participants and adjusting for the dependence between taxa.jsJ participants in each matched set. Let j in matched set s at time point tThe probability for participant We set mentclass2pt{minimTo account for the sum-to-one restriction on non-zero relative abundance  of microbiota (Step 2: The mean frequency s at time point t by Step 3: At each time point, the heterogeneity between matched sets is the overdispersion estimated by DM MLE based on the samples per time point in TEDDY, denoted by j in set s at time t by Step 4: The between-subject heterogeneity within each matched set was the median DM MLE of overdispersion per matched set based on the real data, that is Step 5: The library size for each sample is simulated by a Poisson distribution Additional file 1:Appendix. Gauss-Hermite quadrature approximation for marginal likelihood.Additional file 2:Supplementary Table S1. The list of OTUs detected by JMR, JMR-NC, LMM-N, individually, as shown in Figure 7.


Using color Doppler flow imaging or high-definition flow imaging with three-dimensional volume or spatio-temporal image correlation (STIC) in the glass-body mode allows displaying both gray-scale and color information of the heart cycle-related flow events and vessel spatial relationship. Conventionally, STIC in the glass-body mode has been used to examine the fetal heart and assess heart defects. Recently, a novel application of STIC in the visualization of abdominal precordial veins and intraplacental vascularization in singleton pregnancies has been reported. The aim of this present review is to discuss the use of color Doppler with three- and four-dimensional ultrasonography in the evaluation of extracardiac, placental, umbilical cord and twin abnormalities with examples. The glass-body mode is complementary to conventional 2D ultrasonography. Further studies are required to investigate use of the glass-body mode in the assessment of intraplacental vascularization in singleton and twin pregnancies. It is a common practice to add color flow imaging (CFI) to gray-scale ultrasonography in obstetric scans to detect abnormal blood flow in fetal and placental abnormalities ,4,5,6,7.Using CDFI or HDFI with three-dimensional volume or spatio-temporal image correlation (STIC) in the glass-body mode allows displaying both gray-scale and color information of the heart cycle-related flow events and vessel spatial relationship ,9. WhileA bibliographic search was performed in November 2022 in the Medline/PubMed virtual library using the following descriptors: three-dimensional, Doppler ultrasound, four-dimensional ultrasound, spatiotemporal image correlation technology, and pregnancy. Articles in English, published between 2008 and 2022, available in full text in which the methodologies were case reports, observational studies or literature reviews were reviewed. There were no randomized clinical trials, meta-analyzes or systematic reviews on the use of the glass-body mode in pregnancy.While CDFI with STIC in the glass-body mode can evaluate abnormal anatomy of the fetal heart defects , the glaThe glass-body mode with HDFI can display the origin, course and branching pattern of the normal variants of pericallosal artery with the callosomarginal artery  Figure  after a A 3D volume acquisition along the transvers plane allows the reconstruction of the middle cerebral artery and the circle of Willis. 3D power Doppler measurement of cerebral blood flow is significantly altered in growth restricted fetuses . Flow inVein of Galen aneurysmal malformation is a rare and complex anomaly. Use of 3D power Doppler rendering can well demonstrate the malformation, its feeders and dilatation of straight sinus . HoweverWith transvaginal approach, 3D PDI/ HDFI can demonstrate small medullary vessels in normal and abnormal fetuses including malformations, hypoxia or infection . These fThe use of STIC combined with color Doppler may help evaluate the involvement of the vascular system in lung dysplasia, which is associated with neonatal mortality . In a feCongenital diaphragmatic hernia is associated with pulmonary hypoplasia. Various fetal ultrasound parameters have been used to predict neonatal outcome in isolated congenital diaphragmatic hernia. It seems that measuring contralateral pulmonary vascularization by 2D color Doppler or 3D power Doppler is the most accurate ultrasound parameter to predict neonatal death and severe pulmonary arterial hypertension . After fWhen a targeted examination of the fetal umbilical-portal venous system is required , a syste2D/3D ultrasonography with power Doppler can demonstrate renal artery. One and both renal arteries are not identified in fetuses with unilateral and bilateral renal agenesis, respectively. Variations of the origin of renal arteries are commonly found in normal pregnancies. However, renal malformations including horseshoe kidney and pelvic kidney can be associated with such vascular variations .Sacrococcygeal teratomas (SCT) are usually highly vascularized and thus can cause high-output cardiac failure, and even fetal death. Evaluation of the severity of SCT can guide appropriate management. In addition to SCT ratio measured by 2D ultrasonography, 3D power Doppler vascularization index % may be a promising prognostic indicator .Prediction of preterm delivery remains difficult. It is well known that a short cervical length measured by 2D ultrasonography is associated with preterm delivery. Addition of 3D transvaginal power Doppler ultrasound may improve the prediction . There aPlacenta accreta spectrum disorder (PAS) is a potentially life-threatening condition associated with severe postpartum hemorrhage. 2D ultrasonography with the use of color Doppler is a major modality to diagnose PAS and assess its severity. Addition of 3D Doppler ultrasound may help detect PAS and predict those PAS requiring hysterectomy . The larCFI is helpful in assessing the vascularization in various conditions of placental lakes or cysts which can be benign  or are aRecently Leung KY described a new use of STIC volume acquisition in HDFI, displayed in the glass-body mode, to show blood flow in the intraplacental branches of the umbilical artery (IPB) and the spiral artery jets in a cardiac cycle  Figure A. ComparPre-eclampsia is a major cause of maternal and fetal morbidity and mortality. 3D power Doppler whole placental volume scanning at 11–14 weeks’ gestation was studied to predict pre-eclampsia . The plaFetal growth restriction is common and is associated with adverse perinatal outcomes including mortality. It remains difficult to differentiate a growth restricted fetus from a small but normal fetus. The use of 3D power Doppler placental vascular flow indices in the prediction or identification of fetal growth restriction was reviewed . DetectiUsing CDFI or HDFI can facilitate the detection of a velamentous, marginal or furcate insertion of the umbilical cord into the placenta or vasa previa, which are associated with adverse pregnancy outcomes  Figure A. The glWith the advance in ultrasound technology, it is feasible to use 2D and 3D ultrasonography with HDFI to detect placental anastomoses in a monochorionic twin pregnancy, which are the underlying cause of the development of twin–twin transfusion syndrome (TTTS) . The glaSimilar to FGR in singleton pregnancies, the glass-body mode can be used to show differences in intraplacental vascularization between a normal twin and a FGR twin in a monochorionic twin pregnancy A,B. The The glass-body mode in HDFI can facilitate the detection of reverse arterial perfusion through the umbilical cord of the recipient twin in a case of Twin reverse arterial perfusion (TRAP) . BesidesIn general, the glass-body mode, a CFI, can be adversely affected by partial volume effect, limited temporal and velocity resolution, inappropriate angle of insonation, and aliasing . These aA recent review highlighted the challenges of 3D/4D CFI . The resImage quality in the glass-body mode can be improved by the followed steps. First, a good-quality image of the target structure, for example, the umbilical cord insertion site into the placenta, should be visualized by 2D ultrasonography. Before using color Doppler, it is useful to reduce beam width, depth of penetration and the number of focal points, and hence increase the frame rate and contrast resolution. Appropriate color flow imaging mode should be selected. In general, CDFI is chosen to assess high flow, and HDFI to assess medium or low flow .The angle of insonation is important. To acquire a 3D or STIC volume, an appropriate plane should be chosen. For examples, a cross-sectional plane close to the center of the placenta is used to visualize intraplacental vessels while the sagittal approach is used for visualization of the precordial venous system including ductus venosus. While the region of interest (ROI) should be large enough to include all the targeted vessels, the ROI and the acquisition angle should be kept as narrow as possible to maximize the frame rate during a volume acquisition. Selecting an acquisition time is a trade-off between enhancing the spatial resolution and reducing motion artifacts. The latter can be reduced by acquiring a 3D or STIC volume while the mother is holding her breath and there is no fetal or placental movement. Typical technical settings for a volume acquisition can be set to improve reproducibility.The acquired 3D or STIC volume is then stored in the ultrasound machine to enable subsequent processing. In the glass-body mode, the rendered image can be displayed in the glass-body fetal heart or the high-definition mode. While 3D volume allows display of static images, STIC volume allows assessment of the targeted vessels during a ‘cardiac cycle’ using a cine loop. The pulse rate of the vessels can be subjectively assessed. During the assessment, it is important not to rotate along the z-axis of a 3D or STIC volume so that the displayed red-blue color signals correspond to the direction of blood flow of the targeted vessels. To locate a pathological condition such as an arterio-venous anastomosis in a twin–twin transfusion syndrome, use of TUI can facilitate a systematic assessment. While adequate diagnostic ultrasound information is to be obtained, the ALARA principle  should be observed during a 3D or STIC volume acquisition on a developing fetus especially in the first trimester.The glass-body mode demonstrates vessel spatial course and its relationship with adjacent non-vascular structures by displaying both gray-scale and color information. The gray-scale information is important for the interpretation of the displayed image, especially in cases of fetal abnormalities. This mode can be used to examine the whole vascular system of the fetus, the intraplacental vessels and the umbilical cord vessels. Both the placenta and the umbilical cord are usually not affected by fetal movements, and thus are less prone to motion artifacts.Abnormalities of the fetal vascular system are often complex, requiring gray-scale imaging and color or power Doppler ultrasound to assess the abnormalities. 2D, 3D or 4D ultrasonography are non-invasive without radiation or contrast media. Although 2D ultrasound can allow examination of fetal vascular abnormalities, there are limitations in the examination when the examined vessels do not follow a straight course or lie in a 2D plane. In that case, the examiner needs to reconstruct mentally a 3D image of the examined vessels. On the other hand, 3D ultrasound rendered mode can allow display of complex vascular structures, whereas TUI can facilitate a systematic examination along multiple parallel sagittal, transverse or coronal planes.Displaying the glass-body mode after 3D or STIC volume acquisition in CDFI or HDFI can facilitate evaluation of various fetal non-cardiac, placental, umbilical cord and twin abnormalities, in addition to cardiac abnormalities . In part


Salmonella Typhi in Mumbai, linked to the acquisition and maintenance of a previously characterized IncX3 plasmid carrying the ESBL gene blaSHV-12 and the fluoroquinolone resistance gene qnrB7 in the genetic context of a triple mutant also associated with fluoroquinolone resistance.We report the persistent circulation of third-generation cephalosporin resistant  Salmonella enterica serovar Typhi (S. Typhi), which disproportionately affects populations in locations with limited sanitation and hygiene. There remains a significant burden of typhoid fever in India  differences) in comparison to the other 4.3.1.2 sequences  compared to ceftriaxone-susceptible isolates  is notable in this context but not significant  and ciprofloxacin (qnrB7) resistance in Mumbai, nested successively within a high-risk clone of chromosomally encoded ciprofloxacin resistance found in several countries in South Asia and associated with fluoroquinolone treatment failure [S. Typhi carrying the ESBL gene blaCTX-M-15 in an IncY plasmid in neighboring Pakistan [The World Health Organization classified third-, fourth-, and fifth-generation cephalosporins as critically important for human medicine. We characterized an emergent high-risk lineage of  failure , the 4.3 failure . The eviPakistan  and bioiPakistan  locally Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ciab897_suppl_Supplementary_MaterialsClick here for additional data file.


PTEN were unassociated with any DEM or DMG in contrast to DAs in all other drivers, which were associated with several DEMs and DMGs. This contrasting PTEN-related property of being unassociated with differential gene expression or methylation in IDH1-W-GBs was unaffected by concurrent DAs in other common drivers or by the types of DAs affecting PTEN. From the lists of DEMs and DMGs associated with some common drivers other than PTEN, enriched gene ontology terms and insights into the co-regulatory effects of these drivers on the transcriptome were obtained. The findings from this study can improve our understanding of the molecular mechanisms underlying gliomagenesis with potential therapeutic benefits.During oncogenesis, alterations in driver genes called driver alterations (DAs) modulate the transcriptome, methylome and proteome through oncogenic signaling pathways. These modulatory effects of any DA may be analyzed by examining differentially expressed mRNAs (DEMs), differentially methylated genes (DMGs) and differentially expressed proteins (DEPs) between tumor samples with and without that DA. We aimed to analyze these modulations with 12 common driver genes in Isocitrate Dehydrogenase 1 wildtype glioblastomas (IDH1-W-GBs). Using Cbioportal, groups of tumor samples with and without DAs in these 12 genes were generated from the IDH1-W-GBs available from “The Cancer Genomics Atlas Firehose Legacy Study Group” (TCGA-FL-SG) on Glioblastomas (GBs). For all 12 genes, samples with and without DAs were compared for DEMs, DMGs and DEPs. We found that DAs in  EGFR is altered by different types of mutations or amplifications  were concurrently downregulated in association with DAs in TP53 (and vice versa).Several genes were found to be concurrently upregulated (and downregulated) in association with DAs in PTEN’s lack of association with DEMs and DMGs is unaffected by concurrent DAs in other common drivers or by the types of DAs in PTEN. These findings indicate the ability of DAs in PTEN to get established within the heterogenous tumor landscape of IDH1-W-GBs without affecting its transcriptome or methylome. We showed that DAs in other drivers, such as CDKN2A, EGFR and TP53, are associated with fairly distinct sets of DEMs and DMGs with distinct contributions toward tumor phenotype, as apparent from the enriched GO terms and pathways. However, some common DEMs were noted among some of the sets. Analyzing these DEMs revealed some potential transcriptional co-regulatory mechanisms associated with these drivers. The transcriptional co-regulatory effects of the DAs in CDKN2A and EGFR were cooperative. In contrast, DAs in both EGFR and CDKN2A showed opposing transcriptional co-regulatory effects compared with DAs in TP53.Our main objective was to determine how DAs in common driver genes modulate the transcriptome, methylome and proteome in IDH1-W-GBs by examining the DEMs, DMGs and DEPs associated with them using Cbioportal. We observed that DAs in PTEN are unique because, unlike other DAs, they are unassociated with DEMs or DMGs. Different DAs are known to affect the transcriptome and methylome differently. However, such a complete lack of association with DEMs and DMGs has not been previously reported in association with any other DA or tumor, to the best of our knowledge. Moreover, The insights obtained from the findings in our study can improve our understanding of the molecular mechanisms underlying gliomagenesis with potential therapeutic benefits.


The emergence of artemisinin resistance is a major obstacle to the global malaria eradication/elimination programs. Artemisinin is a very fast-acting antimalarial drug and is the most important drug in the treatment of severe and uncomplicated malaria. For the treatment of acute uncomplicated falciparum malaria, artemisinin derivatives are combined with long half-life partner drugs and widely used as artemisinin-based combination therapies (ACTs). Some ACTs have shown decreased efficacy in the Southeast Asian region. Fortunately, artemisinin has an excellent safety profile and resistant infections can still be treated successfully by modifying the ACT. This review describes the pharmacological properties of ACTs, mechanisms of artemisinin resistance and the potential changes needed in the treatment regimens to overcome resistance. The suggested ACT modifications are extension of the duration of the ACT course, alternating use of different ACT regimens, and addition of another antimalarial drug to the standard ACTs (Triple-ACT). Furthermore, a malaria vaccine  could be added to mass drug administration (MDA) campaigns to enhance the treatment efficacy and to prevent further artemisinin resistance development. This review concludes that artemisinin remains the most important antimalarial drug, despite the development of drug-resistant falciparum malaria. Plasmodium falciparum parasites in Southeast Asia , intermittent preventive treatment (IPT)). The duration of post-treatment prophylactic effect depends on both the terminal elimination half-life of the drug and the susceptibility of the parasite to the drug, which determines the MIC. ACTs containing a partner drug with a long terminal elimination half-life can be used effectively in the prevention of malaria. The most promising combination is dihydroartemisinin-piperaquine, due to the long biological half-life of piperaquine (20–30 days), which allows monthly treatment to prevent malaria .de novo drug resistance is more likely to arise in patients with high parasitemia. The probability of drug resistance development would therefore be higher in patients with hyperparasitemia (>1012 parasites), compared to patients with uncomplicated acute symptomatic infections (108–1012 parasites), compared to patients with asymptomatic infections (<108 parasites). When/if such an event occurs, exposure to another drug with a different mechanism of action would eliminate the mutated parasite and prevent the drug resistant parasite to establish a patent infection. This is the main reason why combination therapies prevent drug resistance development. The ideal combination of drugs would be two (or more) highly potent compounds with different mechanisms of action, and matching pharmacokinetic properties . This would allow drug concentrations to be maintained above the MIC value for a similar duration of time in order to prevent emerging resistant parasites to establish a patent infection.Parasite drug resistance is the result of genetic changes that confers reduced drug susceptibility. This allows a mutated parasite to multiply when exposed to otherwise suppressive drug concentrations. Such a survival advantage, compared to wildtype parasites, would quickly be selected for during high drug pressure in a region, allowing the drug resistant strain to spread. Genetic mutations are by definition random events, and the absolute number of genetic mutations during an infection is therefore expected to increase with increasing parasitemia. Thus, 3–104-fold reduction in parasitemia per parasite life-cycle of 48 h, while the marketed ACT partner drug is less potent, resulting in approximately 101–103-fold reduction in parasitemia per parasite life-cycle of 48 h. It would be highly advantageous with novel partner drugs that are more potent than the currently available antimalarials. This rapid reduction in parasitemia associated with ACT reduces the likelihood of resistance development. However, decades of preceding artemisinin-based monotherapy resulted in the development of artemisinin-resistant parasites in Cambodia, which is now widespread throughout mainland Southeast Asia, resulting in a slower parasite clearance of P. falciparum Kelch13-mutated parasites compared to wildtype parasites (3-fold reduction in parasitemia per life-cycle)  is the most potent and rapidly acting antimalarials on the market. Artemisinin commonly result in approximately 10arasites . Artemisarasites , in Soutarasites , and on arasites . Artemise-cycle) . This ree-cycle) . We and e-cycle) .ACT-related elimination of a malaria infection results in an exponential decay of parasites. The time it takes to clear an infection is dependent on two variables (1) starting parasitemia and (2) rate of decline . Thus, iin vitro susceptibility tests, i.e., the trophozoite inhibition maturation assay (TMI) with an IC50 value >5 ng/ml  gene are associated with a prolonged clinical parasite clearance , and in vivo parasite clearance half-life indicate that PfK13 mutations are an important molecular marker of artemisinin resistance  and mutated PfK13  reduced ring stage drug susceptible compared to the mature stages , 2) enhaed PfK13 , 5) otheed PfK13 , and 6) ed PfK13 .P. falciparum parasites after artemisinin exposure have been reported in vitro . This could be a result of dormant parasites or the differences between humans and monkeys in host-parasite relationships. The possibility of artemisinin-induced dormancy emphasizes the importance of partner drugs in ACTs.Dormant in vitro  and in v campaigns in low transmission settings have been suggested to accelerate the elimination of falciparum malaria.Although resistance to artemisinin and its derivatives has emerged among  regions , ACTs arfalciparum malaria, parasites are potentially exposed to artemisinin for only one full parasite life cycle during the treatment course  during 2001–2004 showed >90% treatment efficacy, i.e., 28-day PCR-corrected rates of adequate clinical and parasitological response (ACPR)  . ASMQ wae (ACPR) . Howevere (ACPR) . This ree (ACPR) . In the his area . These dfalciparum malaria. Several alternative ACT regimens are also available. A recent systematic review and meta-analysis on the efficacy of ACTs used in Sub Saharan Africa from 2010 to 2020 reports an overall high success rate in the treatment of malaria using artesunate-amodiaquine, artemether-lumefantrine, and dihydroartemisinin-piperaquine in this region . There are several rationales behind this approach. Using multiple drugs with different mechanisms of action can increase treatment efficacy by targeting multiple components of the organism and reduce the dose of the individual drugs for a more acceptable side effect profile. There might also be pharmacological interactions between the drugs resulting in the additive or synergistic treatment effects. For infectious diseases, combination therapy has shown to also delay or prevent the development of drug-resistant microbes .P. falciparum parasites. Triple artemisinin-based combination therapies (TACTs) were suggested to treat multi-drug resistant falciparum malaria treatment, by adding a second slowly eliminated partner drug to a standard ACT. Mefloquine and amodiaquine were proposed to be paired with piperaquine and lumefantrine, respectively, based on their potentially counterbalancing resistance mechanisms and similar pharmacokinetic profiles. A study in healthy adult Thai volunteers showed that DP + mefloquine was safe and well tolerated  has been considered as a potential intervention to reduce the infectious reservoir in asymptomatic populations and thus accelerate malaria elimination  prolonged ACT treatment course, which is easy to apply with existing regimens but concerns have been raised on possible side effects of the long half-life drugs and patient compliance; 2) alternate sequential use of different ACT regimens in a region, which can also be applied with existing regimens but additional information are needed on optimal drug rotation timing; and 3) triple ACTs, which has additional benefits over the first two methods of increased efficacy and the ability to delay the development of drug resistance but drug-drug interaction data are needed to support the global implementation of TACTs. Another potential use of ACTs is in combination with malaria vaccine in MDA efforts to accelerate the elimination of malaria in low transmission setting.


Asthma is the most common chronic disease in childhood, yet little is known about rates of asthma and wheezing in children with congenital anomalies. This study explored the prevalence and risk of receiving anti-asthmatic prescriptions in children with congenital anomalies compared to children without anomalies in six European regions/countries.This was a EUROlinkCAT population-based linkage cohort study involving children from 0-9 years of age born between 2000-2014. Congenital anomaly data from six EUROCAT registries were linked to births data in national/vital statistics and to electronic prescription databases. Prescription/pharmacy dispensing records across regions were standardised to a Common Data Model. Anatomical Therapeutic Chemical classification codes beginning with R03 were used to identify anti-asthmatic prescriptions. Random-effects meta-analyses were performed to identify both the relative risk (RR) of receiving >1 anti-asthmatic prescription in a year relative to the reference group, and the heterogeneity of prevalence rates across registries and age group.A total of 5.1% of children with congenital anomalies and 4.9% of reference children were dropped from the study as they were not linked. Children with congenital anomalies  had a higher prevalence of >1 anti-asthmatic prescription and a significantly higher risk of being prescribed anti-asthmatics  compared to reference children . The increased risk was consistent across all age groups. Children with congenital anomalies were more likely to be prescribed beta-2 agonists  and inhaled corticosteroids . Children with oesophageal atresia, diaphragmatic hernia, genetic syndromes and chromosomal anomalies had over twice the risk of being prescribed anti-asthmatics compared to reference children. Regional differences in prevalence and risk of anti-asthmatic prescriptions were identified.Children aged <10 years with congenital anomalies consistently had higher prevalence and risk of receiving >1 anti-asthmatic prescription across age group and across European regions. This study demonstrates that information on the prevalence of anti-asthmatic prescriptions issued/dispensed can be obtained through data linkage to monitor changes in prevalence over time.


Novel immunotherapies continue to be developed and tested for application against a plethora of diseases. The clinical translation of immunotherapies requires an understanding of their mechanisms. The contributions of antibodies in driving long-term responses following immunotherapies continue to be revealed given their diverse effector functions. Developing an in-depth understanding of the role of antibodies in treatment efficacy is required to optimize immunotherapies and improve the chance of successfully translating them into the clinic. However, analyses of antibody responses can be challenging in the context of antigen-agnostic immunotherapies, particularly in the context of cancers that lack pre-defined target antigens. As such, robust methods are needed to evaluate the capacity of a given immunotherapy to induce beneficial antibody responses, and to identify any therapy-limiting antibodies. We previously developed a comprehensive method for detecting antibody responses induced by antigen-agnostic immunotherapies for application in pre-clinical models of vaccinology and cancer therapy. Here, we extend this method to a high-throughput, flow cytometry-based assay able to identify and quantify isotype-specific virus- and tumor-associated antibody responses induced by immunotherapies using small sample volumes with rapid speed and high sensitivity. This method provides a valuable and flexible protocol for investigating antibody responses induced by immunotherapies, which researchers can use to expand their analyses and optimize their own treatment regimens. Knowledge of the immune system’s intrinsic ability to recognize and destroy infectious pathogens and malignant cells has paved the way for attempts to control numerous diseases by immunological means, thereby advancing the field of immunotherapy to what it is today. Many immunotherapies are predicated on exploiting the specificity and selectivity of host immune responses to fight disease, and function by improving the quality and/or quantity of immunological effector mechanisms against a desired target to reduce the severity of clinical disease. The armamentarium that broadly fulfills the definition of immunotherapeutic agents is extensive, including but not limited to, cancer vaccines, virus-vectored vaccines, oncolytic viruses (OVs), monoclonal antibodies, immune checkpoint inhibitors and more, with each uniquely positioned to enhance the built-in protective mechanism of host immune responses against several public health concerns –8.The means by which the immune system identifies and eliminates pathogens or neoplastic cells are complex, and include the involvement of cellular and humoral effectors to drive protective responses –11. HistGiven the potential involvement of antibody responses in mediating disease outcomes, it is important to identify therapy-induced subclasses of immunoglobulins (Igs) that contribute towards a tailored and maximally protective effect following immunotherapy. As such, methods that measure antibody responses to various classes of immunotherapies may prove key to improving efficacy. We previously developed a flexible methodology for detecting antibody responses generated by antigen-agnostic immunotherapies . Here, wThe protocol herein provides an advantageous method for assessing broad endogenous antibody responses against multiple antigenic targets following immunotherapy. By facilitating detection of a variety of isotypes of therapy-induced antibodies, the presented methodology can be used to dissect primary antibody responses, which are often of low magnitude to tolerized tumor antigens following many cancer immunotherapies, and subsequent secondary antibody responses in patients that may have pre-existing antibodies at the time of initial treatment or that receive multiple rounds of treatment. Additionally, this protocol can be extended to analyze immunoglobulin class switching and type 1 versus type 2 immune response biases elicited by a given immunotherapy platform throughout the course of the antibody response. In turn, this can improve the current understanding of both the quantitative and qualitative nature of immunotherapy-induced antibody responses.o Heparinized microhematocrit capillary tubes to collect plasma . Alternatively, serum from clotted blood can be used, but this would restrict analyses to serum-derived factors only. Use of unclotted blood facilitates simultaneous cellular analyses.o 1.5 mL microfuge tubeso Gauze padso Eye Lubricanto Container filled with ice pelletso Gauze padso 3 mL syringeso BD PrecisionGlide™ Single-use Needles o 1.5 mL microfuge tubeso Phosphate-buffered saline o Container filled with ice pelletso DF-1 , Vero  and ID8  cellso Complete Dulbecco’s modified eagle’s medium (DMEM)  or media specific to target cell line of interesto 10% fetal bovine serum o Penicillin/Streptomycin cocktail o 1x non-essential amino acids o 0.25% Ethylene-diamine-tetra-acetic acid o PBSo Gibco™ Trypan Blue Solution, 0.4% o MycoAlert PLUS Mycoplasma Detection Kit 5 target cells per well of a 96-well U bottom plate is optimal for antibody analysis. The total number of cells required will depend on the chosen sample dilution series and the total number of samples. Total target cell numbers needed should be determined ahead of time to prepare the appropriate number of T75cm2 flasks to facilitate target cell growth.o Cell culture-treated flasks and 96-well U-bottom plates . 1x10o 50 mL conical tubes o Multi-channel and standard 10-50 µL and 30-300 µL pipetteso Fluorescence-activated cell sorting (FACS) tubes o Bovine serum albumin o FACS buffer (PBS + 0.5% BSA)o Hank’s Balanced Salt Solution o Fixation buffer o Intracellular staining permeabilization wash buffer o Quantum Molecule of Equivalent Soluble Fluorophore (MESF) Bead Kit . The choice of kit will vary depending on the fluorochromes of the secondary antibody conjugates being utilized. Manufacturer recommendations should be followed to set up for analysis.o Murine AntibodiesIgG1 - Alexa Fluor 488 IgG1 - Allophycocyanin (APC) IgG2a/c - Alexa Fluor 488 IgG2b - Phycoerythrin cyanine 7 (PE-Cy7) IgM - Peridinin chlorophyll protein complex (PerCP-Cy5.5) IgA – Brilliant violet (BV) 421 o Biological safety cabinet – for all steps which involve sterile cell culture and sample processing2 and 37.0°C)o Humidified incubator  was used to generate the data shown here. Manufacturer recommendations should be followed to set up the flow cytometer for multi-color analysis.o FlowJo  and GraphPad Prism  software was used to analyze and graph, respectively, the flow cytometry data presented here.o For an experimental workflow, refer to Culture target cells in flasks or plates to 80%-90% confluencyo Tissue-culture treated flasks and plates were used for adherent cell lines.o Cell lines for this research were either gifted or obtained directly from ATCC. To assure reproducibility, cell lines were expanded in isolation from other cell lines immediately upon arrival, and many aliquots were frozen to create a low-passage lab stock from which project-specific stocks were made. All cell lines were confirmed to be mycoplasma-free using a MycoAlert PLUS Mycoplasma Detection Kit.de novo to be represented. This facilitates detection of the full breadth of the antibody response induced by the immunotherapy.o When using this assay to assess tumor-associated antibody responses, using autologous tumor cells as the target cell type allows for every relevant tumor antigen not generated o For determining non-tumor antigen-specific therapy-induced antibodies, non-tumor permissive cells expressing the relevant target antigen(s) were obtained by infection with an antigen-expressing viral platform distinct from the viral-vectored vaccine platform used for treatment. Where applicable, a standard immunofluorescence assay (IFA) can be performed to determine if there is a sufficient concentration of the target antigen expressed on cells.e.g., 10 mL per T75cm2 flask)Remove culture medium and wash cells in a large volume of PBS  and/or peritoneal lavage fluid in 1.5 mL microfuge tubes containing 5 μL of heparin (3 μg/mL diluted in HBSS) to prevent clotting and put on iceo It is recommended that the volume of sample harvested be enough to allow for preparation of all sample dilutions required, based on the maximum volume of sample allowed by the institutional animal care committee guidelines.The work presented here was approved by the University of Guelph Animal Care Committee  and adhered to the policies published by the Canadian Council on Animal Care.o Transfer blood or peritoneal lavage fluid to FACS tubes and record the volume for each sample to facilitate normalizing data across samples on a per μL or per mL basis.g for 10 min at 4°CCentrifuge at 500xo Centrifugation of samples will separate plasma, or cell-free ascites fluid to the top layer. Collect clarified top layer without disturbing the cell pellet and aliquot into new 1.5 mL tubes.Heat-inactivate samples at 56°C for 30 min and then store on ice if continuing to step 3 or store the samples in an ultra-low temperature freezer, if using the pause point.This step enhances the sensitivity of antibody detection by eliminating complement proteins that could interfere with target antigens.o Pause point: Samples can be stored long-term at -80°C for future detection of antibody responses utilizing this procedure. Samples should be thawed on ice or at 4°C overnight prior to preparing sample dilutions.If analyzing antibody responses directly following sample collection and heat-inactivation, samples should be kept on ice before and during preparation of sample dilutions.o g for five minutes and discard supernatant by rapid inversion of the plate followed by blotting on absorbent paper, and then re-suspend the cells by gently tapping the side of the upright plate.Centrifuge plates containing fixed and permeabilized antigen-expressing target cells at 500xWash cells in 50 μL of PBS per well, repeat centrifugation and discard supernatantBlock cells in 100 μL of 1% BSA-PBS per well and incubate for 20 min at 4°CPause point: blocking can be done overnight at 4°CoPrepare dilutions of samples in 1% BSA-PBS in a 96-well plate for easy transferA range of dilutions should be tested for each experiment to identify one that is optimal . A six-dilution series was chosen in the experiments herein to generate accurate mean areas under the curve.o g for five minutes and discard supernatantRemove the blocking solution by centrifuging plates containing target cells at 500xAdd diluted samples in 100 μL to target cell wells and incubate for one hour at 4°CPause point: Antibody-containing samples can be incubated with target cells overnight at 4°Co Dilute wells with 100 μL of 1% BSA-PBSg for five minutes and discard supernatantCentrifuge at 500xWash with 200 μL of 1% BSA-PBSRepeat the centrifugation and wash stepAdd all secondary detection antibodies at the recommended manufacturer or optimized dilutions in 1% BSA-PBS for a total of 100 μL per well.Antibodies should be titrated and tested at a range of dilutions to identify one that is optimal for positive signal detection.o Incubate for one hour at 4°C in the darkDilute with 100 μL of 1% BSA-PBSg for five minutes and discard supernatantCentrifuge at 500xWash with 200 μL of 1% BSA-PBS and repeat centrifugation and discard of supernatantResuspend in 200 μL of FACS buffer for analysis on a flow cytometerThe procedure for preparing quantum beads was provided by Bang Laboratories, for use with the Quantum MESF Bead Kit (Cat#488). We advise optimizing the use of the desired microsphere standards in any application based on the manufacturer’s recommended conditions and protocols.Refer to Data were analyzed using FlowJo software, but equivalent programs could be used. Target cells were gated based on forward scatter-area and side scatter-area characteristics. Doublets were then excluded by plotting forward scatter-area versus forward scatter-width. The presence of sample-derived antibodies bound to target cells were determined based on fluorescence emitted by secondary detection antibodies (Data were plotted on an x-y graph as mean fluorescence intensity (MFI) derived from a secondary detection antibody versus the number of target cells. This was done for all six dilutions of the tested sample. These data could then be plotted and used to calculate the area under the curve. The fold-change in area under the curve of treated samples compared to untreated samples could then be determined, following subtraction of the area under the curve for samples used to assess background fluorescence. A data analysis summary from data export to graphical representation of the results is provided in To maximize the sensitivity of this assay, samples of interest should be collected at the peak of the antibody response. Kinetics of peak antibody responses can be determined using this method by sampling on multiple days post-treatment. Following a kinetic analysis of the immune response, we chose day 21 post-treatment to quantify antibodies induced by our particular immunotherapy.Approximate time based on an experiment with 20 mice:Transduction/Plating of target cells: 24 hoursCell fixation and permeabilization: 30 minutesBlood or lavage fluid sampling: one hourPreparing sample dilutions: one hourBlocking and binding of antibody-containing samples: two hours (or overnight)Detection with secondary antibody: one-and-a-half hoursRunning samples on a flow cytometer: two hoursTotal time from sample collection to data analysis: ~32 hours.The outcome of this assay depends on the use of sufficient numbers of cells with preserved surface antigens that serve as reservoirs of the desired target antigens and can be detected on the flow cytometer. It is important to evaluate each target cell line for the optimal initial seeding density, growth kinetics and antigen expression (where applicable) to achieve adequate conditions at the time of the assay. The use of EDTA-PBS instead of trypsin-EDTA to detach adherent cells from plates allows for the preservation of trypsin-sensitive antigens on the surface of target cells. Together, these measures ensure a consistent number of available antigenic targets for all samples and minimizes background fluorescence otherwise caused by non-specific binding of secondary antibodies to the plate.Incomplete monolayers or loss of cells during the experimental procedure can result in variability and artificially low on-target signals due to a reduction in the quantity of target antigens. To maximize adhesion and recovery of target cells to ensure high quality signals, cells were fixed in non-polymer-coated FACS tubes prior to plating to prevent fixing of cells to the sides of polymer-coated 96-well U-bottom plates  can be utilized. These controls are important to include in each experiment to prove that antibody responses are truly therapy-induced and/or antigen or tumor-specific. For example, in the case of utilizing this assay to detect virus-associated antibodies following treatment with a virus-vectored vaccine platform, each experimental sample should be incubated with both virus-infected and uninfected cell controls. In the case of utilizing this assay to detect tumor-associated antibody responses, tumor cells that are different from those used for tumor implantation could be used to identify responses to antigens shared between cancer cells. Normal cell controls can also be used to detect antibodies that exist against non-cancer-specific antigens that may cross-react with target tumor cells. Therefore, it is important to include a sham-treated control group in each experiment to prove that the detected antibody responses are truly therapy-induced. During data analysis, positive secondary signals from the off-target cell controls would be categorized as ‘off-target background’ and should be subtracted from fluorescence values acquired from test wells.The desired sample under investigation acts as the potential source of antigen-specific antibodies which bind to antigens in or on permeabilized target cells. Antibodies bound to antigens that are retained after washing are detected by isotype-specific secondary antibodies conjugated to fluorochromes. Induction of relatively low concentrations of antigen-specific antibodies is expected from many immunotherapies, particularly cancer-targeting platforms as they attempt to reactivate the immune system against self-derived, weakly immunogenic cancer antigens. This can be particularly problematic in scenarios where oncolytic viruses are the chosen therapeutic modality, given that more robust neutralizing antibody responses are frequently seen against the virus backbone-derived antigens. Thus, acquiring maximal volumes of blood from experimental animals will maximize the chance of detecting tumor-directed antibodies while facilitating splitting of samples for simultaneous assessment of antibodies induced against the viral vector.The sensitivity of this assay relies on optimization of the dilution of the samples and secondary detection antibodies. At extremely high sample concentrations, there is an increased risk of non-specific binding of antibodies within samples to target cells, creating a threshold in terms of how much fluorescence can be detected. To resolve this, it is recommended that a range of sample dilutions are included for each sample in each experiment, especially if relatively high-magnitude antibody responses are expected. Non-specific binding of secondary antibodies can also occur, especially when they are used at high concentrations. Each fluorochrome-conjugated secondary antibody of interest should be tested at a range of dilutions with target cells only to determine a dilution that fails to yield a significant fluorescent signal on the flow cytometer. This ensures optimal detection of antigen-specific antibodies in experimental samples that have bound to target cells.To provide a more accurate depiction of the antibody response to an immunotherapy by utilizing this protocol, it is recommended that positive secondary antibody signals be quantified based on MFI. This is because MFI values indicate the density of fluorochrome-conjugated secondary antibodies bound to target molecules on a per cell basis , 29. AttIn this study we used autologous tumor cells as natural sources of undefined tumor-associated antigens or permissive non-tumor cells infected with an antigen-expressing viral vector to induce expression of a pre-specified target antigen. To ensure accurate comparisons of antibody concentrations between different samples, it is important that the target cells in each well express similar quantities of antigens. As such, using a single batch of cells within a given assay is important. Whenever possible it is recommended that a large batch of target cells be stored frozen as single-use aliquots that get thawed, passaged once and then used as is or then infected with an antigen-expressing virus and then used. This will maximize the consistency of results between different experiments. When the antigenic target is known, it is recommended to confirm uniform expression of reasonable concentrations of the antigen in or on cells prior to using this assay in a project. This can be accomplished by western blotting and use of an immunofluorescence assay.) and cell-free ascites fluid  samples collected from tumor-bearing control mice that were treated with PBS yielded lower detection-antibody-mediated fluorescent signals following removal of background fluorescence. This is indicative of low-magnitude, tumor-induced IgG1 antibody responses. In contrast, mice treated with the OV had evidence of higher magnitude tumor-associated IgG1 antibody responses, as evidenced through greater MFI signals and area under the curve values  of three for 16 hours to ensure that most cells would be exposed to the virus to maximize production of viral proteins but without a substantial amount of virus-induced cell death occurring. The method described in this paper was then conducted, with the inclusion of uninfected Vero cells as the off-target cell control. Plasma samples from tumor-free mice treated with OrfV were included in the assessment to control for the potential binding of tumor-specific antibodies present within samples to Vero antigens that may be shared with ID8 cells. Mice treated with OrfV had potent virus-associated IgG1 responses, as depicted by an overall increase in fluorescent values compared to plasma from untreated tumor-bearing or tumor-free mice Figure 5To expand the use of this methodology for evaluating vaccine-induced antibody responses to a pre-defined antigen, female Balb/c and C57BL/6 mice were inoculated either intramuscularly with a recombinant adenovirus expressing the full-length human SARS-CoV-2 spike protein (Ad-FLS), or intranasally with Ad-FLS, or with PBS. To express the known target antigen, in this case the SARS-CoV-2 spike protein, in a cell line that normally lacks expression of the protein, DF-1 cells were infected at an MOI of three with a heterologous recombinant avian orthoavulavirus-1 (AOaV-1) expressing the full length SARS-CoV-2 spike protein [AOaV-1-FLS ] for 16 The experiments presented herein demonstrate the utility of this method as a rapid and effective alternative to other techniques for assessing the broad spectrum of antibody responses induced by vaccination to pre-defined antigens on an isotype-specific basis. In doing so, this method can be used to analyze class-switching and type 1 versus type 2 immune response bias induced by an immunotherapy or vaccine. Both these immunological parameters contribute towards dictating therapeutic outcomes.®, whereby the MESF value of each microsphere population was correlated to the MFI value observed. Within the same experiment, plasma collected 21 days following treatment of tumor-free and ID8 ovarian tumor-bearing C57BL/6 mice with PBS or OrfV was analyzed for evidence of OrfV-associated antibodies. Vero cells were infected with OrfV at an MOI of three for 16 hours and were incubated with serial dilutions of plasma samples following the protocol described in this paper. Gating of target cells on the flow cytometer was done based on FSC-A and SSC-A characteristics. Doublets were excluded by plotting FSC-A versus FSC-W. The anti-mouse IgG1 Alexa Fluor 488-derived fluorescent signal and respective MFI value corresponding to virus-associated antibody binding was then determined for target cells treated with each respective sample  to be represented. The method can also be used following antigen-specific therapies where the pre-defined target antigen is expressed in a cell line that normally lacks expression of the protein. In theory, the procedures provided herein can be used to assess the full breadth of antibody responses elicited by any given immunotherapy. It offers several additional advantages such as rapid high-throughput readout, cost-effectiveness, sensitivity, versatility due to compatibility with several sample sources, isotype-specific Ig detection, and multiplex capabilities. While conventional immunological techniques used to detect antibodies, such as the enzyme-linked immunosorbent assay, can equally be applied for quantification of antibody concentrations, these frequently lack the sensitivity necessary to detect target antigens expressed at low concentrations on cells, to detect antigens that are not pre-defined, or to detect low numbers of cells that express the target antigen. Additionally, these remain time- and sample-consuming, with typically only a single Ig-isotype against one antigenic target able to be measured at a time and at a higher cost. The method described here permits the simultaneous analysis of multiple antibody isotypes against either pre-defined or undefined bulk target antigens from a single, small-volume sample. With as little as 20 µL of input sample, any number of antibody isotypes and subtypes can be tested, depending on how many channels are available on a flow cytometer. In addition to the small volume of samples and reagents required, this assay has a relatively quick staining process and contains fewer overall steps than more traditional techniques. By using automation and a flow cytometer capable of high-throughput data acquisition, the entire repertoire of the antibody response to a given immunotherapy can be rapidly detected from 96 samples in a single experiment, within two hours.This protocol builds on our previously established methodology for detecting and quantifying tumor- and virus-associated antibodies following immunotherapy . This flThrough the detection of multiple subclasses of Ig responses, this method can also be used to analyze class-switching and immune response biases induced by immunotherapies. Identification of type 1 vs type 2 immune response biases is relevant in the context of cancers, given that pre-malignant and malignant tissues have commonly been associated with suppressed T-helper cell-1 responses and enhanced T-helper cell-2 responses that correlate with tumor progression and subdued anti-tumor responses , 39. ThiThe protocol presented here represents a valuable methodology that can be added to the toolbox of researchers to evaluate the role of endogenous antibodies induced by any given immunotherapy. The integration of this assay into routine pre-clinical and clinical assessments can support early detection of malignant cells or viral infections, establishment of prognoses for patients, assessment of treatment efficacy, and help inform the design and optimization of next-generation immunotherapies.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Animal Care Committee, University of Guelph.Conception and design: JAM, JPvV, GAW, AMV-P, KK, JJP, SKW and BWB; development of methodology: JAM, JPvV, SKW and BWB; acquisition of data: JAM, JPvV, JGEY, LC, JJP; analysis and interpretation of data: JAM, JPvV, SKW and BWB; writing, review and/or revision of the manuscript: JAM, JPvV, GAW, AMV-P, KK, JJP, SKW and BWB All authors contributed to the article and approved the submitted version.Funding was provided by Discovery Grants from the Natural Sciences and EngineeringResearch Council of Canada to BB. (#436264) and SW (#499834), anOperating Grant from the Cancer Research Society to BB and SW(#843296), and an Operating Grant from the Pet Trust Foundation to KK(#054725). Stipend funding was from: Ontario Graduate Scholarship andNora Cebotarev & Ellen Nilsen Memorial Scholarship (for JM); CanadianGraduate Scholarship-Doctoral Award (NSERC), Ontario GraduateScholarship and Ontario Veterinary College (OVC) Graduate Scholarship (forJV); OVC Graduate Scholarship (for JY and LC).We thank Campus Animal Facilities, University of Guelph, for animal care services.B.W.B is the Chief Scientific Officer of ImmunoCeutica Inc., which is dedicated to the research and development of immunoceuticals. S.K.W. is a founder of Avamab Pharma Inc., which produces gene therapy vectors that express antibodies, and the senior scientific advisor for Cellastra Inc., which is dedicated to research and development of gene therapies targeting root causes of scarring. ImmunoCeutica Inc., Avamab Pharma Inc., and Cellastra Inc. did not provide any funding, nor did they have any role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Frontiers in Chemistry topic collection on Advances in Mechanically Bonded Molecules is dedicated to progress on the fundamental science of how novel structures and dynamics emerge from molecular entanglements.Mechanically bonded molecules such as rotaxanes and catenanes are utilized increasingly in energy storage,  bio-inteRadial [n]catenanes.Wu et al. report a new synthesis of [3]- and [4]catenanes by way of coordination-driven self-assembly of carboxylated Cu(I) metallo-pseudo [2]rotaxanes with divalent organoplatinum complexes by [2 + 2] or [3 + 3] macrocyclization. Although topologically equivalent assemblies have long been accessed by metal-ligand complexation  is rarely demonstrated.  unit of a symmetrical axle, but it exhibits mechanically planar chirality when alkylation or deprotonation of DBA displaces the ring from center. The resulting left- and right-handed enantiomers interconvert as the ring shuttles between two sides of the dumbbell. This “mechanostereoinversion” slows down as the steric bulk of the N-alkyl group increases, allowing enantiomeric resolution by chiral HPLC in some cases. This work lays a strong foundation for molecular machines that switch between optically active and inactive states.Photodriven molecular shuttles. Switchable molecular shuttles are a canonical archetype of artificial molecular machines (AMMs). Since light is among the most attractive fuel sources , photo-driven molecular shuttles represent one of the largest classes of AMMs. A review by Yao et al. summarizes recent progress on molecular shuttles driven by light, highlighting technological applications and prospects for future development.Rigidity and flexibility in rotaxanes. In a topic not previously reviewed, Fadler and Flood explore the role of flexibility and rigidity on the properties of rotaxanes. Threading rings onto axles leads to their mutual rigidification, the extent of which is highly system-specific, yet it impacts the energy barriers and timescales of mechanical motion and even bulk material properties. These concepts can be leveraged in the design of rotaxanes with highly tailored functions.Since bonding is the foundation of molecular structure and dynamics, mechanical bonds on the molecular scale profoundly impact material properties. The field remains rich in untapped potential for new discoveries, and will no doubt continue to fuel technological progress as the fundamental science of mechanical bonds continues to be nurtured.


Plukenetia volubilis L.) has been adopted as a novel economic crop with well-studied nutritional and bioactive benefits for human health. Sacha inchi seeds and oil have high commercial value but scant research has focused on its leaves. This study investigated and compared phenolic compositions, antioxidant potentials and in vitro health-related properties of both young and mature sacha inchi leaves after freeze-drying and oven-drying processes. Results showed that p-coumaric acid, 4-hydroxybenzoic acid, ferulic acid and gallic acid were predominantly detected in both young and mature leaves that also exhibited similar total phenolic contents (TPCs), while higher TPCs were detected in freeze-dried than in oven-dried leaves. Mature leaves exhibited higher antioxidant potential than young leaves after freeze-drying, while the opposite results were observed for oven-drying. Overall in vitro health-related activities were higher in mature leaves compared to young leaves regardless of the drying process. Knowledge gained from this study can be used to encourage prospective utilization of sacha inchi leaves as a source of health-promoting compounds. This, in turn, will increase the commercial value of the leaves and provide a wider market variety of sacha inchi products.Sacha inchi ( Plukenetia volubilis L.), also known as Inca peanut, sacha peanut or mountain peanut is native to the Peruvian Amazon. It has long been a staple crop in the diets of Latin American populations, and also a herbal remedy used widely by indigenous locals in Peru . Preservation methods, including drying, can alter the natural antioxidant capacity of plant-based food products; hence, the impact of such processes should be considered for the accurate evaluation of product health benefits. Phenolics are well recognized as the major contributor to the antioxidant activities of plants. A strong correlation between TPCs and antioxidant activities was reported in our previous research , which dThe anti-glycation capacity of plant materials is often related to their phenolic compositions and antioxidant activities. A literature review on the inhibitory effect of phenolics on advanced glycation end products (AGEs)  suggeste50 of 92.8 and 71.0 µM, respectively  wex) of 320 nm and an emission wavelength (λem) of 405 nm. The glycation reactions were detected using BSA induced by either MG or D-glucose as previously described [ex 330 nm and λem 410 nm. All chemicals and reagents were purchased from Sigma-Aldrich. The inhibitory assays were performed on the SynergyTM HT 96-well UV-visible microplate reader, and the results were calculated as percentage of inhibition at particular extract concentrations using the following equation:A is the initial velocity of the control reaction with enzyme, a is the initial velocity of the control reaction without enzyme, B is the initial velocity of the enzymatic reaction with the sample, and b is the initial velocity of the reaction with the sample but without enzyme.The inhibitory activity of BACE-1 was determined using BACE-1 assay kit from Sigma-Aldrich . The reaction was detected as end-pointed assay at an excitation wavelength (λn = 3), and the results were presented as mean ± standard deviation (SD). Unpaired t-test was used to compare significant differences between values of two data sets. One-way analysis of variance (ANOVA) followed by Duncan’s multiple comparison test was employed to compare significant differences between values of more than two data sets. Values were considered significantly different when p < 0.05. All data were analyzed using the statistical package for the social sciences .All experiments were conducted in triplicate  and retarding the aging process. Both young and mature leaves of sacha inchi showed promise as a source of bioactive compounds for health-related applications. Hot-water extraction was selected for easy and convenient preparation. Information obtained from this study will be useful for the future development of food/dietary supplement products from sacha inchi plant material, and also increase the commercial value of sacha inchi leaves by providing a wider variety of products to consumers. Since hot-water extraction as a tea infusion was chosen as a model for preparation of sacha inchi leaf extracts in this study, our leaf infusions have great potential for commercialization as a functional drink with further research in product development. Furthermore, sacha inchi leaf extract may be concentrated and used as a complementary food supplement, thus helping to enhance bioactivities of the food products. Other than tea and concentrated leaf extract, whole leaves may be consumed raw in fresh-cut salads or added to cooked recipes. However, further in vivo research is required to confirm our in vitro findings. The aspects of effective dose, bioavailability and pharmacokinetics should also be considered.


A refined FI (FIr) was calculated by using a subset of items, identified using factor analysis of mixed data (FAMD)-based cluster analysis. We developed both FIs for the ADNI1 cohort (n = 819). We also calculated another standard FI (FIc) developed by Canevelli and coworkers. Results were validated in an external sample by pooling ADNI2 and ADNI-GO cohorts (n = 815). Cluster analysis yielded two clusters of subjects, which significantly (pFDR < .05) differed on 26 health items, which were used to compute FIr. The data-driven subset of items included in FIr covered a range of systems and included well-known frailty components, e.g., gait alterations and low energy. In prediction analyses, FIr outperformed FIs and FIc in terms of baseline cognition and future dementia conversion in the training and validation cohorts. In conclusion, the data show that data-driven health deficit assessment improves an FI's prediction of current cognitive status and future dementia, and suggest that the standard FI procedure needs to be refined when used for dementia risk assessment purposes.Frailty is a dementia risk factor commonly measured by a frailty index (FI). The standard procedure for creating an FI requires manually selecting health deficit items and lacks criteria for selection optimization. We hypothesized that refining the item selection using data-driven assessment improves sensitivity to cognitive status and future dementia conversion, and compared the predictive value of three FIs: a standard 93-item FI was created after selecting health deficit items according to standard criteria (FIThe online version contains supplementary material available at 10.1007/s11357-022-00669-2. Frailty is an age-related state of multisystem physiological decline increasing the risk of adverse outcomes such as hospital complications and death –3. The fStandard FIs comprise a selection of several age-related health deficits which fit pre-defined criteria , and, whIn general, one approach to maximize explained variance is by employment of data-driven techniques which decompose and weigh correlated input variables. While the accuracy of a neural-network based machine learning approach outperformed an unweighted  FI in the prediction of mortality , the appr) by applying FAMD-based clustering to a set of health variables selected according to standard criteria .Several studies have employed prediction models of dementia due to AD , but to r HR-estimates for mortality in ADNI were comparable to those in the literature [r was the only FI-variable correlating with mortality in fully adjusted analysis Below is the link to the electronic supplementary material.


Persistent trigeminal artery (PTA) is a rare arterial anastomosis between the basilar artery (BA) and internal carotid artery (ICA). It plays an indispensable role in a number of neurological disorders, including trigeminal neuralgia (TN).We report a unique case of a 58-year-old Han female patient with TN caused by PTA associated with craniosynostosis. Preoperative three-dimensional time-of-flight (3D-TOF) magnetic resonance (MR) and 3D constructive inference in steady state (3D-CISS) imaging showed that the PTA run though Meckel’s cave. Complete pain relief was immediately achieved after microvascular decompression (MVD), without facial numbness and other complications. No recurrence was recorded at the 1-year follow up.Microvascular decompression is a feasible option for the treatment of complex TN combined with other abnormalities. For patients deemed suitable for percutaneous balloon compression, PTA should be ruled out. Preoperative 3D-TOF and 3D-CISS MR imaging were essential to identify PTA neurovascular conflicts. Persistent trigeminal artery (PTA) is the most common abnormal arterial anastomosis between the basilar artery (BA) and internal carotid artery (ICA) , 2, and This study was approved by the Ethics Committee of First Affiliated Hospital of Sun Yat-sen University  and has been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its amendments. Details that might disclose the ID of the patient were omitted. Written informed consent was obtained from the patient for publication of this case report and any accompanying images.A 58-year-old Han woman presented to our department in November 2020 complaining of typical TN of the second and third branches of the nerve in the right side of the face for the past 20 years. The pain started during physical activities. She took carbamazepine and had gradually increased the dose to 1200 mg per day, but severe pain remained and the high doses caused severe side effects. TN had been diagnosed 12 years previously at another hospital, where microvascular decompression (MVD) was carried out but failed to exposed the trigeminal nerve. Neurological examinations showed no other abnormalities of the cranial nerve. Three-dimensional time-of-flight (3D-TOF) magnetic resonance (MR) and three-dimensional constructive inference in steady state (3D-CISS) imaging showed obvious neurovascular conflicts (NVCs) due to PTA and the trigeminal nerve and that the PTA originated from the junction of the C3 and C4 segments of the ICA and went up around inner margin of Meckel’s cave, eventually to terminate into the right superior cerebellar artery (SCA) which anastomosed with the anterior inferior cerebellar artery (AICA) . Teflon patches were inserted between the trigeminal nerve and vessels deemed to be responsible for the pain, to achieve complete decompression and release of all culprit vessels Fig. . The patIn our case, TN was caused by compression of the PTA, which is an abnormal arterial anastomosis between the ICA and BA. As PTA originates from the ICA, it usually runs medially to the first branch of the trigeminal nerve and extradurally beneath the oculomotor and trochlear nerves, following which it passes the petroclinoidal ligament or perforates the dura around the clivus to join the BA. Only rarely does the PTA enter the posterior cranial fossa, either from Meckel’s cave or from the dural foramen, and cause cerebellum vascularization with no anastomoses with the BA . We suspDuring the developmental stage of a 4-mm embryo, the ICA supplies the longitudinal neural arteries via the trigeminal artery, optic artery, hypoglossal artery and proatlantal intersegmental artery. In subsequent developmental stages the arteries normally regress and disappear, but if there is incomplete regression or the ipsilateral posterior cerebral artery does not develop properly, a persisting anastomosis, such as PTA, may occur .Both the REZ and cisternal part of the nerve are reported to be the responsible sites of TN . ChidambHemorrhage has been reported to be a severe complication in percutaneous balloon compression (PBC) procedure . Also, PExposure of the trigeminal nerve, especially the REZ of the nerve, is largely confined to patients with narrow posterior cranial fossa , 19. HowThe CT imaging of our patient indicated that the osteoclasia around the foramen oval on the right side does not form a complete bony boundary, thus greatly increasing the possibility of ICA injury during PBC. The incomplete bone boundary might attribute to craniosynostosis.We report the first case of TN caused by PTA associated with craniosynostosis. MVD was a feasible option for complex TN combined with other abnormalities. For patients awaiting PBC, PTA should be ruled out. Preoperative 3D-TOF and 3D-CISS MR image were essential to identify PTA NVCs.


The role of the human eye in severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) is still under investigation. The pathophysiology of the ocular findings is arduous when dealing with critically ill Covid-19 patients with comorbidities. Multiorgan involvement and the effects of inflammation, infection and systemic treatment on the retina are complex, and comparison of studies is difficult. Most studies in human patients have investigated the anterior segment, whereas few reports deal with the posterior segment of the eye. The present review aims to evaluate the retinal manifestations and imaging features in COVID-19 patients.Studies on the retinal manifestations and retinal imaging in COVID-19 patients published through June 2021 were reviewed. We included cross-sectional and case–control studies, case series, case reports and correspondence in the analysis.Flame-shaped hemorrhages, cotton wool spots, augmented diameter and tortuosity of retinal vessels were found on funduscopic examination. Peripapillary, macular retinal nerve fiber layer and ganglion cell layer thickness alterations were reported on spectral domain optical coherence tomography. Reduced vessel density of the superficial and deep retinal capillary plexus on optical coherence tomography angiography was reported.Retinal complications may arise in COVID-19 patients. Although no consensus on presentation is currently available, retinal funduscopy and imaging has shown neuronal and vascular alterations. Systemic neurological complications and microangiopathy are associated with SARS-COV-2; thus, as the retina has a neuronal and vascular component, funduscopy and retinal imaging on COVID-19 patients can provide further insight to SARS-COV-2 disease and the follow-up of patients. In December 2019 an outbreak of pneumonia occurred in Wuhan, China. The cause at the time was still unknown, but following health authority investigations, a novel coronavirus causing severe pneumonia was isolated. Further genomic research identified the virus as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is principally replicated and propagated in the respiratory system leading to an immune response [Ocular infection by coronaviruses has been well established in the animal model. There is evidence of sight-threatening ocular complication in the feline and murine orders . In feliCellular infection of SARS-CoV-2 occurs through binding of the viral spike protein to the cellular angiotensin-converting enzyme 2 (ACE2), and serine protease TMPRSS2 promotes spike protein priming , 8. In hThe virus can reach the retina through various possible mechanisms. Raony et al. suggested a hematogenous route to the central nervous system , and FigThe literature search was carried out from the PubMed, Web of Science and Scopus electronic databases through June 2021. The keywords used were “COVID-19” OR “SARS-CoV-2” OR “2019-nCoV” AND “retinal” OR “fundus” OR “imaging.” Reference lists of included articles and pertinent reviews were also searched. Three authors  independently screened all the titles and abstracts and reference lists of all included articles. Full manuscripts of relevant articles were evaluated by all authors and subjected to group discussion. The flowchart in figure one shows the retinal features evaluated in the publications included in the study , macular RNFL, GCL and IPL in 90 patients recovered from COVID 19 and compared results to 70 control subjects. The study group presented increased global RNFL thickness and increased superior and inferior nasal peripapillary RNFL thickness. In contrast, macular RNFL thickness was decreased in COVID-19 patients in the superior inner, nasal inner, and nasal outer quadrants. The GCL thickness was increased in volume, in the superior, nasal and inferior outer quadrants. Peripapillary RNFL and macular GCL thickness was increased in patients with anosmia and ageusia . The autSavastano et al. reported a reduction of the radial peripapillary capillary plexus density in 80 recovered COVID-19 patients and compared this with healthy control subjects using OCTA. The perfusion density correlated with lopinavir/ritonavir or antiplatelet therapy . HoweverIndeed, OCTA is an interesting line of research that enables the study of the choriocapillaris and retinal vascular density. As these two structures have the highest vascularization for area unit in the human body, Turker et al. speculated that the consequences of COVID-19 could be studied in a non-invasive and rapid manner using OCTA and that the microangiopathy that characterizes COVID-19 infection can potentially cause changes in the vascular density of the retina and the choriocapillaris . These aAbrishami et al. used OCTA to assess foveal and parafoveal vascular density and the extent of the foveal avascular zone (FAZ) in 31 infected patients compared to 23 healthy controls. They found a significant reduction of mean vessel density in the superficial capillary plexus and deep capillary plexus of the retina in the COVID-19 cohort. Moreover, they described an increased FAZ in the COVID-19 group, but these data were not statistically significant. The authors speculated that this reduction could be related to both direct coronavirus infection of the retina and secondary inflammatory effects .A comprehensive summary of the retinal alterations and imaging of the posterior ocular segment in the current literature is shown in Table The studies analyzed in this review show that there are alterations of the vascular and neuronal components of the retina related to SARS-COV-2 infection. Direct comparison of the studies currently available in the literature is difficult owing to different study designs that included patients in various stages of disease with variable treatment modalities. Given that the eye is a window on numerous systemic diseases characterized by neuronal and vascular alterations, retinal examination, SDOCT, and OCTA imaging can provide further insight on the pathogenetic mechanisms of disease and possibly aid in the follow-up of patients with SARS-COV-2 infection.