To evaluate the incidence of sustained elevation of intraocular pressure (SE-IOP) associated with intravitreal injections of anti-vascular endothelial growth factors (anti-VEGF) bevacizumab and aflibercept in patients with wet age-related macular degeneration (wAMD).A retrospective cohort study consisting of 120 eyes from 120 patients with anti-VEGF treatment for wAMD. Three different anti-VEGF groups were considered: i) 71 cases receiving bevacizumab only, ii) 49 cases receiving bevacizumab before switch to aflibercept, iii) 49 cases after switch to aflibercept. 120 uninjected fellow eyes served as controls. SE-IOP was defined as an increase from baseline ≥5 mmHg on 2 consecutive follow-up visits. The incidence of SE-IOP was analysed using exact Poisson tests and survival analysis. The time course of IOP was evaluated with linear mixed effect modelling.p = 0.43). Furthermore, the incidence of SE-IOP did not differ between the three anti-VEGF groups. Comparing the injected eyes of patients under 70 years to those of patients over 70 years, there was a statistically significant difference in survival without SE-IOP .In total, 6 treated eyes (2.38% incidence per eye-year) and 9 fellow eyes (3.58% incidence per eye-year) developed SE-IOP, and survival analysis showed no statistically significant difference (Intravitreal anti-VEGF injections were not associated with sustained elevation of IOP. These results do not support the claim that repeated anti-VEGF injections could elevate IOP. Age-related macular degeneration (AMD) remains the leading cause of irreversible blindness in people aged over 50 in western countries, the wet form (wAMD) being responsible for approximately 90% of blindness attributable to AMD . The incWith the increasing number of patients receiving anti-VEGF therapy for extended periods , it is iIOP spikes may occur after various intraocular procedures, and precautions are particularly important in patients with glaucoma –8. The tConversely, the long-term effects of anti-VEGF agents on IOP are debated. A report from the IRIS (Intelligent Research in Sight) Registry revealed a clinically and statistically significant sustained IOP rise overall and specifically in eyes receiving bevacizumab or ranibizumab, but not in eyes that received aflibercept . The oveIn contrast, several reports have concluded that anti-VEGF therapies do not have a significant long-term effect on IOP –18. An aThe purpose of this real-world study was to evaluate the incidence of sustained elevation of IOP (SE-IOP) and the IOP kinetics in wAMD patients that were treated either with bevacizumab only or switched to aflibercept.This retrospective cohort study included 120 eyes from 120 patients with intravitreal anti-VEGF treatment for wAMD and 120 fellow eyes which were not treated with any intravitreal anti-VEGF agent (controls). Of the treated eyes, 71 received bevacizumab only (non-switched patients), while 49 received bevacizumab as first-line treatment and were later switched to aflibercept as per the treatment protocol (switched patients). Three study groups were formed: i) 71 patients receiving bevacizumab only, ii) 49 patients receiving bevacizumab before switch to aflibercept, iii) the same 49 patients receiving aflibercept after the switch.The patients were treated at Turku University Hospital between June 2011 and July 2019, and they were identified through the corresponding diagnosis . Patients were included in this study if they received bevacizumab only or bevacizumab and aflibercept consecutively in one eye for wAMD and they were anti-VEGF treatment-naive in both eyes. The study followed the guidelines of the Declaration of Helsinki.Patient records and prescription databases were searched for possible confounding conditions (established glaucoma and ocular hypertension), medications (IOP-lowering drugs) and treatments . In the study, the definition of glaucoma was based on ICD coding: eyes were treated as glaucomatous if they were associated with any sub-code under the main code H40, which includes established glaucoma as well as ocular hypertension.Exclusion criteria were ongoing anti-VEGF treatment in the fellow eye, less than two follow-up visits in any study group, lack of meaningful IOP measurements in the fellow eye  and lack of a baseline IOP measurement in either eye. During the follow-up, if intravitreal anti-VEGF treatment was initiated in the fellow eye, or the patient dropped out of either the bevacizumab or aflibercept treatment protocol, data was included up to this event.The patients were diagnosed with wAMD by typical findings in dilated fundoscopy and optical coherence tomography (OCT) : cystic macular edema (CME) and/or neuroepithelial detachment (NED) and/or pigment epithelial detachment (PED) and/or retinal hemorrhage. Fluorescein angiography was performed in cases when needed in differential diagnosis. Each visit included visual acuity testing and tonometry. Rebound tonometry  was the standard practice. Single measurements from alternate methods were excluded for the consistency of IOP data.Treatment of all patients started with three monthly injections of bevacizumab (induction phase), followed by a control visit 1 month after the last injection. If the patient had full response to bevacizumab , the treatment was continued with three subsequent injections of bevacizumab with 2-month (8 weeks) intervals. The patients were controlled after the first 2-month interval with OCT, and later, 2 months after each series of three bevacizumab injections . Any CME, NED, new hemorrhage and PED increase was considered activity. If activity was observed in any control, the injection interval was tapered between 4 and 8 weeks, depending on the observed activity and response. The treatment always continued for a minimum of 15 months, followed by a 12-month observation period.If a sufficient response was not achieved with six monthly bevacizumab injections, the treatment was switched to aflibercept modified treat-and-extend regimen (TER) protocol. In the aflibercept modified TER protocol, the first injection interval was 4 weeks, and subsequent injection intervals were increased by 2 weeks when no activity was observed . If any activity was observed, the injection interval was decreased by 1 week. If activity was observed repeatedly (2 times) with the same injection interval, the longest interval with which there was no activity, was kept for a period of two consecutive injection series and then extended again.Next injection interval was decided based on the OCT findings during each injection visit. Follow-up visits were planned as follows: after three injections with 4–8-week intervals; after two injections with 9–12-week intervals; after one injection with 13–16-week interval.SE-IOP was defined as an increase from baseline ≥5 mmHg on at least 2 consecutive follow-up visits. The criterion was chosen to take into account variation in baseline IOP and to exclude non-sustained elevation of IOP.Descriptive statistics for continuous variables are presented using mean and standard deviation (SD) when variable is normally distributed, and median with first and third quartiles  otherwise. For categorical variables, frequencies and percentages are used. Normality of variables was assessed with Shapiro-Wilk test. Baseline characteristics between the three study groups were tested for statistically significant differences using analysis of variance (ANOVA) for normally distributed and Kruskal-Wallis test for non-normally distributed continuous variables. Chi-square test was used for categorical variables. For some of the following analyses, the groups were combined to increase the statistical power.p-values are also presented. A generalised linear mixed effects model was created to assess the clinical predictors for SE-IOP. The interaction between each potential predictor and the treatment status of the eye (whether it was a treated or a fellow eye) was analysed first separately and then all statistically significant interactions were combined in a multivariate model. Additionally, the Kaplan-Meier method was used to estimate survival curves, and comparisons were made between the treated and fellow eyes, and between the treated eyes of two age groups (< 70 and ≥ 70 years) using log-rank test.To investigate the rate of SE-IOP, eye-year incidence rates with confidence intervals were calculated with exact Poisson tests. Only the time to first event was considered. Corresponding exact mid-p adjusted In addition to the incidence of SE-IOP, the time course of IOP was analysed. Time from the beginning of treatment was converted to a categorical variable. To compare the time course of IOP between the treated and fellow eyes and between the three study groups, linear mixed effect models were created. Least squares means were obtained from the models and plotted, and the interactions between time from baseline and predictor variables  were analysed using ANOVA.p-values < 0.05 were considered statistically significant. Statistical analysis was performed using r version 4.0.1 .Two-sided tests were used in all statistical analyses and Patient characteristics are summarized in Table During the follow-up, glaucoma treatment was enhanced for one patient with bilateral selective laser trabeculoplasty and an additional IOP-lowering drug for the treated eye . In the univariate models, longer anti-VEGF treatment duration also appeared to be a risk factor (p = 0.036), but it was no longer statistically significant in the multivariate model (p = 0.140), whereas younger age remained a statistically significant risk factor (p = 0.016). A Kaplan-Meier curve is provided, showing this association  and 9 fellow eyes (3.58%) developed SE-IOP, the difference being statistically non-significant  . AnotherReis et al. and Bilgic et al. showed that the incidence of SE-IOP is associated with older age and pre-existing glaucoma among other clinical predictors , 13. VarGlaucoma treatment was changed bilaterally for two patients during the follow-up. Of these treatment modifications, one was symmetrical and one asymmetrical between the treated and fellow eyes. Therefore, the glaucoma treatment modifications are not likely to have any significant confounding effect. Furthermore, as the treatment for other glaucoma patients remained unchanged during the follow-up, and only patients with glaucoma or ocular hypertension received any IOP-lowering treatments, we conclude that IOP-lowering treatments are not significant confounders.Our results are in line with previous reports in that bevacizumab seems to cause slightly higher IOP values than aflibercept , 18, 22.A notable limitation of this study was that the patients received aflibercept only after attempted therapy with bevacizumab. Because of this, it is possible that some of the effects seemingly caused by aflibercept resulted instead from the discontinuing of bevacizumab. However, the difference between the drugs remains. Additionally, the relatively small sample size may have decreased the ability to detect statistically significant effects some other researchers have demonstrated. The known systemic effects of intravitreal anti-VEGF injections  could alTo summarize, our results show a low incidence of SE-IOP during anti-VEGF treatment, and no IOP-related effects attributable to the injections were seen. Younger patients might benefit from a closer follow-up of IOP. Some of these results conflict with past literature, and further controlled studies with larger sample sizes are still needed to investigate the possible long-term effects intravitreal anti-VEGF injections have on IOP.


Bone fractures bear potential risk to promote metastatic relapse in breast cancer. We conducted a population-based cohort study of 84,300 breast cancer patients diagnosed between January 2015 and November 2019. Bone fracture after breast cancer diagnosis was associated with an increased metastasis risk. Fractures may pose an increased risk to developing metastasis. Potential clinical implications for cancer patients are in support of fall prevention programs.Experimental studies suggest that bone fractures result in the release of cytokines and cells that might promote metastasis. Obtaining observational data on bone fractures after breast cancer diagnoses related to distant breast cancer recurrence could help to provide first epidemiological evidence for a metastasis-promoting effect of bone fractures. We used data from the largest German statutory health insurance fund  in a population-based cohort study of breast cancer patients with ICD-10 C50 codes documented between January 2015 and November 2019. The risk of metastasis overall, regional, distant non-bone or bone metastasis related to a fracture was modeled by an adjusted discrete time-to-event analysis with time-dependent exposure. Of 154,000 breast cancer patients, 84,300 fulfilled the inclusion criteria and had a follow-up time of more than half a year. During follow-up, fractures were diagnosed in 13,579 (16.1%) patients. Metastases occurred in 7047 (8.4%) patients; thereof 1544 had affected regional lymph nodes only and 5503 distant metastases. Fractures demonstrated a statistically significant association with subsequent metastasis overall . The highest risk for metastasis was observed in patients with subsequent bone metastasis , followed by distant non-bone metastasis  and lymph node metastasis . Breast cancer is the most commonly diagnosed cancer in women (2.1 million new cases in 2018) and the leading cause of cancer death in women globally  . The GerThe idea that tumor growth and recurrence are evoked by trauma and proximate inflammation or healing processes has existed for more than a century ,7,8. ThiIn the present study, we therefore tested the hypothesis that bone fractures occurring after initial breast cancer diagnosis might have an impact on the risk of regional and distant breast cancer recurrence. We performed a cohort analysis on 84,300 breast cancer patients using claims data of a statutory health insurance sample in Germany, allowing a follow-up of 5 years.A retrospective register- and population-based cohort study of breast cancer was performed using administrative data for claims purpose of the largest German statutory health insurance fund . The TK has on average 10 million insured members. The database included records from ambulant and hospital care on selected diagnostic codes , the extracted TK data were anonymized, and included a non-speaking identifier, subject’s birth year and few other variables (see above), so that backtracking of a person is not possible. Therefore, an informed consent was not necessary . This research was conducted according to the principles of the Declaration of Helsinki.From the original cohort of 154,260 individuals we excluded patients as follows : (i) 31,The diagnosis of bone fracture  simultaneous or subsequent to BCa diagnosis was considered as time-dependent exposure variable. Those with prior fractures were considered as having had “no fracture”, even if they were diagnosed with a second fracture simultaneous or after BC diagnosis. In total 13,579 of patients were diagnosed with a bone fracture at or after BCa diagnosis.The primary outcome was a diagnosis of metastases. We further stratified the outcome into three subgroups. (i) Lymph node metastasis, ICD-10 C77, (ii) distant non-bone metastasis, C78 and C79 without C79.5 and (iii) distant bone metastasis, C79.5. The subgroups were non-exclusive. For example, a patient diagnosed both with a lymph node metastasis and with a distant bone metastasis at different or equal times is included in both analyses as a case with corresponding follow-up times. The occurrence of the other metastasis was then ignored.The TK dataset included information on potential confounders. These are birth year, source of BCa/fracture/metastasis diagnosis , year of diagnosis), ICD-codes for diagnosis of other malignant tumors (C00-C97), and a diagnosis of osteoporosis (M81) as well as prescription of anti-estrogens (L02BA), aromatase inhibitors (L02BG), bisphosphonates (M05BA) and bisphosphonate combinations (M05BA). Any depletion of the peripheral estrogen concentration by anti-estrogen treatment, particularly AI, might be associated with a higher risk for osteoporosis , which iTime-to-event regression analyses were conducted for each outcome of metastasis with a discrete underlying time scale to account for ties. Hospital date specifications were aligned with ambulant dates by assigning them the end of respective quarter date. Observation time was calculated as date of first entry with BCa diagnosis (earliest date is the first quarter 2015) until date of last entry or first date of metastasis diagnosis, whichever came first. The last occurring date was 30 November 2019. Follow-up time was calculated by the reverse Kaplan–Meyer method. Analyses were adjusted for age at breast cancer diagnosis, source of BCa diagnosis , secondary tumors (time-dependent yes/no), other malignant tumor before BCa diagnosis (yes/no), osteoporosis (time-dependent yes/no), prescriptions of anti-estrogen therapy (time-dependent yes/no) and aromatase inhibitors (time-dependent yes/no). All analyses were stratified by year of diagnosis.We performed several sensitivity analyses: (i) we excluded all BCa cases that have a first date of diagnoses in 2015, as the majority of these are likely to be prevalent cases. (ii) We minimized the probability of prevalent metastasis in prolonging the waiting period from >0.5 to >1 year between initial breast cancer diagnosis and occurrence of metastasis or censoring. (iii) We repeated (II) for those with a BCa diagnosis after 2015 and (iv) we restricted the analysis to patients with prescriptions of anti-estrogens and AI as an indicator for estrogen receptor positive tumors, which is, in view of prognosis, a more homogeneous group.Statistical analyses were performed in SAS version 9.4 . All tests were two-sided and confidence intervals, not including one, were considered statistically significant.Characteristics of the study population were shown according to the occurrence of a fracture . At firsUnadjusted hazard ratios (HR) for fractures versus no fracture were statistically significantly higher for overall metastasis and distant metastasis, but not for lymph node metastasis  0.92, 1.14) . After aAfter excluding all patients with diagnosis of BCa in 2015, we observed non-significantly elevated HRs for fractures related to distant non-bone  and bone metastasis , but not overall or in patients with lymph node metastasis . Of noteWhen the waiting period was extended from half a year to more than one year , Model 4A similar pattern of associations was observed in the subgroup of patients with endocrine therapy as an indicator for ER-positive tumors , Model 6The present study demonstrated that patients diagnosed with a bone fracture after or concurrent with the diagnosis of breast cancer may have an increased chance of being diagnosed with a distant metastasis within 5 years after a BCa diagnosis. Fractures were not consistently related to lymph node metastasis, but they remained associated with higher hazard ratios for metastases in distant organs of the bone and other sites in sensitivity analyses, suggesting a systemic mechanism of action.Breast cancer cells are frequently present in lymph nodes and distant organs, such as the bone marrow of early-stage breast cancer patients without any clinical or radiological signs of overt metastasis (TNM-stage M0) ,15,16. AAlthough the presented epidemiologic data cannot prove any causal relationships, it is tempting to speculate how bone fractures may affect dormant DTCs present in lymph nodes and distant organs. Tissue repair after bone fractures is followed by changes in the immune system (and systemic release of cytokines)  that couDespite the large database and the association found between fractures and metastatic relapse, using a database of a statutory health insurance sample has obvious limitations. In Germany, data need to be deleted after 5 years of storage due to data protection regulations. Our database provides, therefore, no information on late relapses, which are more frequent in hormone receptor-positive patients than HER2-positive or triple-negative patients . It is pHealth insurance data have been sporadically used for studies of health (care) conditions; some of which performed time-to-event analyses ,30,31 orIn conclusion, the results of our current analysis provide first evidence that fractures may pose an increased risk to develop distant metastasis in breast cancer. Whether this also applies to other bone-seeking tumors, such as prostate cancer, remains to be investigated. A better understanding of the mechanisms behind a potential influence of bone fractures on distant metastatic progression by future experimental studies might lead to the identification of patients with a higher individual risk of metastasis after fractures and the discovery of new preventive strategies focused on these putative high-risk patients to block or slow down metastatic relapse in cancer patients with fractures.


Filipino Americans show higher thyroid cancer recurrence rates compared to European Americans. Although they are likely to die of this malignancy, the molecular mechanism has not yet been determined. Recent studies demonstrated that small non-coding RNAs could be utilized to assess thyroid cancer prognosis in tumor models. The goal of this study is to determine whether microRNA (miRNA) signatures are differentially expressed in thyroid cancer in two different ethnic groups. We also determined whether these miRNA signatures are related to cancer staging. This is a retrospective study of archival samples from patients with thyroid cancer (both sexes) in the pathology division from the last ten years at Loma Linda University School of Medicine, California. Deidentified patient demographics were extracted from the patient chart. Discarded formalin-fixed paraffin-embedded tissues were collected post-surgeries. We determined the differential expressions of microRNA in archival samples from Filipino Americans compared to European Americans using the state-of-the-art technique, HiSeq4000. By ingenuity pathway analysis, we determined miRNA targets and the pathways that those targets are involved in. We validated their expressions by real-time quantitative PCR and correlated them with the clinicopathological status in a larger cohort of miRNA samples from both ethnicities. We identified the differentially upregulated/downregulated miRNA clusters in Filipino Americans compared to European Americans. Some of these miRNA clusters are known to target genes that are linked to cancer invasion and metastasis. In univariate analysis, ethnicity and tumor staging were significant factors predicting miR-4633-5p upregulation. When including these factors in a multivariate logistic regression model, ethnicity and tumor staging remained significant independent predictors of miRNA upregulation, whereas the interaction of ethnicity and tumor staging was not significant. In contrast, ethnicity remained an independent predictor of significantly downregulated miR-491-5p and let-7 family. We provide evidence that Filipino Americans showed differentially expressed tumor-tissue-derived microRNA clusters. The functional implications of these miRNAs are under investigation. Thyroid cancer (TC) is the most common type of endocrine malignancy, and the incidence of TC has increased in the last few decades ,2,3,4. ARecent studies have identified the link between miRNA expression and cancer, where significant changes in expression have been detected in malignant cells when compared to benign cells . Many camiRNAs are classified as oncogenes or tumor suppressor genes. It is important to know that miRNAs should not always be classified as tumor suppressors or oncogenes; their classification depends on the tissue or cell type in which they are expressed . These gThe deregulation of miRNA profiles has been used not only as a diagnostic marker but also as a predictive marker in various types of malignancies, including thyroid cancer . In thisn = 5) and European Americans (n = 5) for miRNA-seq. We expanded our pilot study by acquiring more archival thyroid tissues and fresh tissues from both Filipino American (n = 50) and European American (n = 50) men and women. The samples were obtained from the biorepository as well as the Departments of Pathology at Loma Linda University Medical Center (LLUMC) and Riverside University Health System (RUHS) and included tissues from the years 2004 to 2019. This study consists of de-identified discarded tissue samples from both Filipino American (1st generation Filipinos who have resided in the USA for the last 20 years) and European American (Europeans who have been residing in the USA) patients with thyroid cancer (ethnicity status was recorded according to their self-identified ethnic group), aged 18–80 years, and from both sexes with adequate clinical information and paraffin blocks for immunohistochemistry (IHC).We initially selected 10 patients with thyroid cancer from both Filipino Americans  staining by our expert pathologists. We included papillary thyroid cancer subtypes (PTC) from both ethnicities to maintain genetic uniformity and tissue specificity. Microdissected cancer samples were obtained after being postmarked by pathologists.We obtained deidentified patient information, including demographic data (age and sex), tumor size, extrathyroidal extension, nodal status, and distant metastases (for determining disease stage) by the independent review of de-identified charts . All samples were obtained under IRB-approved studies according to the university and hospital policies at both LLU and RUHS.For purification of miRNA and total RNA from formalin-fixed paraffin-embedded (FFPE) microdissected samples, an H&E section was consulted in each case to guide the area of interest, as described before . We usedA library of small RNAs was prepared from the FFPE samples of TC using QIAseq™ miRNA Library Kit (QIAGEN). We also established high-quality libraries with RNA Integrity Numbers (RINs) > 8, and RNA quality was checked by running on agarose gel to visualize 28S and 18S RNA A,B with http://www.bioinformatics.babraham.ac.uk/projects/fastqc, accessed on 20 July 2016). Reads shorter than 20 bp or with a Phred quality score (Q score) less than 20 were removed. We first converted the raw sequencing data to unique molecular indices (UMI) and then converted the UMI to fold-change value by directly using the Primary QIAseq miRNA Quantification Data Analysis Output Excel file as the Secondary QIAseq miRNA Library Kit Data Analysis input file. Integrated UMIs enabled the quantification of individual miRNA molecules, eliminating PCR and sequence bias. Primary read mapping and differential expression analysis were performed via GeneGlobe Analysis Center (QIAGEN). The secondary QIAGEN miRNA library kit data analysis software analyzed the UMI counts to calculate changes in miRNA expression. It used automated normalization through geNorm and total UMI count normalization methods.Illumina HiSeq4000 SR flowcell was clustered on cBot with SR cluster kit from 5 uL of 2 nM template. Sequencing was performed on an Illumina HiSeq 4000 platform using a 75-base single-read protocol. We performed the sequencing on an Illumina HiSeq 4000 using single-read 100-cycle SBS reagents. Base call files (*.bcl) were converted to FASTQ and sorted using bcl2fastq scripts (Illumina), with one miss-matched barcode allowed. The quality of the sequenced reads for each sample was assessed using the FastQC program  on the replicate normalized UMI counts. Each miRNA in each test group was compared to the control group. The p-value was calculated by a parametric, unpaired, two-sample equal variance with two-tailed distribution. Any p-value less than 0.05 was indicated in red. Each group (including the control group) contains at least three samples for the software to calculate the p-value. Samples were assigned to controls and test groups. The average ratios of normalized UMI counts (or relative miRNA expression) between the control group and each test group were used to calculate the fold change. To identify differential expression, a widely used and accepted cut-off of 2-fold change was used. We analyzed the biological pathways in supplemental attachments using both the Ingenuity Pathway Analysis (IPA) and KEGG map. For the geNorm and total UMI count normalized methods, To identify the top ten miRNAs that were up- or downregulated in the Filipino Americans set when compared to the European Americans set, we used a cut-off of a 2-fold change before sorting them based on highest to lowest fold change (both +ve and −ve). We used the entire set of up- and downregulated miRNAs separately as inputs for the Ingenuity Pathway Analysis (IPA), a web-based software from QIAGEN © 2021–2021, to analyze the relation to diseases, their functions, and to identify the putative targets for these miRNAs. We used the IPA diseases and functions analysis and the target filter analysis tools with default parameters. IPA miRNA Target Filtering allowed us to deduce the genes targeted by miRNAs along with identifying potential relationships between miRNAs and their target genes. Overall, this helped in visualizing which mRNAs are being targeted by a given miRNA.TMmicroRNA cDNA synthesis mix . We used an equal number of samples from both ethnicities (Filipino Americans = 50 and European Americans = 50). RT-qPCR for miRNA was performed using iQSYBR(R) Green PCR Supermix  with specific primers for both miR-4633-5p, miR-6775-3p, miR-4749-3p, miR-323b-3p, miR-491-5p, and let-7 genes and probes on a Stratagene Mx3005P instrument . U6 was used for miR normalization. The results were analyzed using the ΔΔ cycles to threshold (ΔΔCt). Quantification of miRNA expression and miRNA-seq data validations were performed by RT-qPCR assay. cDNA was synthesized with 100 ng of total RNA (including miRs) using MystiCqP values of less than 0.05 were considered to indicate statistical significance.Student’s t-test and one-way ANOVA test with Bonferroni correction were used to assess continuous data. Logistic regression analysis was performed using R software, version 4.0.5. Univariate analysis was performed to determine if patient age, sex, Filipino American or European American ethnicity, pTNM, and/or BMI were significant predictors of upregulation of miR-4633-5p. Once the statistical significance of the predictors was determined, multivariate logistic regression analysis was performed, and the interaction model was tested for significance. p values. For the calculation of the odds ratio, we used the unconditional maximum likelihood estimate (MLE), and for the CI we used the normal approximation . The p values for the odds ratios were based on the chi-square statistics and declared significant for p values less than 0.05.For the significant variables, odds ratios and their 95% confidence interval (CI) were calculated using the package “epitools”, version 0.5.10.1. For miR-4633-5p, we calculated the odds ratios, 95% CI, and Our sequencing data showed that the vast majority of miRNAs are downregulated and unchanged, whereas only a few are upregulated, in Filipino Americans when compared to European Americans as positive controls . Using tIn our study, we observed that the number of miRNAs that were significantly downregulated in Filipino Americans when compared to European Americans was approximately nine times higher than that of the miRNAs that were significantly upregulated. There were 51 upregulated and 448 downregulated miRNAs in the Filipino American samples compared to the European American samples and the rest were unchanged in Filipino Americans when compared to European Americans. We ranked these miRNAs in the order of highest to lowest differential expression, and the top ten miRNAs that were down- or upregulated in the Filipino American patients when compared to the European American patients ranged from −97.34 to −444.88 folds  and 2.74IPA (QIAGEN), mapped 50 of the 51 miRNAs that were upregulated. The one that was missing in the IPA knowledgebase was hsa-miR-3653-3p. Similarly, the downregulated dataset contained 448 IDs, out of which 444 were mapped; hsa-miR-1254, hsa-miR-1273a, hsa-miR-3669, and hsa-miR-548-3p did not map with those in the IPA knowledgebase. Most of the differentially expressed miRNAs were linked to organismal injury and abnormalities, reproductive system disease, and cancer. The molecular and cellular functions associated with these miRNAs were cellular growth and proliferation, cell cycle, and cellular development .The lists of up- and downregulated miRNAs were used as input for IPA for the next phase of the analysis. We were able to detect the putative targets for almost all of the miRNAs that were differentially regulated in Filipino Americans compared to European Americans, using IPA target filter analysis that uses TargetScan as an inbuilt web-based miRNA target prediction tool. As mentioned before, 50 upregulated miRNAs out of 51 were mapped to the IPA knowledgebase. Using the “microRNA target filter analysis” option, 48 miRNAs were found to target 14,090 mRNAs. Next, we selected the thyroid cancer signaling pathway and found that 41 out of the 48 miRNAs were targeting 44 mRNAs within the thyroid cancer signaling pathway . SimilarNext, we added the thyroid cancer signaling pathway molecules in the “my pathways” tool for visualizing the direct and indirect relationships among the miRNAs and the molecules from the pathway. We used the “grow” tool and selected the confidence level to be either experimentally observed or predicted with high confidence. We found seven miRNAs from the upregulated set connected as the upstream or downstream of ten molecules from the thyroid cancer signaling pathway in humans. p < 0.01) to PTC  to PTC .p < 0.05), and two were found to be linked to the proliferation of thyroid tumor cell lines (p < 0.05). We also observed that a number of miRNAs were linked to thyroid hormone metabolism pathways  via IL-10, IL-6, IL12RB2, JAK1/STAT3, PIK3R1, CD40, IFNAR1, and IFNA1/IFNA13, as shown in n = 50) compared to European American (n = 50) samples and correlated the expression of selected miRNAs to disease stages. Interestingly, miR-4633-5p was consistently upregulated in Filipino American versus European American samples  the expressions of the top three up and downregulated miRNAs  from RNA-seq data in Filipino American  was shown to correlate in Filipino Americans with thyroid cancer (A stage-dependent significantly higher miR-4633-5p expression (* d cancer .p = 0.0145), pTNM stage II (p = 0.0162), and pTNM stage III (p = 0.0298) were significant factors predicting miR-4633-5p, miR-6775-3p, and miR-4749-3p upregulation. When including these factors in a multivariate logistic regression model, ethnicity (p = 0.0262), pTNM II (p < 0.0441), and pTNM III (p < 0.0125) remained significant independent predictors of miRNA upregulation, whereas the interaction of staging and ethnicity was not significant. p = 0.013. For patients with pTNM stage I compared to pTNM stage II–IV, the odds ratio was 3.97 , p = 0.011. In the univariate analysis, ethnicity (Upregulated miR-4633-5p target genes are shown in p < 0.05) in the Filipino American compared to European American samples in all stages of cancer. Target genes for miR-491-5p and let-7 genes are shown in We confirmed that miR-491-5p and let-7 gene families were downregulated A,B in FiAlthough miR-6775-3p and miR-4749-3p were variably upregulated and showed no difference in ethnicity, staging and upregulation of miR-6775-3p and miR-4749-3p showed an independent predicting factor . AlthougIn this study, we applied small RNA-seq to examine tissue-specific miRNA profiles from Filipino American and European American patients with thyroid cancer to understand the differential expression patterns and to evaluate their abundance, targets, and relation to different biological pathways. We were able to prepare a clean miRNA library from the archival samples. Using IPA, we have shown that relevant pathways are likely regulated by the targets of highly conserved miRNAs, which are differentially expressed in Filipino and European Americans. We were able to detect the putative targets for most of these miRNAs by IPA. We have analyzed the miRNA target genes that are related to human diseases and are involved functionally in disease progression. The targeted pathways involve kinases and transmembrane receptor molecules  , which are involved in the cell cycle and cellular differentiation/cell proliferation. We have shown a large number of downregulated genes that are involved in organismal injury and abnormalities, cancer, gastrointestinal disease, cellular development, and immunological disorders. The upregulated clusters were fewer in number and are known to be involved in immunological disorders, neurological development (cell cycle), and urological disorders. We detected seven of the known downregulated miRNAs  linked to papillary thyroid cancer or cell lines (miR-17-5p) with tumor suppressor or oncogenic function reported in the literature. IPA canonical pathway analysis was used for the prediction of pathways that are activated or inhibited. Pathway maps were used to understand the correlation between miRNA target genes and different diseases so that this can lead to new targets for therapeutic agents in the future. Additionally, we have detected that the targets for the differentially expressed miRNAs are involved in pathways related to thyroid hormone metabolism. Although thyroid hormones regulate the physiological processes of normal cells, thyroid hormones and their receptors are dysregulated in thyroid cancer. Therefore, it will be interesting to see whether the influence of miRNAs targeting the metabolic pathways affect thyroid cancer health disparities. It has been shown that inflammation and immunity alter the prognosis in many cancers, including thyroid cancer. Recent data showed that the ratio of immune cells plays an important role in the prognosis of high-risk PTC. Interestingly, our data showed that the differentially regulated miRNAs with their targets are linked to T cell receptor signaling pathways . Further study is needed to elucidate the roles of Th1 and Th2 pathways in thyroid cancer progression in Filipino Americans with aggressive PTC. We also found that the targets of two upregulated and eight downregulated miRNAs were linked to the T-helper type 17 (Th17) activation pathway. Th17 cells are known to influence anti-tumoral CD8+ T cell responses. Although Th17 cells play controversial roles in cancer due to their dual roles, either good or bad in cancer progression, it is important to determine whether they are cancer type-specific or influenced by the tumor microenvironment. Further studies are needed to reveal the significance of these pathways.Out of all miRNAs, we chose the top three upregulated ones—miR-4633-5p ,25,26, mPrevious studies showed lower expressions of miRNA-491-5p and let-7 in individual tumors ,41,46,47In conclusion, we have detected both up- and downregulated miRNA signatures in Filipino Americans when compared to European Americans by Hiseq4000 and confirmed the differential expressions by RT-qPCR in archival tissue samples. We have also identified target genes for these miRNAs and the pathways to which those are related by IPA. Out of the top ten up- and downregulated miRNAs, we found only a few of them are significantly differentially expressed with advanced thyroid cancer staging. Future study is underway to validate the functional implications of the upregulated miR-4633-5p/miR-323-3p genes and downregulated miR-4915p/let-7 family genes and their involvement in the pathways related to thyroid cancer progression, not only in Filipino Americans but also in other ethnic groups.


Exploring biomarkers monitoring latent tuberculosis infection (LTBI) treatment effectiveness would benefit optimizing the therapeutic regimen. This study aims to identify potential mycobacteria-specific antigen-induced cytokines associated with host responses to preventive treatment.Based on a randomized controlled trial on LTBI treatment among individuals with chest radiography abnormalities suggestive of prior tuberculosis (TB), the dynamically changed cytokine levels in QuantiFERON-TB Gold In-Tube (QFT) supernatants were estimated during the treatment by bead-based multiplex assays and enzyme-linked immunosorbent assay.p = 0.028) among treated participants, while such a difference was not found for untreated controls (p = 0.292).In total, 63 treated participants and 32 untreated controls were included in the study. The levels of 13 background-corrected mycobacteria-specific antigen-stimulated cytokines  were found to be statistically different between before and after treatment in treated participants, while no statistically differences were observed in untreated controls. Among these 13 cytokines, the level of IL-8 was significantly lower in the QFT reversed group than that in the non-reversed group (Our results suggested that the lower level of mycobacteria-specific antigen-induced IL-8 might be associated with the host’s positive response to LTBI treatment. Mycobacterium tuberculosis (M.tb), and there were more than 7 million new cases of active tuberculosis (TB) being reported in 2019 -positive result (TB Ag-Nil ≥ 0.35 IU/ml) and without current active TB at baseline survey were included in the RCT. They were randomly classified into two groups  were used. The levels of cytokines in the Nil supernatants (unstimulated) and the TBAg supernatants  were analyzed by magnetic bead suspension array using the Bio-Plex Pro Human Cytokine panels according to the manufacturer’s instructions. Prior to measuring the samples, the supernatants were diluted 4× according to the manufacturers’ instructions. As the level of IL-8 was above the detection limit when measured for 4×-diluted supernatants by the Bio-Plex Pro Human Cytokine panels, the supernatants were diluted 100× and measured by an enzyme-linked immunosorbent assay (ELISA) . Cytokines with >50% of the samples below the lower detection level (LDL) or above the upper limit of the assay will be excluded from further statistical analysis. Additionally, cytokines with occasional values (<50%) below the LDL were assigned an averaged value between 0 and the lowest detectable level in each assay plate . Cytokin2) test and Fisher’s exact test were used to compare the distribution of categorical variables across groups. The level of background-corrected mycobacteria-specific antigen-stimulated cytokine concentrations was presented with median (Q25–Q75). Wilcoxon rank sum test was used to compare responses between different participants at the same time point. Wilcoxon signed rank test was used to evaluate the responses for the same person at different time points. To assess the ability of the cytokines to reflect infection status, receiver operating characteristic (ROC) analysis was conducted, and the area under ROC curves (AUCs) was calculated. A two-tailed p-value < 0.05 was considered statistically significant.Statistical analyses were performed using SAS 9.4 version  and GraphPad Prism 9 . Participants who completed ≥90% doses of the therapy were defined as completed the regimens. In order to reflect the change of host infection status, a more strict definition of QFT reversion was used in the present study as IFN-γ level declined from >0.70 IU/ml at T0 to <0.20 IU/ml at T1 . Chi-squA total of 32 untreated controls and 63 treated participants were included in this study. The definition used to identify background-corrected mycobacteria-specific antigen-stimulated cytokines related to preventive treatment was shown in In order to further explore the relationship between the selected cytokines and host infection status (the level of released IFN-γ in the QFT test was used to reflect infection status), the treated and untreated participants were both divided into two groups according to their QFT retesting results at T1 (reversed or not reversed) . As showTo further evaluate the performance of background-corrected mycobacteria-specific antigen-stimulated IL-8 in reflecting infection status reflected by a more strict definition of reversion, the ROC analysis was performed and the AUC was calculated among treated participants at T1. As shown in Based on a randomized controlled study, the level of 13 cytokines  induced by mycobacteria-specific antigen was changed significantly after LTBI treatment. Notably, IL-8 might be a potential marker to reflect the change of TB infection status, indicating that it might play an important role in host immune protection against TB infection.There have been several studies illustrating the importance of cytokines as a biomarker for monitoring the anti-TB or LTBI preventive treatment. A prospective study by M.tb infection status is one way to reflect the response to preventive treatment, facilitate treatment monitoring, and individualize treatment regimen adjustment. A review has suggested a clearance model of M.tb based on animal and ex vivo human experiments (M.tb could be eliminated from host tissue by anti-TB drugs. A subsequent study used an in vitro model of treated M.tb infection coupled with gel-enhanced liquid chromatography–tandem mass spectrometry (GeLC MS/MS) to identify and quantify peptides that may indicate M.tb infection clearance and found that several activated proteins were associated with several mechanisms against M.tb infection and could potentially be used as biomarkers for TB treatment monitoring  to reflect infection clearance, misclassification still could not be excluded.Our results suggested that the level of mycobacteria-specific antigen-induced IL-8 decreased along with preventive treatment and might be used to identify infection clearance suggested by a strict definition of QFT reversion. It provides a clue for exploring prognosis biomarkers to evaluate the performance of LTBI treatment.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the protocol of the present study has been approved by the Ethics Committees of the Institute of Pathogen Biology, Chinese Academy of Medical Sciences (No: IPB-2018-1). All participants have signed the written informed consent. The patients/participants provided their written informed consent to participate in this study.LeG and QJ designed the study. XC, HX, HZ, SP, ZL, DW, JY, BF, ZQ, YD, BZ, JL, LiG, FS, and XG were in charge of data management. XC, HX, and LeG did data analysis and wrote the report. QJ commented on the report and improved English writing. SP, HX, ZL, DW, JY, BF, ZQ, YD, JL, BZ, LiG, FS, and XG organized investigations at the study sites. All authors contributed to the review and revision and have seen and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


The Coronavirus Disease 2019 (COVID-19) has affected over 100 million cases worldwide. Children accounted for 1–5% of all cases with less reported symptoms and better prognosis compared to adults. This study aimed to describe the epidemiological characteristics and outcomes of pediatric COVID-19 cases in Saudi Arabia in addition to identifying risk factors associated with disease severity.This was a multicenter, cross-sectional retrospective study that included confirmed SARS-CoV-2 infection among pediatric patients (< 14 years) from the time of initial identification in March 2020 to the end of July 2020 in 6 centers across the country. Patients were classified based on clinical severity. Study outcomes included time to recovery, need for invasive ventilation, and mortality. Multivariate logistic regression analysis was conducted to explore factors associated with increased disease severity.p = 0.006 and 0.030, respectively).The study enrolled 567 children with (51.5%) were males, and (44.6%) aged from 6 to 14 years old. Asymptomatic patients accounted for 38.98% of the cases: while 319 patients (56%) had mild disease, and 27 patients (4.76%) had moderate-to-severe disease. Only 10 patients (1.76%) required Pediatric Intensive Care Unit admission. The calculated case-fatality was 0.7%. After performing multivariate regression analysis, chronic lung conditions  and decreased red blood cells (RBCs) count  were found to be significant predictors for moderate-to-severe disease (Most COVID-19 cases in the current study had a benign course of illness and carried an excellent prognosis. Children with chronic lung conditions or low RBCs count are at higher risk to develop moderate-to-severe COVID-19 disease.The online version contains supplementary material available at 10.1186/s12887-021-02959-8. Coronaviridae, it is transmitted by either direct contact or respiratory droplet. Infections caused by viruses from the Coronaviridae family mainly affect the respiratory and gastrointestinal systems, causing symptoms that vary from simple common cold or pneumonia to acute respiratory distress syndrome (ARDS), multi-organ failure, or death  and moderate-to severe  disease groups (p = 0.345).Table Only two complications due to COVID-19 with recovery were observed in the study. One patient aged 12 years and was a known case of epidermolysis bullosa and cardiomyopathy. He presented with fever, cough, and mild shortness of breath with evidence of myocarditis on echocardiography. He was treated in the general ward and recovered after receiving Intravenous Immunoglobulin (IVIG) and Dexamethasone. The other patient aged 6 years and was medically and surgically free. He presented with MIS-C and evidence of bilateral consolidation in chest x-ray and cardiac tamponade on echocardiography. The patient required PICU admission and received IVIG, Dexamethasone, Favipiravir, Hydroxychloroquine, and Azithromycin. He was later discharged in good condition after being hospitalized for 10 days.A total of four patients (14.8%) in the moderate to severe group died with an overall case-fatality rate of 0.7%. One patient aged 17 months and was having severe aplastic anemia and pulmonary tuberculosis on treatment. He presented with severe pneumonia that was complicated by ARDS; he subsequently died from septic shock. The second patient aged 4 months and was under investigation for inborn error of metabolism. He was tested positive for COVID-19, but his death was most likely attributed to his metabolic disease. The third patient aged 6 months and was diagnosed with spinal muscular atrophy (SMN1-EXON7 deletion); he was severely hypotonic and was on limited code status before developing COVID-19. The fourth patient aged 11 years and was medically and surgically free. He presented with a picture of MIS-C. The patient later developed ARDS and subsequently died.p = 0.006] and low RBCs count  were significant predictors for moderate-to-severe COVID-19 in the model.Chronic lung conditions, chronic heart conditions, hemoglobin level, RBCs count, and CRP level were entered into a multivariate regression analysis to identify risk factors/predictors for moderate-to-severe COVID-19 Table . After cThis multi-center study described the epidemiology, clinical presentation, and outcomes of pediatric COVID-19 in Saudi Arabia. The majority (95%) of the patients were asymptomatic or had mild disease. Almost half of the patients were >  6 years of age, including those with moderate-to-severe disease. Most patients received supportive care only; few patients required admission to the ICU, and the fatality rate was very low. The study also found that having an underlying chronic lung condition and reduced RBCs are risk factors for developing moderate-to-severe disease.n = 56) had uncomplicated COVID-19; of which 13% were admitted to the PICU. Only one patient required invasive mechanical ventilation, and no mortality was reported [The epidemiology of COVID-19 in Saudi Arabia and the Gulf region was described in multiple studies. For example, Al Yazidi et al. found in a multicenter cohort from Oman that 68% of the documented cases  receptor , 19. AnoThe role of underlying medical conditions as risk factors for severe COVID-19 in children was investigated in few studies. Graff et al. reported that immunocompromising conditions, gastrointestinal conditions, diabetes, and asthma are predictors for hospital admission. In addition, asthma and gastrointestinal conditions were associated with increased odds of requiring respiratory support. The current study found that those with underlying lung conditions, for which the majority had asthma/hyperactive airway disease, were more likely to develop moderate-to-severe disease. However, we could not demonstrate any further association with any other comorbidities. This could be explained by the very small number of cases with such comorbidities .A meta-analysis also looked at the effect of anemia and iron metabolism in COVID-19 and found that severe cases, compared to moderate COVID-19 cases, had significantly lower hemoglobin and RBCs count, and higher ferritin level. It is hypothesized that the innate immune system in the acute phase of infection may aim to decrease iron bioavailability in order to prevent an expanding viral load. This results in the activation of hepcidin, sequestration of intracellular iron, increased ferritin levels, drop-in hemoglobin level, and resultant hypoxia . These fIn addition, the prognostic role of elevated inflammatory markers and Prothrombin time was reported in many studies. A meta-analysis conducted by Elshazli et al. reported that elevated CRP, procalcitonin, ESR, and prothrombin time were significantly associated with increased odds of severe disease progression . These fGracia Salido et al. reported 74 patients with PICU admissions due to severe COVID-19; 60% fulfilled the criteria of MIS-C diagnosis. Lopinavir-ritonavir was used in 40% of patients. No difference was noticed in the outcome if antiviral treatment was given . In the Most COVID-19 cases in the current study had a benign course of illness and carried an excellent prognosis, which is comparable to most studies worldwide. Chronic lung disease and decreased RBCs were found to be significant predictors for moderate-to-severe COVID-19 presentation. No recommendation could be made regarding the use of antivirals and immunomodulatory therapy due to the low number of severe cases and mortality. There is still a need for further studies identifying the contributing risk factors and best therapeutic modalities in pediatric COVID-19.Although the current study provided a sufficient sample size, it was limited by the retrospective design, the limited number of patients with moderate-to-severe disease, and the low mortality rate. This led to major constraints in the analysis due to missing laboratory investigations and diagnostic workup. In addition, the study could not assess the effect of using antivirals or immunomodulatory agents on the outcome. Future large prospective studies examining risk factors for severe COVID-19 in children are warranted to understand the full picture of pediatric COVID-19. In addition, more robust data on the best treatment modalities are strongly indicated.Additional file 1.


Reports have suggested a reduction in exacerbations of chronic obstructive pulmonary disease (COPD) during the coronavirus disease 2019 (COVID-19) pandemic, particularly hospital admissions for severe exacerbations. However, the magnitude of this reduction varies between studies.Electronic databases were searched from January 2020 to May 2021. Two independent reviewers screened titles and abstracts and, when necessary, full text to determine if studies met inclusion criteria. A modified version of the Newcastle-Ottawa Scale was used to assess study quality. A narrative summary of eligible studies was synthesised, and meta-analysis was conducted using a random effect model to pool the rate ratio and 95% confidence intervals (95% CI) for hospital admissions. Exacerbation reduction was compared against the COVID-19 Containment and Health Index.A total of 13 of 745 studies met the inclusion criteria and were included in this review, with data from nine countries. Nine studies could be included in the meta-analysis. The pooled rate ratio of hospital admissions for COPD exacerbations during the pandemic period was 0.50 (95% CI 0.44–0.57). Findings on the rate of community-treated exacerbations were inconclusive. Three studies reported a significant decrease in the incidence of respiratory viral infections compared with the pre-pandemic period. There was not a significant relationship between exacerbation reduction and the COVID-19 Containment and Health Index .There was a 50% reduction in admissions for COPD exacerbations during the COVID-19 pandemic period compared to pre-pandemic times, likely associated with a reduction in respiratory viral infections that trigger exacerbations. Future guidelines should consider including recommendations on respiratory virus infection control measures to reduce the burden of COPD exacerbations beyond the pandemic period. Chronic obstructive pulmonary disease (COPD) affects more than 250 million people around the world and is the third leading cause of death . People The coronavirus disease 2019 (COVID-19) pandemic has been challenging for people living with COPD. Despite the low prevalence of COPD patients in most hospital series reporting COVID-19, COPD has been associated with increased severity and mortality of COVID-19 , 5. As aMost exacerbations of COPD are caused by respiratory viruses . A previA number of small studies have reported an apparent reduction in the rate of COPD exacerbations during the COVID-19 pandemic, yet the precise relationship between COVID-19 preventative measures and COPD exacerbation reduction, particularly hospital admissions for severe exacerbations remain unclear. This is an important area to explore because exacerbations have been identified as the most disruptive aspect of COPD by patients , currentWe prospectively registered this systematic review and meta-analysis at PROSPERO (ID: CRD42021249522), and followed the Preferred Reporting in Systematic Reviews and Meta-Analyses (PRISMA) guidelines see  15]..15].st January 2020 to 17th May 2021 ; I: intervention (COVID-19 -related restrictions); C: comparison (During COVID-19 pandemic versus pre-pandemic times); O: outcome (admissions for COPD exacerbation).Two independent reviewers used a modified version of the Newcastle-Ottawa Scale (NOS)  to evaluhttps://ourworldindata.org/grapher/covid-containment-and-health-index) for each country and averaged the score over the reported study period. The Containment and Health Index is based on 13 measures: school closures, workplace closures, cancellation of public events, restrictions on public gatherings, closures of public transport, stay-at-home requirements, public information campaigns, restrictions on internal movements, international travel controls, testing policy, extent of contact tracing, face covering and vaccine policy. The index has a value between 0–100 and is calculated on a daily basis, a higher score indicating a stricter response. These data were extracted from Oxford Coronavirus Government Response Tracker (OxCGRT) ..32].2) [The reduction in respiratory viruses associated with residual COPD exacerbations 30) suggests that a major mechanism driving exacerbation reduction is a reduction in acquisition of respiratory viruses, likely through hand hygiene, face coverings and physical distancing in the general population, and shielding in those with COPD. However, there are alternative explanations. The absence of a correlation between COVID-19 Containment and Health Index and exacerbation reduction may suggest that individual behaviour in people living with COPD is more impactful than the changes instituted at societal level. Anxiety in association with COVID-19 might have resulted in an increase in medication adherence , 30. Air suggests2)  and inde2)  were rep2) . In Germ2) . This alDespite the high burden of COPD exacerbations on society, infection control measures against respiratory viruses associated with exacerbations are not currently included in COPD clinical management guidelines. Guidelines primarily focus on optimisation of pharmacological and non-pharmacological interventions, and recommendations to actively reduce viral transmission are not provided. A Cochrane review has highlighted the efficacy of simple, low-cost physical interventions such as hand hygiene and face coverings to reduce respiratory virus transmission in the general population, although endorsing routine use was noted to be potentially difficult in a non-epidemic period . A recenThese analyses have strengths and limitations. This is the first systematic analysis of the impact of COVID-19 pandemic restrictions on COPD exacerbations, especially the most severe COPD exacerbations requiring hospital admission. The results from nine countries were consistent, and we were able to pool data to estimate the weighted rate ratio, considering variation between studies. There was heterogeneity between studies in location and follow-up time while some studies did not report the rate ratio with 95% CI and could not be included in the meta-analysis. Restrictions within individual countries changed with time and we were not able to confirm that jurisdictions with the greatest restrictions saw the greatest reduction in exacerbations. Our DAG permits identification of confounders in the relationship between the coronavirus pandemic and reduced admissions to hospital for COPD exacerbations. Key confounders are age, sex, prior exacerbation frequency and prior disease severity. These confounders have not been accounted for in studies published to date.In conclusion, this systematic analysis and meta-analysis reports a 50% reduction in hospital admissions for COPD exacerbations during the COVID-19 pandemic, likely through reduction in transmission of respiratory viruses. Data for community treated exacerbations were inconclusive and this requires further examination. This work has important implications for future decision-making regarding the prevention of COPD exacerbations. Future guidelines should consider including recommendations on respiratory virus infection control measures to reduce the burden of COPD exacerbations beyond the pandemic period.S1 Checklist(PDF)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.


The COVID-19 pandemic has ushered a globally focused vaccine development program that produced multiple successful vaccines within a year. Four SARS-CoV-2 vaccines have been approved for use in Canada, using two different technologies, all of which have shown excellent efficacy in reducing the rate of symptomatic COVID-19 infection and 100% efficacy in preventing death from COVID-19. People with inflammatory bowel disease (IBD), like many others with immune-mediated chronic diseases, were excluded from the pivotal trials of these vaccines, leading to early hesitancy by regulatory bodies to endorse administering the vaccines to these groups. However, recent data has shown that the adverse event rate to SARS-CoV-2 vaccine among people with IBD is similar to the general population. Early data has further shown that people with IBD are capable of mounting a robust immune response to SARS-CoV-2 vaccines, particularly following a second dose, whereas the response to the first dose is blunted in those receiving anti-TNF therapy or conventional immunosuppressants . Based on these data and evidence from previous vaccine programs among people with IBD, multiple national and international expert panels have recommended that individuals with IBD receive complete vaccination against SARS-CoV-2 as soon as possible. Although people with IBD were excluded from the SARS-CoV-2 vaccine trials, real-world evidence suggests that these vaccines are safe and elicit robust immune responses and protect against SARS-CoV-2 infection following two doses of mRNA vaccine.The effectiveness of the first dose of vaccine (for a two-dose vaccine) is limited (reduced immunogenicity) for people with IBD receiving anti-TNF therapy or conventional immunosuppressants .National and international expert panels recommend that all people with IBD receive a complete series SARS-CoV-2 vaccine at the earliest opportunity, irrespective of vaccine type, disease status, or treatment.COVID-19 Vaccines: Authorized vaccines: Canada.ca).In response to the COVID-19 pandemic, international pharmaceutical companies have rapidly developed, tested and produced highly effective vaccines against the Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) virus. More than 200 COVID-19 vaccine candidates are under development or in clinical trials, using both traditional (inactivated or live attenuated vaccines) and newer  technologies , were excluded from the clinical trials that ultimately led to the approval of these vaccines. Thus, there was initially some uncertainty regarding efficacy and safety of these vaccines in people with IBD. This article reviews the immunology, mechanisms and evidence for currently available  COVID-19 vaccines and discusses the potential risks and benefits of people with IBD being vaccinated.Natural COVID-19 infection often results in exaggerated and dysfunctional innate and adaptive immune responses ,4. InflaControlling COVID-19 requires effective and safe vaccines that provide disease-attenuating immunity, including robust T-cell and B-cell memory responses with the development of germinal centers and high-affinity neutralizing antibodies against COVID-19 and any variants. As variants of concern circulate in the population, several types of vaccines may become important in achieving herd immunity (when 70–95% of the population is immune) , providiCOVID-19 Vaccines: Authorized vaccines - Canada.ca): 1) Pfizer-BioNTech BNT162b2 mRNA vaccine ; 2) NIH-Moderna mRNA-1273 vaccine ; 3) AstraZeneca ChAdOx1 nCoV-19 adenovirus vector vaccine  and 4) Janssen Ad26.COV2.S adenovirus vector vaccine . Large clinical trials have demonstrated these vaccines to be highly efficacious and safe  has recently received approval for use in persons aged 12 and older  and the NIH-Moderna vaccine  use a lipid nanoparticle delivery system to transport modified SARS-CoV-2 genetic material (messenger ribonucleic acid [mRNA]) encoding the virus’ spike protein to host cells ,12. The A large multinational RCT in 43,548 persons, 16 years of age or older who were healthy or had stable chronic disease (excluding immune-mediated inflammatory diseases) demonstrated 95.3% efficacy of the Pfizer-BioNtech vaccine in reducing symptomatic COVID-19 infections at least seven days after the second dose and 90% efficacy in reducing severe disease leading to hospitalization or death  . A similThe AstraZeneca vaccine  and the Janssen Ad26.COV2.S vaccine  use a non-replicating adenovirus vector to deliver SARS-CoV-2 DNA encoding the spike protein to human cells, which uses the host machinery to produce viral spike protein . The AstA pooled interim analysis of 11,636 healthy adults randomized to either active vaccine or control across four RCTs held in the UK, Brazil and South Africa showed 66.7% efficacy of the AstraZeneca ChAdOx1 nCoV-19 vaccine in preventing symptomatic COVID-19 infection at least 14 days after the second dose and 100% efficacy in preventing hospitalization and/or death from SARS-CoV-2 . FurtherRecent rare reports of cerebral and systemic venous thromboembolic events (VTEs), associated with thrombocytopenia and hemorrhage with both the AstraZeneca and Janssen vaccines have raised concerns regarding the risk-benefit ratio of these vaccines, particularly among younger individuals who are at very low risk of dying from COVID-19 . Termed In light of the risk of VITT, NACI has issued a statement that mRNA vaccines are preferred over non-mRNA vaccines. These facts have also prompted several provincial health authorities to suspend administration of the AstraZeneca vaccine as the first dose until more data are available and to consider administering an mRNA vaccine for the second dose among those who received the AstraZeneca vaccine for the first dose ,23. ThesImportantly, the background risk of VTE in the Canadian population is roughly 0.1% , and theCanadian Health Policy, December 2020. ISSN 2562-492). However, this led to a lag in Canadian vaccine procurement and vaccination efforts when Moderna and Pfizer reduced vaccine production to build capacity for larger-scale production. Further, transport and storage logistics of existing vaccines coming from Europe and the US to a central repository in Canada  has been challenging for some of the COVID-19 vaccines, particularly those requiring storage at −20°C to −80°C.For many years, Canada was a leader in vaccine production . While pThe Canadian government plans to increase the domestic production capability of COVID-19 vaccines . First, People with chronic immune-mediated diseases such as IBD were excluded from the trials that evaluated the currently approved SARS-CoV-2 vaccines; therefore, concerns have been raised by healthcare providers and the IBD community regarding the safety and efficacy of SARS-CoV-2 vaccines for people with IBD, as well as the influence of drug therapy on vaccine immunogenicity and safety. Importantly, as none of the approved vaccines are live attenuated vaccines, there is no reason to suspect that individuals with IBD receiving immunosuppressive therapy would be at increased risk of virus reactivation. Moreover, multiple observational studies have now been published on outcomes of SARS-CoV-2 vaccination in people with IBD with no cause for concern indicated by any.In a study of 246 people with IBD who received a SARS-CoV-2 vaccine, the overall rate of adverse events was similar to the general population, while biologic therapy was associated with fewer adverse events, possibly due to blunting of an aggressive immune response . A studyImportantly, in the CLARITY-IBD study, seroconversion was robust following the second dose of vaccine and in individuals who received a dose of vaccine following recovery from COVID-19 infection. Furthermore, a national US cohort study of nearly 15,000 predominantly white males with IBD receiving a wide spectrum of medications in the Veterans Health Administration System reported that the risk of infection with SARS-CoV-2 was 1.34% in those who were unvaccinated and 0.11% among those who were at least seven days from their second dose of mRNA vaccine (80.4% vaccine effectiveness) .NACI updated COVID-19 vaccine statement, May 28, 2021: Summary—Canada.ca).Canadian , EuropeaRegardless of vaccination status, individuals with IBD should remain vigilant with practicing recommended public health measures to prevent SARS-CoV-2 infection and transmission. Given the impaired serological responses to SARS-CoV-2 among people on immunosuppressive treatment, serological testing and virus surveillance should be considered to detect suboptimal vaccine responses, infection persistence and viral evolution to inform public health policy.Vaccine recommendations for special populations with IBD  are provided in articles dedicated to those populations .The collective efforts of scientists, physicians, politicians and industry have led to the fastest ever vaccine development program against any infectious disease known to mankind. Moreover, efficacy and safety of the approved vaccines have been extremely high. While SARS-CoV-2 variants are on the rise across the globe, we are closer to achieving herd immunity with the development and mass rollout of these efficacious vaccines. Despite people with IBD being excluded from the pivotal COVID-19 vaccine trials, emerging evidence and past evidence from other vaccination programs suggest that these vaccines should be similarly safe and effective in those with IBD. As such, the overwhelming recommendation by all societies is that individuals with IBD should receive any of the available vaccines at the earliest opportunity, without delay or interruption of their IBD treatment. Future research is needed to determine if vaccine efficacy wanes among those receiving anti-TNF therapy or conventional immunosuppressive agents and if a timely booster dose is warranted to increase protection. It is important that people with IBD are not excluded from existing vaccination programs and that they do their part to procure timely vaccination to contribute to the ultimate goal of achieving herd immunity.


Cells are complex systems with concurrent multi-physical responses, and cell physiological signals are often encoded with spatiotemporal dynamics and further coupled with multiple cellular activities. However, most existing electronic sensors are only single-modality and cannot capture multi-parametric cellular responses. In this paper, a 1024-pixel CMOS quad-modality cellular interfacing array that enables multi-parametric cell profiling for drug development is presented. The quad-modality CMOS array features cellular impedance characterization, optical detection, extracellular potential recording, and biphasic current stimulation. The fibroblast transparency and surface adhesion are jointly monitored by cellular impedance and optical sensing modalities for comprehensive cell growth evaluation. Simultaneous current stimulation and opto-mechanical monitoring based on cardiomyocytes are demonstrated without any stimulation/sensing dead-zone. Furthermore, drug dose-dependent multi-parametric feature extractions in cardiomyocytes from their extracellular potentials and opto-mechanical signals are presented. The CMOS array demonstrates great potential for fully automated drug screening and drug safety assessments, which may substantially reduce the drug screening time and cost in future new drug development. Understanding complex cell physiology, such as cell function, proliferation, apoptosis, and morphology, is a fundamental step to advance biological scientific research and bio-technology development.16 Compared to traditional biochemical assays or electronics-only sensors, cell-based assays provide physiologically relevant information and offer accurate representations of real-life models. Moreover, they can achieve high sensitivity, selectivity, and fast sample-in-answer-out response time, which can be potentially improved by further genetic engineering the “front-end” cells. In practice, cell-based assays are widely used in the pharmaceutical industry to screen and down-select drug leads or test new drugs for efficacy, pharmacodynamics, pharmacokinetics, and cytotoxicity.Cell-based assays employ living cells as “sensor front-ends” to perform label-free biochemical sensing through cellular physiology that converts biochemical signals to easily measurable physical signals by underlying electronic sensors.21 electrical impedance sensors for cell-growth assay, cardiac beating measurements, and myocardial ischemia detection,24 ion-sensitive field effect transistor (ISFET) arrays for cell metabolism assay,26 magnetic sensor arrays for capturing cardiac beating or molecular detection,29 and optical sensors for DNA sensing and sequencing.34Most existing cell-based assays utilize single-modality electronic sensors, each of which only measures one cell physiological property. Examples include microelectrode arrays (MEAs) with extracellular potential amplifiers and stimulators for recording neuron activities and action potential conduction,However, cells are highly complex systems with concurrent multi-physical activities, and thereby holistic understanding of such complex cellular physiological responses remains a daunting task. For example, cells perform various metabolic activities such as anabolism and catabolism, exhibit electrical activities with excitable membranes, and experience mechanical activities such as chemotaxis, phototaxis, and geotaxis. Also, cells communicate with each other through a wide variety of chemical and physical signals. Therefore, real-time and multi-parametric cell profiling and modelling are of paramount importance to enable decoding and decoupling complex cellular signals and identify target pathways for comprehensive understanding of cell physiology.37 In the early stages of drug development before clinical trials, phenotypic drug screening is often performed, so that large numbers of drug compounds are screened using many panels of disease-relevant cell lines to identify target pathways and potential drug candidates. Furthermore, extensive cell-based assays are utilized to assess the efficacy and toxicity of the selected compounds. The drug development cost increases exponentially at the later stages of new drug development largely due to expensive and low throughput animal testing and clinical trials.39 It has been recently reported that due to late stage failures, the return on investment (ROI) for pharmaceutical companies may drop to as low as 5%.40 Moreover, the cost of drug withdrawal from the market due to drug-induced organ toxicity is even higher.43 For example, terfenadine was withdrawn from the market due to cardiac toxicity with an estimated loss of US $6 billion.42 Therefore, it is essential to down-select failure drug leads and identify drug-induced problems at the early stage.39 As a result, it is indispensable to use cost-effective cell-based assays to investigate the cytotoxic effects of drug compounds with a wide variety of healthy and disease-relevant cells, so that expensive animal testing and clinical trials are only used on the most promising drug leads. However, due to the massive combination of compound libraries and disease-relevant cell lines, as well as extensive drug cytotoxicity test sets, i.e., 5–6 million tests,8 phenotypic drug screenings are often extremely time-consuming and labour-intensive. In parallel, kinetic and multi-parametric cell profiling is essential to capture unknown drug effects or real-time cellular behaviours, which exacerbates the complexity and labour cost of phenotypic drug screening using conventional single-modality sensors. Thereby, a fully automated and low-cost drug screening platform supporting real-time multi-parametric cell profiling is necessary, so that the cost and time for new drug development can be substantially reduced.Multi-parametric cell profiling and modelling are particularly critical for drug screening and drug safety/toxicity assessment.in situ biphasic current stimulation. The detailed operations of the CMOS chip, its electrical characterization, and the biocompatible packaging techniques are explained in references.45 Fibroblasts and neonatal rat ventricular myocytes (NRVM) are successfully cultured on-chip. The time-lapse cell transparency/density and cell-to-surface adhesion of the on-chip cultured fibroblasts are measured by two distinct sensing modalities: static optical opacity detection and cellular impedance characterization. This joint two-modality measurement comprehensively monitors cell migration, proliferation, and viability. Additional time-lapse fibroblast detachment assays with trypsin enzyme are performed for further verification. In addition, simultaneous biphasic current stimulation and optical response monitoring based on cardiomyocytes are demonstrated without any monitoring dead-zone area caused by electrical stimulation artefacts. The real-time optical transparency modulation of on-chip cultured cardiomyocytes is due to their cardiac muscle contraction and relaxation events and is readily measured by in-pixel photodiodes, enabling cardiomyocyte stimulation and response monitoring at the same time and location without any monitoring dead-zone. Furthermore, this real-time optical transparency modulation reveals critical physiological parameters related to the mechanical beating of cardiomyocytes. The multi-parametric isoproterenol dose-dependent feature extractions based on extracellular potential signals and optical signals are also presented with a statistical summary.To address these challenges, in this paper, a 1024-pixel CMOS quad-modality cellular interfacing array to enable real-time multi-parametric cell profiling is presented . The proThe quad-modality CMOS joint sensor/simulator array features extracellular potential recording, static and dynamic optical recordings, cellular impedance sensing, and biphasic current stimulation for on-chip cultured cell samples . The arr21 The selected two electrodes, i.e., vertically adjacent electrode pairs or horizontally adjacent electrode pairs, are scanned through the entire CMOS chip to achieve complete 2-D cellular impedance characterization. For static optical imaging, the entire 4096 optical recording sites are scanned through the CMOS chip, while for dynamic optical recording, one optical recording site per pixel group is selected, and the real-time received light intensity is measured. For the cellular stimulation, two arbitrary electrodes in the same pixel group are selected and connected to the current stimulator that sends fully programmable charge balancing biphasic current pulses.The operations of each modality are briefly explained as follows. For extracellular potential recording, two electrodes, one for sensing and the other for reference, are selected from two arbitrary pixels and a differential amplifier is configured for these two electrodes to enable differential extracellular potential recording with a large suppression (>60 dB) of common-mode noise or perturbations. For cellular impedance sensing, the electrodes of the two adjacent pixels are selected; one for voltage excitation and the other for current termination, and the cellular impedance including the cell-surface contact impedance in the vicinity of the two electrodes is measured. Since two adjacent electrodes with a fixed pixel pitch size of 58 μm are selected, there is no geometry-dependent or location-dependent impedance variations.2) is 1.18 seconds and 9.6 seconds for optical and impedance sensing, respectively.The CMOS chip supply voltage is 3 V and the peak DC power consumption for optical sensing, impedance detection, cellular potential recording, and stimulation are 84 mW, 30 mW, 6 mW, and 9 mW, respectively. The measurement time to scan the entire sensing area . The I/O pads of the chip are wire-bonded to the PCB traces using gold bonding wires with a diameter of 28 μm. The bonding wires are first encapsulated in medical epoxy (EPOTEK) for electrical isolation, while the pixel area is exposed without epoxy to allow for on-chip cell culture and contact-based cellular sensing. Then, an additional PDMS layer is deposited on the cured medical epoxy for improved biocompatibility. Finally, a standard 35 mm cell culture plate with a laser-cut bottom is mounted on the PCB to hold the cell samples and culture medium.73 NRVMs are transduced with an Ad-GFP vector (MOI = 1–2) for 2 hours in suspension at room temperature (RT) and then seeded overnight on the CMOS chip. NRVM spheroids are formed using AggreWellTM . A total of 0.5 mL of rinsing solution  is added to each well and the plate is centrifuged for 5 min at 2000 × g. The plate is washed with cell culture medium prior to use, and 0.5 mL of medium is added to the sample well and centrifuged for 3 min at 2000 × g to remove bubbles. Cell samples containing 1.2 × 106 NRVMs in a 0.5 mL volume is added to each well. The AggreWell plate is centrifuged for 5 min at 200 × g. The plate is incubated overnight, and its medium is replenished by removing 0.5 mL from the edge of the well and adding 0.5 mL fresh medium into the well. After three nights, the spheroids have formed and can be recovered from the plate by gently jetting them out with a pipette.Neonatal rat ventricular myocytes (NRVMs) and cardiac fibroblasts are isolated from 1 to 2 day-old neonatal rats and cultured as monolayers as previously described.Cardiac fibroblasts are formed using a 12-well Corning plate  coated with 2 mL of 1% autoclaved agarose gel. Their hydrophobic coating allows the cardiac fibroblasts to spontaneously form spheroids of different sizes ranging from 30–500 μm. Cardiac fibroblasts are maintained in DMEM supplemented with 10% FBS , 2 mM GlutaMAX, penicillin–streptomycin, and MEM non-essential amino acids (ThermoFisher Scientific). The cardiac fibroblasts are also stained for fluorescence imaging using calcein-AM (ThermoFisher Scientific). Trypsin–EDTA 0.05% (ThermoFisher Scientific) was used to detach the spheroids. The cardiomyocyte spheroids and fibroblast aggregates are pipetted directly onto the CMOS chip. Experiments with murine subjects were performed in compliance with the relevant laws and institutional guidelines and were approved by the IACUC of Emory University.The I/O of the quad-modality CMOS cellular interfacing array are accessed through pin headers in the PCB. The digital control codes are generated by the field-programmable gate array (Measurement Computing USB 1616HS) and streamed into the chip through the on-chip SPI at a clock frequency of 2 MHz. The cellular output signals of the CMOS chips are acquired by the FPGA board and processed in MATLAB. The chip power can be supported by D-type batteries to minimize the 60 Hz power line noise, and the electrical and cell-based measurements are performed in a custom-designed Faraday metal cage for proper electromagnetic shielding.51 Cell-to-cell and cell-to-extracellular-matrix (ECM) adhesive properties are closely related with important physiological processes, such as tissue organization, cell viability and proliferation,49 and cell migration.50 Moreover, cell adhesion assays are essential to investigate the activation of membrane-bound proteins and interactions with extracellular microenvironments,51 which play central roles in disease pathogenesis, such as cardiac fibrosis and cancer metastasis, wound healing, and therapeutic target identification. In parallel, optical opacity offers an additional and orthogonal modality for monitoring cell viability, proliferation, and migration, while providing unique information about cell morphology, density, and contractile function.53 While immunohistochemistry and microscopy are routinely used to assay cell viability, proliferation, and migration, the required labelling procedures and complex imaging set-ups preclude long-term, repeated, and kinetic observation and manipulation, especially at the scale required for high-throughput drug testing platforms. Furthermore, repeated application of fluorescent stains could result in photo-toxicity and cell damage. In this section, time-lapse cellular impedance images and optical opacity images based on the same on-chip cultured fibroblast sample are demonstrated to track cell density and surface adhesion for holistic cell growth assay.Cell-to-surface adhesion and cell transparency are critical phenotypic parameters for functional drug screenings and drug toxicity/safety assessments.t = 0 (the dotted line in t = 39 h (the dotted line in 54 The time-lapse stereo-microscope images (2). The measured opacity images in t = 0 h) is captured in the measured optical opacity image in t = 36 h  after the fibroblast aggregate is pipetted on the top side of the chip. By comparing it to the parallel measured optical opacity image at t = 42 h  are cultured on a fibronectin coated quad-modality CMOS cellular interface array chip. The stimulation capture rates of on-chip cultured rat cardiomyocytes are first characterized 64 The measured average time to reach the contraction peak after stimulation is 190.3 ms with a standard deviation of 13.2 ms  throughout the CMOS chip with the electrical current stimulation at the centre of the chip at t = 0 ms , showingA real-time synchronized collective cardiac muscle contraction and relaxation behaviour is observed, notably without any monitoring dead-zone. The on-chip cultured cardiomyocyte monolayer first contracts upon stimulation until it reaches its peak and then relaxes back to its baseline. Upon stimulation at the chip centre, the cardiomyocyte movement starts from the periphery and then propagates to the centre for both contraction and relaxation. This could be due to the stress build-up at the centre that strains the less constrained periphery first. Interestingly, this is distinct from action potential propagation that is initiated at the centre by stimulation and then propagates to the chip periphery.In this section, physiologically relevant parameters are defined and extracted based on parallel measured extracellular potentials and dynamic optical signals. This dual-modality framework will then be used to investigate the dose-dependent effects of isoproterenol, a known beta-adrenergic agonist, on the excitation–contraction dynamics of cardiomyocytes.Tap), action potential spike amplitude, and field potential duration (Td) can be extracted with respect to the stimulation time, which are closely related to intracellular calcium transients. In addition, since the extracellular potential signal and optical signal are measured on the same pixel with concurrent current stimulation, it achieves a good biological correlation. These two orthogonal domain signals and relevant parameters can be directly processed and extracted, such as the time delay between action potential initiation and muscle contraction initiation, which is particularly useful for drug screening to evaluate arrhythmogenic drug side effects.69duration  and c caσ) of 9.1 ms; the measured relaxation slope peak time (RX′_PKS) is 308.9 ms with a σ of 16.5 ms; the measured optical cycle duration (Td) is 478.6 ms with a σ of 23.6 ms. With an increased isoproterenol concentration of 3 nM (Td decrease to 226.2 ms, 207.4 ms, 302.4 ms, and 424.6 ms, respectively. These parameters further decrease with the isoproterenol concentration of 10 nM (70Finally, the multi-parametric dose-dependent response of cardiomyocytes is studied using isoproterenol and feature extractions from their extracellular potentials and opto-mechanical signals are performed.  of 3 nM , the meaof 10 nM . These mIn this paper, a CMOS quad-modality cellular interfacing array chip supporting multi-parametric and holistic cellular characterization for fully automated high-throughput drug screening and drug safety/toxicity assessments is presented. The CMOS quad-modality cellular interfacing array features extracellular potential recording, cellular impedance measurement, static/dynamic optical recording, and biphasic current stimulation. It contains 1024 multi-modal pixels with a 58 μm pixel pitch size in a total chip area of 2 mm by 3 mm, including all the front-end circuits and signal conditioning electronics.i.e., the optical sensing modality and the impedance sensing modality. The measured optical opacity image shows the location and projected 2-D shape of the fibroblast aggregate as well as the opacity gradient within the fibroblast aggregate. The time-lapse optical opacity image tracks the cell opacity changes over time and captures that fibroblasts exhibit increasing optical transparency with time, due to desired cell proliferation and migration. In parallel, the measured cellular impedance monitors the cell-to-surface adhesion, and the time-lapse impedance measurement tracks the expansion of fibroblast adhesion on the CMOS chip surface over time. By comparing the optical opacity and impedance measurements, the cellular growth process and surface adhesion behaviour are monitored comprehensively.With our CMOS quad-modality chip, label-free time-lapse cell transparency and cell-to-surface adhesion measurements based on on-chip cultured fibroblasts are demonstrated for cell growth and viability monitoring using two orthogonal modalities, Secondly, using on-chip cultured cardiomyocytes, simultaneous electrical current stimulation and cellular optical response monitoring are demonstrated without any monitoring dead-zone. The simultaneous stimulation and cellular response monitoring are essential to study electro-mechanically active cells such as cardiac cells. However, conventional electrical stimulation and extracellular potential recording systems suffer from a large dead-zone area around the stimulation site where the potential recording is prohibited due to the potential recording amplifier saturation by the stimulation artefacts. To address these challenges, simultaneous electrical current stimulation and optical cellular response monitoring are proposed. Upon stimulation, the synchronized cardiac muscle contraction and relaxation modulate the light transparency of cardiomyocytes and this light transparency modulation is captured by in-pixel photodiodes of the CMOS array. Simultaneous electrical current stimulation and optical recording on a single pixel at different stimulation rates are first demonstrated. Extracellular potential recording at a distant site is used for reference. Then, 1024 optical sensing pixels are sequentially scanned to measure the real-time collective behaviour of the on-chip cultured synchronized cardiomyocyte cluster upon stimulation. The measured transient optical signals at 1024 pixels show that upon stimulation at the chip centre, the cardiac muscle movements start from the chip periphery and then propagate to the centre both for contraction and relaxation, which could be due to the build-up of stress from the centre that strains the less constrained periphery first.Finally, multi-parametric feature extractions are presented. Several critical physiological parameters such as the contraction slope peak time, the contraction peak time, the relaxation slope peak time, and the optical cycle duration are defined based on time derivatives of the recorded real-time optical signals of the cardiomyocytes, which are closely related to their intracellular calcium transients. Then, isoproterenol as an example drug is administrated to show the dose-dependent multi-parametric feature extractions. At different isoproterenol concentrations, the extracellular potentials and optical signals are measured at the same pixel to ensure a good biological correlation. The physiological parameters are extracted from extracellular potentials and optical signals and show that the action potential cyclic processes are consistently shortened with increased isoproterenol concentrations. Our CMOS quad-modality cellular interfacing array enables measurements of orthogonal physiological processes such as electrochemical and opto-mechanical responses on the same cellular sample with high biological correlation, supporting holistic cellular characterization and high throughput drug screening. As future work, new machine learning algorithms for multi-parametric feature extractions and classifications can be investigated and integrated into our CMOS quad-modality cellular interfacing array to facilitate drug screening/development and the discovery of dose-dependent patterns and relations among various physiological parameters.