This case is shared to reiterate and confirm the principles of ensuring the safety of the surgical team caring for COVID-19-confirmed patients, thus, preventing the spread of infection within the hospital.A 54-year-old male, COVID-19-confirmed patient complaining of abdominal pain since two days prior was transferred to our hospital. Perforated appendicitis with a periappendiceal abscess was diagnosed by computed tomography. Laparoscopic appendectomy was performed in a negative-pressure operating room. The surgical team wore enhanced personal protective equipment. Electrocautery was not used during surgery and no other special instruments were applied to reduce aerosol generation. No special instruments or filters were used for the removal of intra-abdominal gas. The operation was completed successfully and no immediate surgical complications occurred. The patient advanced to a normal diet on the 4th postoperative day. The patient was treated with antibiotics for bacteremia and antiviral therapy for underlying pneumonia in the setting of COVID-19 with most symptoms dissipating by the 7th postoperative day. The patient was discharged on the 30th postoperative day without any complications.A well-designed manual, a well-trained surgical team, and a negative-pressure operating room are essential for safe laparoscopic appendectomies in COVID-19 patients.When surgery is performed in a negative-pressure operating room by a well-trained surgical team, a laparoscopic appendectomy can be successfully performed under the principles of obtaining optimum clinical outcomes while faithfully ensuring the safety of healthcare providers and the hospital environment. We inteThe hospital involved was a 550-bed tertiary referral hospital, which operates a Level 1 emergency center and a 12-bed negative-pressure isolation ward including five negative-pressure isolation ICU beds . It operThis case report was drafted according to the SCARE Guidelines 2018 .2A 54-year-old male patient was transferred with diagnosed acute perforated appendicitis. The patient was admitted to a public hospital after being confirmed for COVID-19 by real-time polymerase chain reaction (RT-PCR) analysis four days before the transfer. The patient had no complaints including respiratory symptoms at the time of admission. Lower abdominal pain and diarrhea began two days prior to transfer, and he was diagnosed with perforated appendicitis on a non-contrast abdominal computed tomography (CT) scan. The public hospital mainly treated mild COVID-19 cases, and because it was not suitable for performing the emergency surgery, the patient was transferred to our hospital facility, dedicated to treating complicated COVID-19 cases and equipped with a negative-pressure operating room.The patient was taking anti-hypertensive medication that did not contain anticoagulants and had no significant surgical, family, or social history. The patient was an immigrant construction worker.The patient complained of only abdominal pain and did not report any respiratory symptoms such as coughing or sputum. The patient was 176 cm tall and 75 Kg with a body mass index (BMI) of 24.2. The physical examination was significant for tenderness and rebound tenderness throughout the lower abdomen. The vital signs showed 80/52 mmHg blood pressure, a pulse of 98 beats/min, a respiratory rate was 20 times/min, a body temperature of 36.2 OC, and 96 % oxygen saturation on room air.Laboratory investigations revealed a white blood cell (WBC) count of 15.3 × 10³/㎕ and a C-reactive protein (CRP) value of 18.30 mg/dL. The other laboratory findings were not specific. Since information from the non-contrast CT scan that accompanied the patient was limited, a contrast-enhanced abdominal CT scan was ordered to establish a more precise surgical plan. The CT scan showed perforated acute appendicitis with a periappendiceal abscess and widespread pelvic peritonitis . There wBased on the above findings, upon written informed consent for the procedure and notification of the risks and benefits, a faculty surgeon with nine years of experience performed an emergency laparoscopic appendectomy. The CT scan findings showed inflammatory processes spread throughout the pelvic cavity, so it was felt that laparoscopic appendectomy was less invasive and could be completed within a short time, and thus, considered more favorable for the treatment outcome without the risk of further infection spread.The patient entered the operating room on a negative-pressure cart, which was placed in the operating room (OR) during the surgery .Fig. 2A)General anesthesia was performed through endotracheal intubation using a video laryngoscope by an anesthesiologist wearing a Level 4 protective suit. The operation was initiated by the surgical team wearing enhanced personal protective equipment (PEE). The enhanced PPE included an isolation gown, surgical gown, head cover, face shield, two pairs of surgical gloves, shoe covers, and an N95 mask [2 flow rate of 40 L/min. A closed-circuit smoke evacuation system was not used. The perforated appendix and the spread of inflammation to the surrounding organs were observed, and suppurative fluid was accumulated in the pelvic cavity. Electrocautery was not used during surgery. An ultrasonic scalpel for dissection, a plastic clip for vascular ligation, and an endo-loop for stump ligation of the appendix were all used [A 12-mm port was applied to the umbilicus, and a 5 mm port was applied to the upper pubis and the left para-midline. Pneumoperitoneum was formed to 14 mmHg abdominal pressure at a COall used . DisposaPersonnel exiting the OR disposed of their PPE in the OR under supervision and showered immediately thereafter. Dedicated well-identifiable containers for infectious-risk waste were used.The patient was monitored in a negative-pressure cart in that OR until the vital signs stabilized and the patient was fully conscious before being transferred to a negative-pressure isolation ward. The patient was subsequently transferred to the department of infectious disease for postoperative care.The operation lasted 45 min, anesthesia was applied for 60 min, and the operating room retention time was 90 min.The operating room was maintained under negative-pressure air conditioning for two hours after the patient was moved out. Following three hours of fumigating disinfectant, surface disinfection with diluted chlorine bleach (500 ppm) was applied. Five hours after the operation was completed, the operating room was ready for use again. The entire process of patient transfer, preparation of the operating room, the surgical procedure, and postoperative disinfection was monitored and recorded by an infection specialist nurse.All participating members of the surgical team were monitored actively for two weeks. There were no signs of medical staff infection nor the spread of infection within the operating room.Staphylococcus capitis infection detected on blood cultures, persisted until the 6th postoperative day with resolution following antibiotic therapy. On the 7th postoperative day, the respiratory and abdominal symptoms improved, the WBC count normalized, and the CRP level decreased to 1.43 mg/dL.On the 4th postoperative day, the patient started a normal diet. Pneumonia was found on a chest CT scan, prompted by subjective complaints of coughing symptoms, and hydroxychloroquine therapy was started . The draNo postoperative complications were observed, and after two consecutive negative results for COVID-19 by RT-PCR, the patient was discharged on the 30th postoperative day. Follow-up was not possible because the patient returned to his home country immediately after discharge.3Before this case, we experienced eight cases of emergency laparoscopic appendectomies performed before COVID-19 tests were confirmed. As a hospital dedicated to infectious diseases, a multidisciplinary team had developed an instruction manual for emergency surgery for confirmed COVID-19 patients. The manual was improved through training and debriefing via several simulations ,8. It isOne of the concerns in laparoscopic surgery for COVID-19-confirmed patients is the proper care of aerosols containing viruses that could be produced by these patients. In this case, no electrocautery was used, and no special device was used for intra-peritoneal gas leaking out during surgery or for intra-peritoneal gas discharge following surgery . The negThere is a risk of infection to the surgical team during surgery, but there is a higher probability of contamination, especially when PPEs are removed after surgery . TherefoIn this case report, there was a limit to reviewing the overall considerations for laparoscopic appendectomy for patients with confirmed COVID-19. Therefore, follow-up analysis and research are warranted for all surgical cases suspected of a positive diagnosis as well as the confirmed patients.4When surgery is performed in a negative-pressure OR by a well-trained surgical team, a laparoscopic appendectomy can be performed under the principle of obtaining optimum clinical outcomes while faithfully ensuring the safety of healthcare providers and the hospital environment.The authors report no declarations of interest.No funding involved in this case study.On the basis of this being a case report, the Institutional Review Board of the Myongji hospital does not mandate that ethical approval is required. Thus, this case report is exempt from the Institutional Review Board Approval process .Written informed consent was not obtained from the patient for publication of this case report because the patent was leaving the country. A letter stating that the hospital president will take full responsibility for the patient's damage caused by publication is available for review by the Editor-in-Chief of this journal on request.Major contributors to writing the manuscript: Dongkyu Oh and Yu Min Kang.Searching the literature, contributor to writing and reviewing the manuscript: Jin Yong Choi.Reviewing the manuscript for important intellectual content: Yu Min Kang.Study concept and final review: Wang Jun Lee.All authors read and approved the final manuscript.NA.Wang Jun Lee, M.D., PhD.Not commissioned, externally peer-reviewed.


Current guidelines for COVID-19 management recommend the utilization of various repurposed drugs. Despite ongoing research toward the development of a vaccine against SARS-CoV-2, such a vaccine will not be available in time to contribute to the containment of the ongoing pandemic. Therefore, there is an urgent need to develop a framework for the rapid identification of novel targets for diagnostic and therapeutic interventions. We analyzed publicly available transcriptomic datasets of SARS-CoV infected humans and mammals to identify consistent differentially expressed genes then validated in SARS-CoV-2 infected epithelial cells transcriptomic datasets. Comprehensive toxicogenomic analysis of the identified genes to identify possible interactions with clinically proven drugs was carried out. We identified IFITM3 as an early upregulated gene, and valproic acid was found to enhance its mRNA expression as well as induce its antiviral action. These findings indicate that analysis of publicly available transcriptomic and toxicogenomic data represents a rapid approach for the identification of novel targets and molecules that can modify the action of such targets during the early phases of emerging infections like COVID-19. Coronaviruses are a large family of viruses that were first described over 50 years ago . Since thttps://www.ncbi.nlm.nih.gov/geo/). Only microarray gene expression datasets with the word “SARS-COV,” virus, or modified strain infected vs. mock-infected and no more than 48 h after the infection. Twelve datasets fulfilled the criteria, as detailed in http://metascape.org/gp/index.html#/main/step1). The shortlisted genes expression was then explored in another dataset (GSE147507), where RNA-Sequencing of primary human lung epithelium (NHBE) mock-treated or infected with SARS-CoV-2 was done to examine whether there is a difference in the response of SARS-CoV-2 from other strains in terms of DEGs (Publicly available transcriptomic datasets were retrieved from Gene Expression Omnibus (GEO)  between mock-infected and virally infected models in the 12 studies. Thirty-eight genes that were DEGs in at least 9 out of 12 studies (75%) were considered common DEGs due to SARS-COV infection of the lungs in the first 48 h post-infection. These genes are listed in In order to identify DEG in SARS-CoV infected lung tissue-specific to each of the models used and those which are shared, we intersected the DEGs from datasets that use the same model. Human epithelial cells datasets , mice datasets , Ferret (GSE11704) and Cynomolgus macaques (GSE23955) were all intersected with the COVID-19 infected epithelial cells dataset as shown in As expected, the top genes identified are involved in innate immune responses against RNA viruses. These include the cytosolic DNA-sensing pathway, Toll-like receptor signaling pathway, and negative regulation of binding. Interferon (IFN) response to viral infections such as type I interferon signaling pathway, defense response to the virus, the antiviral mechanism by IFN-stimulated genes, regulation of type I interferon production, response to interferon-alpha, and regulation of defense response to virus and Influenza A, were also upregulated. Genes that play significant roles in activating immune systems such as regulation of response to cytokine stimulus, negative regulation of immune response, myeloid cell homeostasis, and positive regulation of the multi-organism process are also upregulated .p < 0.05), while two other genes OAS2 and MX1 showed a trend of enhancement, although it was not statistically significant . IFITM3 mRNA levels were one of the highly expressed genes compared to the other identified genes at baseline in mock-infected HBE and were further induced by the virus, which results in overall high mRNA levels.The identified genes expression levels were higher in human bronchial epithelium infected with SARS-CoV-2 compared to those mock-infected . Howeverhttp://ctdbase.org/) to identify drugs/chemicals that might affect the mRNA expression of IFITM3 in at least two reference studies  compared to healthy controls  was extracted and reanalyzed. Healthy controls were compared to responders and non-responders patients on valproic acid therapy. We found upregulation of the mRNA expression of IFITM3 in patients, and the difference was significant in the responder group only , to activate antiviral mechanisms and effector cells like natural killers . In miceOur reanalysis of transcriptomic data showed that although the IFN pathway is upregulated consistently in SARS-CoV related infection, SARS-CoV-2 showed specific upregulation of the gene for a unique interferon-induced protein, namely IFITM3. IFITM3 is a 15-kDa protein that localizes to endosomes and lysosomes and is possibly acquired by mammalian ancestral cells via horizontal gene transfer .Interferon-induced transmembrane proteins  are innate immune responders to virus infections as they regulate the fusion of invading virus and endocytic vesicles and direct it to the lysosomes , 20. IFIThe epithelial cell and resident leukocytes in lung upper and lower airways that constitutively express IFITM3 can withstand viral infections, and this is vital to decide viral tropism as viruses favor cells with low IFITM3 expression . IFITM3 Interestingly, IFITM3-rs12252-C/C SNP prevalence in the Chinese population is 26.5%, and recent research confirmed that SNPs in IFITM3 could change the severity of influenza infection, as was shown in one case with COVID-19 . IFITM3 Chiroptera), which act as natural hosts for many viral infections, use IFITM3 as an antiviral mechanism if there is S-palmitoylation of the protein; however, if this modification is disturbed, the bat can develop viral infection (Expressing the gene is not the only prerequisite to the antiviral action of IFITM3, as it was found recently that within the protein, an amphipathic helix is critical for its blocking effect of viral fusion of similar pathogenic viruses like influenza A virus and Zika virus . Anothernfection . Based oOur toxicogenomic analysis showed that valproic acid increased the mRNA expression of IFITM3, supporting a new report that the SARS-CoV-2-human protein-protein interaction map showed that valproic acid might be a potential repurposing drug for COVID-19 . VirtualOne limitation of the study is that it is based on the publicly available transcriptome dataset, which is limited in number, partly because this is a novel disease, but also because ongoing lockdowns have made it challenging for scientists to carry out the extensive laboratory work required.Our evaluation showed that the analysis of publicly available transcriptomic data could be a reasonable approach to identify the novel target and suggest drugs that can modify the action of such targets during the early phases of emerging infections like COVID-19 until a complete understanding of the disease become clear. This can justify the experimental use of clinically approved drugs and guide the clinicians in their limited options against such lethal disease.All datasets presented in this study are included in the article/supplementary material.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Due to the time-consuming experimental methods for proposing useful treatments, computational approaches facilitate investigating thousands of alternatives simultaneously and narrow down the cases for experimental validation. Herein, we conducted four independent analyses for RNA interference (RNAi)-based therapy with computational and bioinformatic methods. The aim is to target the evolutionarily conserved regions in the SARS-CoV-2 genome in order to down-regulate or silence its RNA. miRNAs are denoted to play an important role in the resistance of some species to viral infections. A comprehensive analysis of the miRNAs available in the body of humans, as well as the miRNAs in bats and many other species, were done to find efficient candidates with low side effects in the human body. Moreover, the evolutionarily conserved regions in the SARS-CoV-2 genome were considered for designing novel significant siRNA that are target-specific. A small set of miRNAs and five siRNAs were suggested as the possible efficient candidates with a high affinity to the SARS-CoV-2 genome and low side effects. The suggested candidates are promising therapeutics for the experimental evaluations and may speed up the procedure of treatment design. Materials and implementations are available at:  Recently a new coronavirus (CoV) named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged from China and globally outbreak  with oveCOVID-19 infection causes multiple frequent symptoms such as fever, dry cough, difficulty breathing or shortness of breath, dyspnea, fatigue, headache, diarrhea, and lymphopenia . IndividBecause of the recent outbreak of COVID-19, our knowledge about its pathogenesis, molecular mechanisms, prevention, and treatment strategies is deficient . BesidesWe aim to prevent virus activities through inhibiting viral replication and translation, or by prohibiting the viral-host binding. To this aim, we target the most functional regions that are responsible for viral replication and translation, or viral-host binding. The evolutionarily conserved regions in SARS-CoV-2 have more potential to play critical roles in viral replication and translation because these regions were selected and conserved during evolution. Therefore, these regions are promising candidates for targeting by antiviral and oligonucleotide therapies . MoreoveRNA interference (RNAi) is an alternative therapeutic strategy when current treatment technologies fail to obtain a promising result . RNAi-bamiRNAs are small non-coding RNAs with usually 22 nt lengths, which have vital functions in post-transcriptional regulation of target genes . They plsiRNA has high specificity and is an efficient strategy for suppressing specific genes . Since sNevertheless, suggesting candidate miRNAs and siRNAs that potentially have high affinities to interact with the SARS-CoV-2 genome is a promising idea that requires significant attention. One of the challenges in using RNAi-based treatments is the competition between the body mRNAs and SARS-CoV-2 RNA for binding to the miRNA/siRNA . If miRNSeveral databases have gathered the experimentally-validated miRNA-mRNA interactions . MoreoveBesides the miRNAs available in the human body, the miRNAs available in other species can have a promising mechanism against SARS-CoV-2. Among all species, bats (Chiroptera) are the most favorable, since bats are the most probable origin of SARS-CoV-2 and some other CoVs . MoreoveThis study aims to target the unstructured conserved regions (UCRs) of SARS-CoV-2 using the available miRNAs or novel siRNAs. In this study, some candidate miRNAs from human, bat, and other species are suggested that potentially inhibit SARS-CoV-2 replication and translation while having less affinity to bind other human body mRNAs. Using these miRNAs aids in maximizing treatment efficiency and minimizing side effects. The affinity binding of miRNAs to the UCRs of SARS-CoV-2 is computed to ensure a high potential treatment. To further investigate the effects of using suggested miRNAs, the biological functions of their targets in UCRs of SARS-CoV-2 are analyzed. Moreover, the potential side-effects are investigated by examining the functionalities of mRNAs that may computationally interact with the suggested miRNAs. In addition to recommending efficient miRNAs, five efficient and target-specific siRNAs are proposed for silencing SARS-CoV-2 genes. The suggested siRNAs satisfy in the required sequence constraints , the list of miRNAs and their interactions were collected from CLASH . This daIn the second analysis , the sequences of 539 bat-specific miRNAs were obtained from the previously published article by In the third analysis , the known miRNAs of 286 species were downloaded from the miRNA registry, mirBase . mirBaseThe fourth analysis  is conducted for proposing new potential siRNAs, for which siDirect 2.0  tool is In this study, we conducted three independent analyses for suggesting candidate miRNAs and one analysis for designing efficient siRNAs. The miRNAs and their interaction information in theses analysis are obtained from the following sources:All analyses in this study are conducted with the aim of either recommending potential miRNAs or proposing efficient siRNAs. These two procedures are described in the “Recommending Potential miRNA” and “Proposing Efficient siRNA”. The overall workflow is shown in The conserved regions were obtained by Computing the free binding energy for the hybridization of miRNAs of each dataset and UCRs of SARS-CoV-2: Binding energy is one of the important factors for measuring the affinity of binding . EffectiAnalyzing the efficiency of utilizing the suggested miRNAs by investigating the biological functions related to the target UCRs of SARS-CoV-2.Finding the most probable target mRNA in the human body that is a rival for target UCR and analyzing its GO terms and pathways.Investigation for promising miRNAs is conducted according to the following steps:We considered a window of length 23-mer and slide it on the target UCR in SARS-CoV-2. In this way, all possible 23-mer subsequences in the target sequence are considered, and the corresponding siRNA is designed with a 21 nt complementary guide and a passenger strand with two nt overhang at the 3’ end. Then, the list of all possible siRNAs was refined to the siRNAs that have the following three sequence conditions :– with “A” or “U” nucleotide at the 5′ end of the guide strand– with “G” or “C” nucleotide at the 5′ end of the passenger strand– with more than 4 “A” or “U” residues in the 5′ terminal seven bp of the guide strand– without contiguous substring of G/C of length larger than four bp– with less than 30% GC contentCalculating melting temperature (Tm) for the seed-target duplex located at 2–8 from the 5′ end of the siRNA guide strand and its target . Tm calcRemoving siRNAs that have near-perfect complementary to other human mRNAs, rather than the selected target location. siDirect analyzed 19 nt lengths from 2 to 20 positions in both strands via BLAST and pre-computed hits to find near-perfect match candidates . FinallyProviding efficient and target-specific siRNA design for mammalian RNAi is done by siDirect 2.0 , which hTo evaluate the efficiency of available miRNAs in the human body for targeting UCRs, we analyzed the CLASH database. CLASH is a gold standard dataset that contains experimentally validated interactions between human miRNAs and human mRNAs.The goal of this analysis was to find appropriate available miRNAs that have efficient interactions with UCRs and low-affinity to human mRNAs. For each miRNA . The entry of SARS-CoV-2 into cells is mediated by spike (S) glycoproteins, which binds to human angiotensin-converting enzyme 2 (ACE2) for cell entry . The spiTo analyze the possible side effect of using the candidate bat miRNAs in human body, we enriched their predicted targets in GO using UniProt . Figure The previous analyses suggested the favorable candidate miRNAs from human and bat that have a potential role in the treatment of COVID-19. Nevertheless, other species may contain miRNAs that reveal promising antiviral activities against SARS-CoV-2. To investigate this idea, we considered the miRNA sequences of all species available in mirBase . To chooTribolium castaneum) and targets a UCR in ORF9ab, which encodes nucleocapsid protein in SARS-CoV-2. It binds to the viral genome and provides stability for virus , sea vase , red junglefowl , human (homo sapiens), and Lotus japonicus. These miRNAs target UCRs in nsp3, nsp4, and spike protein. As it is mentioned in “Suggesting Candidate miRNAs from the Human Body for the Treatment of COVID-19”, these regions are of high importance for viral transcription, replication, and binding to host cells. The suggested miRNAs from various species are potentially promising alternatives for targeting these important regions in SARS-CoV-2 and lowering its functionality and spread.Other candidate miRNAs are from gray short-tailed opossum  foldings of these five siRNAs were obtained by RNAfold in the ViennaRNA package . The obtThe recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) became pandemic and international concern. Currently, no vaccine or specific drug has been proposed for SARS-CoV-2 or other zoonotic coronaviruses. Therefore, it is essential and emergency to look deeper into potential treatments. RNA interference (RNAi) is a therapeutic strategy that is profitable in the treatment of viral infections when other conventional approaches fail to obtain promising results. This approach is based on delivering small RNA duplexes, including miRNA or siRNA, to the body for triggering the inhibition of specific genes. In this study, we investigated the potential RNAi-based therapy that may help in the treatment of COVID-19. We analyzed the efficiency of the available miRNAs as well as newly designed siRNAs for downregulating and silencing the regions in SARS-CoV-2 that are both conserved and unstructured with high probability. To this aim, the unstructured conserved regions in SARS-CoV-2 were obtained using multiple sequence alignment and computing the unpaired probability using a computational method. Fifty-eight unstructured conserved regions were obtained and considered as the desirable regions for targeting by miRNA/siRNA, due to their evolutionary conservation, disinclination to develop resistance, high affinity to bind by hybridization and more accessibility to therapeutic interventions .Four independent analyses were conducted to investigate the application of RNAi-based therapies in the decline of SARS-CoV-2 survival. Three analyses aim to suggest potential miRNAs that exist in human, bat and 286 other species for downregulating the unstructured conserved regions of SARS-CoV-2. The investigation of efficient miRNAs was done using computing the free binding energy of the hybridization of miRNAs and the regions in SARS-CoV-2. The ones with the least free binding energy were considered as the candidate miRNAs. Among the investigated miRNAs, four human miRNAs, six bat-specific miRNAs, and six other miRNAs from other species were extracted that have a high affinity to bind essential and functional regions in SARS-CoV-2 genome. The candidate miRNAs were further examined to check their possible side effects. In order to do this, the most probable human mRNA targets for the candidate miRNAs were obtained from experimentally validated databases or computational tools. Moreover, the Go terms , as well as Reactome pathways related to the most probable targets, were extracted. Among the recommended miRNAs, tca-miR-6014-3 is very promising because its side effects prevent viral activity in the body and reduce viral infections.In addition to three mentioned analyses for suggesting potential miRNAs, another analysis was conducted to design efficient siRNAs for silencing the unstructured conserved regions of SARS-CoV-2. Five efficient siRNAs were identified for targeting four critical regions in SARS-CoV-2. These regions play essential roles in viral replication and the attachment of the virus to host cells. Therefore, the application of proposed siRNAs may inhibit the replication and entry of SARS-CoV-2. Moreover, the most potent human mRNA targets of these siRNAs were obtained using computational tools, and their GO terms and pathways were analyzed for investigating their possible side effects.All in sum, this study recommends 16 potential miRNAs and five efficient siRNAs that may help in the treatment of COVID-19. The candidate miRNAs and siRNAs are promising cases to be validated experimentally in cultured cells, or mouse models can reduce the time and costs of trying non-promising cases.10.7717/peerj.10505/supp-1Supplemental Information 1Sheet 1: The UCRs of SARS-CoV-2 including their regions, sequences, corresponding proteins, and unpaired probabilities.Sheet 2: The computed free binding energy between human miRNAs and mRNAs.Sheet 3: Comparison of binding energy for binding human miRNAs to mRNAs or UCRs.Sheet 4: The computed free binding energy between bat miRNAs and human mRNAs.Sheet 5: The list of possible mRNA targets for candidate bat-specific miRNAs.Sheet 6: The computed free binding energy between miRNAs of all species and human mRNAs.Sheet 7: The list of possible mRNA targets for candidate miRNAs in other species.Click here for additional data file.


We currently find ourselves in the midst of a global coronavirus disease 2019 (COVID-19) pandemic, caused by the highly infectious novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we discuss aspects of SARS-CoV-2 biology and pathology and how these might interact with the circadian clock of the host. We further focus on the severe manifestation of the illness, leading to hospitalization in an intensive care unit. The most common severe complications of COVID-19 relate to clock-regulated human physiology. We speculate on how the pandemic might be used to gain insights on the circadian clock but, more importantly, on how knowledge of the circadian clock might be used to mitigate the disease expression and the clinical course of COVID-19. Chronobiology of COVID-19 CARE Conference). Convened in July 2020, this virtual workshop brought together 250 researchers and clinicians worldwide. They engaged in a wide-ranging dialogue to consider how chronobiology and COVID-19 pathology might relate, what kinds of model systems are needed, and how clinical data might best be harnessed to explore such connections. Here, we provide a brief overview of COVID-19 with an emphasis on elements potentially sensitive to circadian regulation based on the existing pre-COVID literature. We conclude with a review of how critical illness and the intensive care setting influence circadian output, which is relevant to patients with severe COVID-19.Coronavirus disease 2019 (COVID-19) is a new pandemic respiratory illness with a highly variable clinical presentation, ranging from asymptomatic infection to profound respiratory and multiorgan failure . With ovThe causative agent of COVID-19, SARS-CoV-2, is a novel beta-coronavirus that bears genetic and clinical resemblance to two prior endemic coronaviruses of global concern: SARS, severe acute respiratory syndrome, which appeared in 2002 and MERS, Middle East respiratory syndrome, which appeared in 2012 . While aSARS-CoV-2 infection begins in the upper respiratory tract with differentiated airway epithelial cells being the initial sites of infection, particularly multiciliated cells and secretory (club) cells . InfectiTwo forms of systemic pathology bear special mention in severe COVID-19. The first is macrophage hyperactivation, leading to the elaboration of high levels of circulating pro-inflammatory cytokines such as interleukin 6 IL-6; . The terThe prognosis of COVID-19 largely depends on the severity of respiratory failure. Patients with mild to moderate disease appear to clinically recover within 2 weeks, although viral RNA can be detected in many patients for longer periods . At presSARS-CoV-2  is an enThe genome of SARS-CoV-2 consists of a single positive-strand RNA roughly 30 kb in length that is dominated by a single open reading frame (Orf1a/1b) taking up two thirds of its length . Orf1a/1The next step in the SARS-CoV-2 replication involves the establishment of a specialized organelle in continuity with the endoplasmic reticulum (ER) called a double-membrane vesicle (DMV) structure . The DMVViral RNA synthesis by coronaviruses generally involves the production of a complementary negative-strand RNA template starting from the 3’ end of the viral genome . Full-leThe final step in the SARS-CoV-2 life cycle is the assembly and release of virions. For coronaviruses, structural proteins of the viral envelope  are cotranslationally inserted into the ER membrane and traffic to the ER-Golgi intermediate compartment ERGIC; . There, To summarize, SARS-CoV-2 engages infected hosts at multiple levels during severe infection, inserting itself into the host’s physiological processes, immune responses, and fundamental cellular machineries. All these layers of biological organization are influenced by circadian rhythms, whose basic structure in mammals is summarized in the next section.Circadian clocks impose a 24-h temporal structure on more processes than we are able to count . In 19722+ could synchronize and phase shift clock gene oscillations in Rat-1 fibroblasts. That this is relevant for human entrainment and clock-regulated gene expression was shown with administration of glucocorticoids once a day, in the afternoon, resulting in reentrainment of clock gene expression in peripheral blood mononuclear cells  pathology to how the circadian clock may contribute to containment or exacerbation of symptoms to considerations in the clinical critical care environment. Any of these aspects may lead to novel opportunities for interventions.Concerning viral biology, are ACE2, TMPRSS2, and FURIN clock regulated within cells? We interrogated the database that describes gene expression in 64 tissues over 24 h in young, healthy, male baboons . These gACE2, angiotensin-converting enzyme 2, catalyzes the hydrolysis of angiotensin 2 into angiotensin 1-7, which has important anti-inflammatory properties . As a reBiological functions involved in viral processing within cells  commonly exhibit circadian oscillations in activity . In geneConcerning how the circadian clock may contribute to exacerbation of pathology in critically ill patients with COVID-19, several symptoms are particularly relevant. These patients may experience respiratory failure, microcoagulation events, and cytokine storm. The molecular mechanisms involved in each of these are regulated by the circadian clock. For instance, human respiratory function shows clock regulation in mechanics (airway resistance and flow characteristics), ventilatory response to hypercapnia, and airway responses to inhaled allergens . ConcernWith regard to the expression of cytokines, both the transcription of cytokine-encoding genes as well as stimulation of cytokine release show strong time-of-day regulation. Clock proteins are known to be activators of some cytokines and suppressors of others . Clock rThe development of therapies to mitigate COVID-19 is an intense area of focus. Current therapies available to critically ill patients include anti-inflammatory drugs like glucocorticoids and tocilizumab, direct antivirals like remdesivir, and SARS-CoV-2–neutralizing antibodies conferred by convalescent plasma. Whether time of day plays a role in the efficacy of these drugs in COVID-19 patients is unclear. However, the pharmacokinetics and pharmacodynamics of many drugs are known to change over the 24 h. Accordingly, so do the efficacy and tolerance to chemotherapy , and antRecently, a prospective meta-analysis from the WHO Rapid Evidence Appraisal for COVID-19 Therapies (REACT) Working Group pooled data from seven trials totaling 1703 patients and showed that administration of systemic corticosteroids is associated with a lower 28-day mortality in critically ill COVID-19 patients . CommonlThe potential effect of the ICU environment on a patient’s circadian clock has been acknowledged for years. This may be especially relevant in critically ill COVID-19 patients who require a prolonged ICU stay with limited exposure to normal environmental zeitgebers. ICUs are well known to feature low, noncycling or weakly cycling light environments and excessive noise due to equipment and alarms, conditions which disturb normal sleep . FurtherCertain severe illnesses may lead to disrupted circadian clock regulation independent of the environment. Patients with neurologic or systemic critical illness have been shown to rapidly enter a state of profound behavioral quiescence with the onset of illness, during which rest-activity rhythm showed severe temporal disorganization and dissociation from melatonin rhythms . DampeniInterestingly, strong physiologic stressors can induce a state of sleep-like behavioral quiescence in animal models, which occurs in tandem with protective mechanisms to restore cellular homeostasis and recover from cellular stress . During Nearly 30% of patients admitted to an ICU develop delirium, which increases the mortality risk . PatientAlthough it is clear that the circadian clock is challenged in the ICU, the origin of circadian manifestations in critically ill patients cannot be definitively attributed to the ICU environments. For example, severe illnesses may have direct effects on the circadian clock, regardless of the ICU environments. Furthermore, we have been operating with a bias that an intact circadian system is always optimal. This is partly due to observations of disruptions of metabolism with short-term clock disruption and increased chronic pathologies with long-term disruption . Stress-Chronobiologists are often biased to think that the presence of a circadian rhythm is the preferred state. Here, we have identified an ambiguous situation with a testable hypothesis. Our alternative hypothesis is that supporting the circadian clock with zeitgebers will lead to entrainment and daily rhythms, and this in turn will lead to a better outcome. The null hypothesis would be that the presence of rhythms is an exacerbation and that the suppression of rhythms sometimes seen in serious illness is adaptive. These hypotheses could be tested descriptively via collection of continuous data from ICU stations and analysis for rhythmicity and appropriate entrained phase as correlated with outcome. The data routinely collected in the ICU—e.g., temperature, oxygen saturation, and heart rate—show circadian rhythms in normal individuals. It represents a huge amount of information if it can be harnessed and analyzed. The hypotheses could also be tested experimentally, for example, by examining whether the circadian phase, amplitude, or robustness in temperature rhythms is associated with patient outcomes from severe COVID-19.Given that the mediators of the most common complications in COVID-19  are extensively regulated by the circadian clock, it may be expected that they occur at discrete times of day. Understanding this would allow for preventive treatments. Without deeper knowledge of the endogenous circadian clock characteristics specifically in the ICU, correct determination of clock regulation of these complications is not possible. What is needed is a comprehensive atlas of potential circadian outputs in ICU patients that include minimally invasive measurements such as blood pressure, heart rate, body temperature, as well as serial peripheral blood sampling over at least two cycles. From this dataset, ICU-appropriate biomarkers of the circadian clock can be derived to determine the circadian profile of the patient  and to assess the internal time of greatest risk for complications and that of optimal medication dosing schedules.It can be challenging to administer a high-amplitude zeitgeber, whether light/dark or administration of corticosteroids with a <24-h half-life, to sedated or septic patients. Glucocorticoid administration should be weighed against its potentially life-threatening side effects including immunosuppression, and creating a dark environment for the patient during the night may interfere with patient care. On the other hand, modifying timing of nutritional support is feasible in the ICU for many patients and could potentially entrain peripheral clocks. We know of no efforts to administer food in the ICU intermittently, mimicking normal eating behaviors, in sedated patients. How is feeding accomplished in the ICU and what does nutrition therapy look like? Clinical trials and guidelines on nutrition therapy during and after critical illness have largely focused on optimal timing (early vs late initiation), route of delivery , and caloric/protein target. Enteral nutrition (EN) is the preferred route of artificial nutrition therapy in critically ill patients. Initiation of nutrition support therapy in the form of early EN within 24-48 h is recommended in the critically ill patient who is unable to maintain volitional intake, unless there are reasons to delay EN such as enteral obstruction, active gastrointestinal bleeding, and compromised splanchnic circulation .Several methods of EN administration exist, including continuous, cyclic, intermittent, and bolus techniques. Continuous feeding involves hourly administration of EN over 24 h assisted by a feeding pump; cyclic feeding involves administration of EN over a time period of <24 h generally assisted by a feeding pump; intermittent feeding involves administration of EN over 20-60 min every 4-6 h via pump assist or gravity assist; and bolus feeding involves administration of EN over a 4- to 10-min period using a syringe or gravity drip.In practice, pump-assisted continuous feeding is generally acceptable for critically ill patients to reduce EN-related complications such as aspiration, feeding intolerance/high gastric residual, underfeeding, and diarrhea. However, a limited number of studies have been conducted to support this practice . Small rTime-restricted feeding (TRF) is an approach to resetting biological rhythmicity via metabolic entrainment of peripheral tissues. It may be time to explore the possibility of pursuing circadian realignment by optimizing the timing of feeding in relation to day-night cycle for critically ill patients receiving EN . CurrentTaken together there are numerous aspects of the COVID-19 pandemic that may have a relationship to circadian and other biological rhythms, ranging from behavior of asymptotic carriers, organ physiology in the sick, the viral life cycle within infected cells, and the host immune response. Although biologically plausible, there has been limited attention to the potential protective effects of healthy sleep and circadian rhythms. Interestingly, the consideration of the available data on the ICU environment and critically ill patients points out the lack of data demonstrating if and how sleep and a synchronized clock contribute to healing. There are numerous examples of circadian disruption leading to illness but few examples of synchronization leading to healing. With judicious collection of time-stamped data, the current pandemic can be used to better understand how the circadian clock is involved in critical illness. We also propose that knowledge of the state of the circadian clock may be used to mitigate all stages of COVID-19.


Online dating apps are popular platforms for seeking romance and sexual relationships among young adults. As mobile apps can easily gain access to a pool of strangers (“new friends”) at any time and place, it leads to heightened sexual health risks and privacy concerns.This study aimed to evaluate the effectiveness of a peer-led web-based intervention for online dating apps to prepare Chinese college students so that they have better self-efficacy when using dating apps.U test. A post-hoc multiple linear regression model was used to examine the correlates of the GSE and RPS.An open clustered randomized controlled trial was conducted among students from three colleges  in Hong Kong. Students aged 17 to 27 years who attended common core curriculum or general education were randomized into intervention and control groups. The intervention material, developed with high peer engagement, included four short videos, an interactive scenario game, and a risk assessment tool. An existing website promoting physical activities and healthy living was used as a control. Using the information, motivation, and behavioral skills (IMB) approach to design the evaluation, questionnaires covering participants’ sociodemographics and dating app characteristics, as well as the general self-efficacy scale (GSE) as the primary outcome and the risk propensity scale (RPS) as the secondary outcome were administered before, immediately after, and at 1 month after the intervention. Intention-to-treat analysis was adopted, and between-group differences were assessed using the Mann-Whitney P<.001) in the intervention group. University of Hong Kong students were more susceptible to risk reduction after the intervention when compared with students from the other two institutions.A total of 578 eligible participants (290 in the intervention group and 288 in the control group) participated in the study with 36 lost to follow-up. There were more female participants  than male participants in the sample, reflecting the distribution of college students. Over half of the participants  reported the following reasons for using dating apps: being curious , trying to make new friends , and finding friends with similar interests . Overall, the participants in the intervention group reported favorable experiences when compared with the finding in the control group. There was significant improvement in the GSE score and reduction in the RPS score (The online intervention was effective in improving general self-efficacy and reducing risk tendency among young students. Future work is needed to determine if this approach is cost-effective and such behavioral change is sustainable.ClinicalTrials.gov NCT03685643; https://clinicaltrials.gov/ct2/show/NCT03685643.RR2-10.1186/s13063-018-3167-5 The use of online dating apps has become popular for seeking friendship and romantic and sexual relationships owing to the advancement of mobile technology and increased internet accessibility through smartphones. Many dating apps are free to download and embed a geolocation function that allows users to find nearby like-minded individuals. Through virtual communication platforms, users can easily meet friends and gain access to a wide pool of potential sexual partners at any place and time.In the United States, there was an increase in the use of online or mobile dating apps by 4% in only 2 years (from 11% in 2013 to 15% in 2015)  per target student in the peer-led group vs €11.6 [US $13.9] in the teacher-led arm) . TherefoThis study demonstrated that a peer-led intervention could improve short-term self-efficacy and reduce risk propensity in using dating apps among young Chinese adults. It is important to evaluate the motivations for using dating apps and attempt to understand the underlying mechanism between using dating apps and associated risks. Future work is needed to determine how to maintain behavioral change over a longer duration, how to reach underserved populations using different means, and how to disseminate such an intervention on a large scale.


The Minimum Dietary Diversity for Women (MMD-W) was validated as a proxy of micronutrient adequacy for nonpregnant women, with proposed data collection being either a list-based or a qualitative open recall method. Few studies have compared the performance of these 2 methods.We compared performance in predicting micronutrient adequacy of food group indicators (FGIs) measured by the list-based and the quantitative open recall methods using varying quantity cut-offs. We also examined the agreement between list-based and open recall FGIs.n = 600 pregnant women) and India (n = 655). The performance of different indicators to predict micronutrient adequacy was compared using receiver operating characteristic (ROC) analysis. Correlations between list-based and open recall FGIs were calculated using Spearman's rank test; agreement was assessed by the intraclass correlation coefficient (ICC) and kappa statistics. Food groups that were most often misreported by the list-based method were identified.Data were collected in Bangladesh  with the cut-off of 1 portion. In India, these values were lower: ∼0.4 for correlation, 0.32–0.37 for ICCs, and 0.17–0.22 for kappas. Food groups most susceptible to misreporting using the list-based method were beans/peas in Bangladesh and other vegetables in India.Our study provides initial support for the use of list-based questionnaires in assessing food group diversity or prevalence of MDD-W in pregnant women. Additional and context-specific work may be required to understand the potential of simple methodologies to assess consumption of specific food groups. This trial was registered at clinicaltrials.gov as NCT02745249 (Bangladesh) and NCT03378141 (India). Poor dietary patterns are 1 of the leading risk factors for morbidity and mortality globally, particularly for women and children in low- and middle-income countries , 3. AlthIn response to this demand, a 10 food-group Minimum Dietary Diversity for Women (MDD-W) indicator was developed and has been validated as a proxy measure for assessing micronutrient adequacy in nonpregnant women at the population level . HoweverThe MDD-W indicator was designed for situations where a quantitative 24-h recall (or other quantitative method) is not feasible, and neither the list-based nor the open recall methods require estimation of portion sizes (the quantity of food consumed). Intuitively, quantity, as well as diversity, matters in achieving micronutrient adequacy, which is reflected, for example, in recommended daily (or weekly) portion sizes in food-based dietary guidelines. However, minimum quantities have rarely been examined in the context of evaluating the performance of simple food group indicators (FGIs). To our knowledge, only 4 studies have examined the effect of different minimum quantity criteria on indicator performance . Three o1) compare the performance in assessing micronutrient adequacy of 2 methods of measuring FGIs—the list-based method and the open recall method using varying quantity cut-offs ; and 2) examine the agreement of list-based and open recall FGIs in predicting mean dietary diversity, achievement of MDD-W, and individual food group consumption.To address this gap in the literature, this article uses data from pregnant women in Bangladesh and India to: upazilas) from 4 districts . In India, data were collected between October and November 2017 in 26 rural blocks from 2 districts (Unnao and Kanpur-Dehat) in the state of Uttar Pradesh. Representative samples were selected from each study site, including 600 pregnant women aged 13–43 y in Bangladesh and 655 pregnant women aged 18–40 y in India.We used baseline data collected as part of 2 studies that assessed the feasibility of integrating a package of maternal nutrition interventions during pregnancy in existing health systems in Bangladesh  and IndiThe studies received ethical approval from the Institutional Review Boards of BRAC University in Bangladesh, the Suraksha Independent Ethics Committee in India, and the International Food Policy Research Institute in the USA. Written informed consent was obtained from all study participants.We used both qualitative list-based questionnaires  and a quSupplemental Table 1.In the list-based method, the trained enumerator read a list of foods and beverages from each food group to the respondent and asked her if she had consumed any of them during the previous day or night. The qualitative questionnaire was based on a list of 17 food groups in Bangladesh and 19 food groups in India that reflected the distinctive characteristics of food consumption in each country. Several examples of local foods or dishes made from these local foods were provided for each food group. Details of food-group questions from the list-based method are presented in Minimum Dietary Diversity for Women: A Guide to Measurement  starchy staple foods, 2) beans and peas, 3) nuts and seeds, 4) dairy products , 5) flesh foods , 6) eggs, 7) dark green leafy vegetables, 8) vitamin A-rich fruits and vegetables, 9) other vegetables, and 10) other fruits. A list-based FGI was obtained by summing the number of food groups consumed by each woman.For the list-based method, the food groups were recategorized into the 10 food groups used in the MDD-W measurement guide : 1) star1) any quantity consumed, 2) a minimum quantity consumed of 15 g, or 3) a minimum of 1 portion, as defined in the respective dietary guidelines of Bangladesh , based on the qualitative list-based and on the quantitative open recall methods for each country. We performed a receiver operating characteristic (ROC) analysis to compare how well the 2 methods predicted micronutrient adequacy as measured by MPA (using an MPA cut-off of ≥0.60) . The areTo examine the agreement between list-based FGI and the different open recall FGIs, we first used scatter plots to show the distribution of list-based FGI compared with those of the 3 open recall FGIs . We calcNext, we reported the proportion of women who had consumed each of the 10 food groups based on the qualitative list-based or the quantitative open recall methods. We tested the differences between these proportions using an ANOVA test, adjusting significance levels for multiple comparisons across classifiers via Bonferroni's correction. We then calculated the frequency of misreporting for each food group and identified the food groups that were most often misreported by the qualitative list-based method compared with the quantitative open recall method with different cut-offs.P value of < 0.05 and all tests were 2-sided. Analyses were performed using Stata 15.1 (Statacorp).Statistical significance was set at a P > 0.05).The performance of FGIs to predict micronutrient adequacy of the diet was similar for the list-based and open recall methods in both countries (AUCs ranged from 0.76 to 0.82 in Bangladesh and from 0.69 to 0.78 in India) .The distribution of FGI scores, by method and country, are presented in The mean differences among open recall and list-based FGIs were positive for the open recall FGIs using any quantity and 15 g minimum cut-offs, and negative for the open recall FGI with the 1 portion cut-off (The share of women achieving MDD-W (consumed ≥5 food groups) in Bangladesh was 65% according to the list-based FGI; according to the open recall FGIs, it was 81% using the any quantity cut-off, 74% using the 15 g minimum cut-off, and 31% using the 1 portion cut-off. The share was much lower in India (18% for the list-based method). The agreement in classifying women as achieving or not achieving MDD-W was ∼80% in Bangladesh when comparing the open recall FGIs using the any quantity or 15 g cut-offs with the list-based FGI, but the agreement was lower (60%) when comparing the open recall using the 1 portion cut-off with the list-based FGI . Both the list-based FGI and open recall FGIs performed adequately and similarly in predicting micronutrient adequacy of the diet; AUCs ranged from 0.69 to 0.82 and were not statistically significantly different among the various indicators. FGIs and MDD-W indicators measured by the list-based and the quantitative open recall methods had moderate agreement in Bangladesh and fair agreement in India. In both countries, the list-based method tended to misreport the consumption of specific food groups compared with the open recall method, particularly with the 1 portion cut-off.In Bangladesh, misreporting exceeded 10% for beans and peas, flesh foods, and vitamin A-rich fruits, but was less frequent for other food groups when comparing the list-based FGI and the open recall FGIs using the any quantity or 15 g cut-offs. For other food groups, overreporting was large only when comparing the list-based FGI and the open recall FGI using the 1 portion cut-off. In India, the pattern was less consistent; underreporting was found for dairy products and other vegetables, whereas overreporting was observed for other food groups when comparing the list-based FGI and open recall FGI using the 1 portion cut-off.We have tried to identify the specific foods that were responsible for the misreporting of food group consumption by looking in more detail at the frequency of specific foods reported with the open recall approach. The underreporting of the beans and peas food group in Bangladesh could be due to foods made of soybeans that were not identified as such (when given as examples to the respondent) on the list-based questionnaire. In India, it could be that milk put in tea was not recorded with the list-based method, and that onions and/or tomatoes were not identified in mixed dishes. This is consistent with findings from Burkina Faso, where misreporting was common for foods used in mixed dishes or in small quantities for sauce ingredients . As for The lower correlation and less agreement among list-based and open recall FGIs in India compared with Bangladesh needs further examination. In both countries, we have worked with enumerator teams specially trained in collecting information on dietary intake, with a standardized adaptation process for the list-based questionnaire. The nature of the diet in each country is complex, with several mixed dishes containing multiple ingredients. These were also the focus of special training to ensure that mixed dishes and food items with multiple ingredients were classified in the right food groups. Ingredients with trivial quantities mainly used to add flavor were to be put in the “condiments and seasonings” group.The MDD-W measurement guide suggests 2 qualitative methods  to measure food group diversity , but limTo our knowledge, this is the first study that uses data from South Asian countries to compare the relative validity of 2 measurement methods for MDD-W in pregnant women. Strengths of the study include our access to 2 data sets with well-documented high-quality training and methods, resulting in high-quality dietary data, and with sufficiently large sample sizes. Ours is also the first study to compare list-based and open recall methods for MDD-W using multiple quantity cut-offs. The need for such studies is urgent, given that there is wide uptake of MDD-W.Limitations of the study include that our open recall was quantitative, so we are not comparing the 2 methodologies described for MDD-W, where both the open recall and list-based methods are qualitative. However, the first 2 passes of our quantitative recall are similar to a qualitative recall, and our approach enabled us to also look at the issue of varying quantity cut-offs. An additional limitation of our study is that both sites were in South Asia, and ideally studies would incorporate a wider range of geographies and food cultures.Our AUC results showed similar predictive values among the list-based and the open recall FGIs (even for the different minimum cut-offs), illustrating that the list-based FGI performed as well as the open recall FGIs in predicting micronutrient adequacy of the diet for pregnant women. This has important implications because the list-based method has several advantages, such as lower capacity requirements for enumerators and shorter training time, which in turn reduce the cost of data collection. Our findings confirm the validity of the list-based FGI as a simple tool to measure dietary diversity in pregnant women that may be more feasible than other complex measures in resource-poor settings. Our study thus provides initial support for the use of the list-based FGI in assessing FGIs  or prevalence of MDD-W. However, for assessing prevalence of intake of specific food groups, estimates from list-based FGI and the open recall FGIs differed quite substantially for select food groups in each country, so additional methodological work from a wider range of contexts is required to understand the potential of using simple methodologies to assess consumption of specific food groups.nzz134_Supplemental_FileClick here for additional data file.


In order to improve the mechanical and dielectric properties of radome cyanate, a synergistic reinforcement method is employed to develop a resin-based ternary-composite with high heat-resistance and preferable radar-band transmission, which is expected to be applied to fabricate radomes capable of resisting high temperature and strong electric field. According to copolymerization characteristics and self-curing mechanism, epoxy resin (EP) and bismaleimide (BMI) are employed as reinforcements mixed into a cyanate ester (CE) matrix to prepare CE/BMI/EP composites of a heat-resistant radome material by high-temperature viscous-flow blending methods under the catalysis of aluminum acetylpyruvate. The crystallization temperature, transition heat, and reaction rate of cured polymers were tested to analyze heat-resistance characteristics and evaluate material synthesis processes. Scanning electron microscopy was used to characterize the micro-morphology of tensile fracture, which was combined with the tensile strength test and dynamic thermomechanical analysis to investigate the composite modifications on tenacity and rigidity. Weibull statistics were performed to analyze the experimental results of the dielectric breakdown field, and the dielectric-polarization and wave-transmission performances were investigated according to alternative current dielectric spectra. Compared with the pure CE and the CE composites individually reinforced by EP or BMI, the CE/BMI/EP composite acquires the most significant amelioration in both the mechanical and electrical insulation performances as indicated by the breaking elongation and dielectric breakdown strength being simultaneously improved by 40%, which are consistently manifested by the obviously increased transverse lines uniformly distributed on the fracture cross-section. Furthermore, the glass-transition temperature of CE/BMI/EP composite reaches the highest values of nearly 300 °C, with the relative dielectric constant and dielectric loss being mostly reduced to less than 3.2 and 0.01, respectively. The experimental results demonstrate that the CE/BMI/EP composite is a highly-qualified wave-transmission material with preferences in mechanical, thermostability, and electrical insulation performances, suggesting its prospective applications in low-frequency transmittance radomes. Transmittance materials are primarily applied in radome manufactures to ensure that the antenna device system operates smoothly in remote control and communications, resisting the mechanical, thermal, electrical, and chemical impacts in harsh environments. The stealth function required for recently progressive radomes has been achieved by coordinating the diversified characteristics of wave-absorption at high frequencies and wave-transmission at low frequencies, which is intriguing for developing next-generation transmittance materials ,2,3. At Cyanate ester (CE) is a thermosetting resin polymer with relatively high thermal stability and a characteristic molecular structure containing two or more cyanate functional groups –(OCN). CE materials show minimal water absorption (<1.5%) and higher bending and tensile strengths than that of epoxy resin. The glass-transition of CE resin generally occurs at the temperature range of 240~300 °C, which can be increased to the maximum value of 400 °C after being thermally modified ,9,10. CEPrevious studies for developing CE-based modified materials have always concentrated on the improvement of an objective performance for radome applications by individually blending or combining specialized reinforcement agents to the CE matrix. These research schemes are incompetent at taking into account the comprehensive characteristics of the modified materials, which sets substantial limitations on the practical applications of CE resin. For instance, breakable chain segments produced from the group reactions of amino-terminal polypropylene and CE trimers can be embedded in the backbone of the cured network to decrease the crosslinking density and increase the tenacity of CE resin, which will however degrade electrical resistance and dielectric properties ; CE/carbThe fundamental raw materials used in the material synthesis experiments were as follows: white powdery crystals of bisphenol A cyanate ester  as the monomer matrix; viscous liquid of epoxy resin monomer  as a reinforcement agent; light yellow crystalline powder of bismaleimide monomer  as a coordination reinforcement agent; and white powder of aluminum acetylpyruvate  as the resin-curing catalysis. Reinforcement agent in 10% mass percentage (10 wt%) was adopted to individually prepare the CE-based composites modified with EP (CE/EP), BMI (CE/BMI) and EP together with BMI (CE/BMI/EP), as shown in Curing reaction of the CE monomers (pre-polymers) with reactive (–O–C≡N) functional groups is addition polymerization without producing any by-products, in which the ring trimerizations will occur to form a three-dimensional cross-linked network when being heated, as shown in BMI monomers also undergo self-polymerization when being heated, with the hydroxyl in one imide ring as the electron donor and the electron-deficient double-bond in another imide as the electron acceptor, in which two independent radicals are produced by the formation of electron donor-to-acceptor complex to initiate the chain growth reactions, as schematically shown in In order to characterize the fractured micro-structures related to mechanical properties, the cross-sections of the quick-frozen brittle fractures, which had been obtained by treating the demolded sheet samples of 30 mm × 30 mm × 1 mm in liquid nitrogen, were observed by an ultra-high-resolution cold field emission scanning electron microscope  with a magnification of 2k under the accelerating voltage of 0.5 kV~30 kV. The tensile stress-strain characteristics, by which the tensile strength, Young modulus, and breaking elongation can be evaluated, were measured according to the GB/1447-2005 standard with a mechanical elongation testing machine  to estimate the material tenacity improved by the composite modifications. By employing the dynamic mechanics analysis (DMA) method, the viscoelasticity of the resin-based composites is evaluated by measuring the energy-storage modulus at different temperatures as implemented on a dynamic thermomechanical analyzer  under a nitrogen atmosphere with the samples being scaled as 50 mm × 10 mm × 1 mm and a heating rate of 5 °C/min at the testing temperature range of 30~300 °C.According to the differential scanning calorimetry (DSC) method, the variation curves of heat-flow vs. temperature before and after the resin curing process were tested using a thermal analyzer  under a nitrogen atmosphere at the temperature range of 30~300 °C with a heating rate of 10 °C/min. The exothermic-peak and glass-transition temperatures were evaluated to investigate the curing process and heat-resistant characteristics of the prepared materials.Direct current (DC) electrical insulation performances of the circular film samples with a diameter of 80 mm and a thickness of 0.1 mm were tested with the film breakdown strength tester  by measuring the dielectric breakdown voltage according to the ASDTM149 standard, in which the voltage-increasing rate and dielectric capacitance are controlled to 500 V/s and 6 kV·A, respectively. The tested results of the dielectric breakdown field were analyzed though Weibull 2-parameter statistics. Alternative current (AC) dielectric polarization behaviors (complex dielectric permittivity spectrum) of the circular film samples with both sides being vacuum evaporated by aluminum film electrodes with a 25 mm diameter were tested in a scanned frequency range of 10 kHz~10 GHz at ambient temperature utilizing a wide-frequency dielectric spectrometer  to obtain the dielectric constant and loss spectra in a wave-region comprising x-band (radar band), by which the dielectric properties and wave-transmittance of CE-resin composites are studied.The fracture cross-sections of pure CE, CE/BMI, CE/EP, and CE/BMI/EP composites after being cured were characterized by SEM, as seen in It is indicated from The tensile strength, Young modulus, and break elongation of the CE resin and composite materials were evaluated from the elongation stress-strain tests with the results being shown in Tg), the transformation of molecular-chain motions from localized vibrations to random global displacements results in the nosedive ability to store elastic deformation energy [Tg ~ 270 °C, the polymer molecules of all the three composites will exist in a loose or free aggregation state when elastic energy storage promptly tends to almost zero [The DMA testing results of pure CE and CE-based composites are shown in n energy ,27. Afteost zero .With the combination of SEM micro-morphology, mechanical tensile experiment, and DMA analysis, it can be reasonably summarized that the fully blended and polymerized (after curing) CE/BMI/EP composite with a tight molecular aggregation possesses excellent mechanical properties including the quintessential ductile fracture and high tenacity. Therefore, we have successfully developed a deformation-stable CE/BMI/EP composite with significantly improved performances of tensile resistance and elastic energy storage.Curing temperatures of CE resin and its composites were measured employing DSC dynamic tests performed in resin-curing processes, as per the results shown in Tg is an essential thermal property of resin materials, which indicates the thermal stability and determines the working temperature range in thermodynamic environments. The cured materials of CE and modified composites were also tested for the DSC temperature spectra by which Tg can be effectively evaluated, as seen in the results shown in Tg), which is lower than the CE-based composites due to the mixture effects on the melting point of a multiple-phase system. Otherwise, the ternary CE/BMI/EP composite shows an approximate Tg of 289 °C, which is definitely higher than the other binary composites, implying that the preferred enhancement of heat-resistance has been achieved in concomitance with mechanical modification.Glass-transition temperature Tg. Nevertheless, in the extended curing process of the CE/BMI/EP composite, the copolymerizations of EP and BMI with CE monomers can produce various heterocyclic derivatives of pyrimidine, which will substantially intensify intermolecular entanglements. Meanwhile, the formation of CE network structures is expedited by BMI reinforcement with a minority of BMI self-polymerization reactions, which will affirmatively improve the crosslinking density of curing networks and the aggregation of polymer products. Therefore, a larger kinetic energy of molecular segments is required to destroy the polymeric aggregating configurations for the qualitative transformation from the elastomeric state to amorphous glassy morphology. Moreover, the more flexible oxazolidone groups will be generated from the reactions of EP with CE monomers, which hinders the segment movements of molecule chains and will raise material viscosity. Accordingly, a higher temperature is required for qualitatively exacerbating molecule vibrations to break the viscous resistance of the CE/BMI/EP composite, resulting in the evident inhibition on glass-transition and the excellent heat-resistance retained from the CE resin.The polymerization structures of the ternary composite obtained by simultaneous EP and BMI modification in curing reaction process were more complex than the other resin materials, leading to the qualitative changes in polymer aggregation. The self-polymerization reaction occurs in the CE monomer matrix after being heated to curing temperature to form the regularly reticulated configurations of densely distributed triazine-rings, which are capable of restricting the molecular segments to a stable state below a specific high temperature , whereasEb and shape-parameter β from the fitted Weibull distributions of DBS data are listed in Eb and 19% reduction of β, which indicate the remarkable improvements of DBS and high-voltage resistance stability, while the two binary composites show only 11% DBS enhancement with degraded high-voltage resistances. The polymerization reactions of the ternary composite in the curing process are more complicated than the pure CE resin and binary composites, which render the multiple heterocyclic structures including the oxazoquinolinone and pyrimidine produced by the CE copolymerizations with EP and BMI, respectively, leading to the abatement of intermolecular polarity and the amelioration of electrical insulation performance [Two-parameter Weibull Statistics was employed to analyze the dielectric breakdown strength (DBS) results being tested at ambient temperature, as shown in formance . Meanwhi6~108 Hz, which comprises a characteristic low frequency x-band of skywave over-the-horizon radar.Dielectric characteristics of wave-transmittance materials determine the electromagnetic-wave transmissivity and is the most important index to evaluate the performance of radome. The relative dielectric constants and dielectric loss of the pure CE and CE/EP, CE/BMI, and CE/BMI/EP composites as a function of AC electric field frequency are shown in AC dielectric spectra indicate that the CE/BMI/EP composite also has earned a promotion in dielectric properties. The low dielectric polarization characteristics of CE resin originate from the polar configuration formed by self-polymerization. After the collaborative modifications of EP and BMI reinforcements, a new stable composite structure with smaller intermolecular voids has been achieved through a variety of copolymerization reactions, which restricts the movements of polar structures and obtains a faster dielectric response around intermolecular voids. In contrast, the dense reticulations of CE resin are partially interrupted by BMI or EP reinforcement to form larger intermolecular spaces and possibly cause two-phase interfaces in the binary composites, leading to a higher activity of polar molecule segments and the interfacial polarization with longer relaxations. Therefore, the synergistic effect of coordinate reinforcement is also valid for improving the dielectric performances of CE resin. Simultaneously, with a higher breakdown-resistance than CE, the low dielectric constant and loss of CE/BMI/EP composite determines the high-quality signal transmission coefficient and the high-efficiency electromagnetic wave transmittance in the radar wave-band, suggesting a preferable wave-transmittance material in prospective applications of antenna radomes. To this end, mechanical, thermal, and dielectric characteristics of the CE/BMI/EP composite are listed in A resin-based ternary-composite with high heat-resistance and preferable radar-band transmission has been developed to ameliorate the mechanical and dielectric properties of a cyanate copolymer used for antenna radomes. The synergistic reinforcement method is suggested to synthesize resin-based transmission materials being capable of resisting high temperature and strong electric field. The CE resin as a fair wave-transmittance material is significantly modified in terms of the mechanical and dielectric properties by a synergistical reinforcement scheme of blending EP and BMI into the CE matrix. The prepared materials have been systematically investigated by the micro-morphology SEM and mechanical elongation experiments in combination with dynamic thermomechanical analyses. The curing process and glass-transition are shown in the DSC tests, and the DC electrical insulation and wave-transmittance performances are evaluated through DBS Weibull statistics and AC complex dielectric spectra. The CE/BMI/EP composite achieved by coordinately blending BMI and EP reinforcements represents a preferable processing character of curing at the temperature of 194 °C with a wide and flat exothermic peak for easy casting. The cured CE/BMI/EP composite shows a higher glass-transition temperature with an improvement in heat-resistance compared with pure CE resin. The fractured surface of the CE/BMI/EP composite illustrates the evident ductile striations, and the tensile strength approaches 96 MPa with a break-elongation of 5.9%, implying that the intermolecular forces have been enhanced by simultaneously blending BMI and EP into the CE matrix to realize a compatibly copolymerized ternary composite. Meanwhile, the CE/BMI/EP composite acquires the considerable increments in mechanical energy-storage and stiffness, confirming the promoted tenacity observed by SEM. Compared with the binary-composite, the highly intensified intermolecular interactions in the ternary composite result in the reduction of free spaces for charge transports, as indicated by the DBS increment of 40%. The relative dielectric constant and dielectric loss of the CE/BMI/EP composite decline to 2.6~2.8 and 0.002~0.004, respectively, in a wide frequency range of 10 kHz~10 GHz, which means an extremely low dielectric polarization specified for high quality transmittance materials. In contrast to the CE resin, the CE/BMI/EP composite as an efficient wave-transmittance material possesses the analogical processability and higher heat-resistance as well as with the mechanical strength being significantly improved. In a wide X-band of radar communications, the CE/BMI/EP composite represents an evident reduction in dielectric permittivity and transmission loss in comparison to the CE resin, demonstrating a notably higher electromagnetic wave-transmittance. In brief, the CE/BMI/EP composite acquires an appreciable amelioration simultaneously in mechanical tensile strength, electrical insulation performance, and heat-resistance, promising its competency in applications for the radar radomes requiring for high mechanical strength and electrical insulation.


Cervical cancer disproportionally affects women in low- and middle-income countries, in part due to the difficulty of implementing existing cervical cancer screening and diagnostic technologies in low-resource settings. Single-board computers offer a low-cost alternative to provide computational support for automated point-of-care technologies. Here we demonstrate two new devices for cervical cancer prevention that use a single-board computer: 1) a low-cost imaging system for real-time detection of cervical precancer and 2) a low-cost reader for real-time interpretation of lateral flow-based molecular tests to detect cervical cancer biomarkers. Using a Raspberry Pi computer to provide real-time image collection and processing, we developed: 1) a low-cost, portable high-resolution microendoscope system (PiHRME); and 2) a low-cost automatic lateral flow test reader (PiReader). The PiHRME acquired high-resolution  a low-cost imaging system for real-time detection of cervical precancer and 2) a low-cost reader for real-time interpretation of lateral flow-based molecular tests to detect cervical cancer biomarkers. I.Cervical cancer continues to disproportionally affect women in low-resource settings. According to the most recent 2018 GLOBOCAN estimates, the incidence and mortality rate of cervical cancer in Low/Medium Human Development Index (HDI) regions are 18.2 per 100,000 and 12.0 per 100,000 respectively, nearly double the incidence rate and triple the mortality rate of that in High/Very High HDI regions To address this disparity, a number of point-of-care technologies to improve cervical cancer prevention, screening, and detection are in development A number of high-resolution imaging technologies have been developed to provide real-time detection of high-grade cervical precancer without the need for biopsy Similarly, a number of lateral flow-based tests have been developed to detect biomarkers associated with cervical cancer ®, have recently proven to be an effective way to reduce the cost and size of medical and scientific instruments, without sacrificing performance Single-board computers, such as the Raspberry PiHere we demonstrate the use of a Raspberry Pi single-board computer to integrate imaging and quantitative computational analysis in two low-cost, portable imaging systems for cervical cancer prevention. The first is a high-resolution imaging system with real-time analysis software and the second is a low-cost, portable lateral flow test reader to automatically read a point-of-care diagnostic test. Using a Raspberry Pi computer and open-source software frameworks, we reduced the cost of the HRME imaging system by half while maintaining image quality and still providing real-time image analysis support. Similarly, we used this platform to develop a low-cost reader for a lateral flow HPV serology test to assess whether a patient had received multiple doses of the HPV vaccine II.A.We developed and evaluated the PiHRME, a High-Resolution Microendoscope designed to capture and analyze high-resolution images of cervical epithelium in real-time; changes in nuclear morphometry are analyzed to detect the presence of high-grade cervical precancer.1)et al.et al.The optical system and Raspberry Pi computer are encased within a 2)Previous versions of the HRME have incorporated image analysis software that relies on image segmentation to quantify changes in nuclear morphometry due to high-grade cervical precancer and early cancer A dataset consisting of 4,053 cervical images from an ongoing clinical study approved by the Institutional Review Boards at The University of Texas MD Anderson Cancer Center, Rice University, and Barretos Cancer Hospital in Brazil was organized with binary class labels (i.e. HRME positive or HRME negative) as defined by the pre-existing HRME algorithm. The class distribution of the dataset was 1,214 (30%) HRME positive and 2,839 (70%) HRME negative images. Open-source code for retraining TensorFlow models was then used to extract a feature vector of each image using the pre-trained network model to optimize the parameters of the final network layer to predict the new label (in this case HRME positive or HRME negative) To evaluate the feasibility of using the model for real-time inference, the PiHRME frame rate was benchmarked with and without model inference on each frame. A simple user interface was developed to display the following information over each frame in the video feed on the PiHRME: image frame number, probability output of the CNN prediction (from 0 to 1.00), and speed of classification .3)Image quality of the PiHRME was compared to the current tablet-based HRME system. Image resolution was determined by imaging a 1951 US Air Force Resolution Target. Image contrast was compared by plotting the intensity changes that occurred across the three vertical bars of Group 6 Element 1 of the same resolution target.To evaluate the ability of the CNN algorithm to support real-time image analysis of images acquired with the PiHRME, we created and imaged fluorescent calibration targets that were representative of normal and precancerous cervical nuclear morphology. The calibration targets consist of small circular patterns (8–23 4)Ex vivo images of LEEP specimens  were obtained using the PiHRME under a protocol approved by the Institutional Review Boards at The University of Texas MD Anderson Cancer Center and Rice University. Before imaging, LEEP specimens were stained with 0.01% proflavine solution, a topical antiseptic solution that has been used to fluorescently stain cervical nuclei for imaging, and Lugol’s iodine, to increase image contrast. Similar staining has been performed previously to acquire high-resolution images of the cervix using HRME imaging systems 5)High-resolution in vivo imaging of the cervix using the PiHRME was performed in a low-resource setting in accordance with the protocol approved by the Comité Nacional de Ética de la Investigación en Salud  in El Salvador and the Institutional Review Boards at The University of Texas MD Anderson Cancer Center and Rice University. Women undergoing a routine colposcopy examination for cervical cancer prevention in El Salvador were imaged with the PiHRME. All visible lesions on the cervix along with one normal area were imaged and all visibly abnormal areas were biopsied. Before imaging, 0.01% proflavine solution and Lugol’s iodine were applied. Following image acquisition, images were analyzed using the CNN image analysis algorithm.B.We developed and evaluated the PiReader, a scanner designed to capture and analyze images of lateral flow-based tests.1)The PiReader image analysis software was developed in Python and incorporates Open Source Computer Vision Library software (OpenCV). When the PiReader is switched on, the home screen appears and allows the user to select the type of lateral flow test they would like to analyze. Once selected, a live view from the microscope is displayed on the touchscreen, and fiducial marks are displayed to indicate that a test has been properly inserted into the reader. Once the test is in place, the user presses the “Start Analysis” button on the touchscreen. Test results appear on the touchscreen within one second.2)The PiReader was initially designed to automatically evaluate the results of a lateral flow HPV serology test previously described by Grant et al, All images generated during image analysis are saved onto the Raspberry Pi in a file identified by the date and time of the reading. The final result, along with SBR calculations, are saved in a text file within the same folder.3)et al.To compare performance of the PiReader to a flatbed computer scanner, test strips were prepared using plasma from a non-vaccinated volunteer spiked with different concentrations of HPV16 antibody and scanned using both systems. The concentrations tested were 0, 0.5, 1, 2, 4, 8, 16, and According to Villa et al, those naturally infected with HPV exhibit plasma concentrations of anti-HPV16 ranging from 50–100 milli-Merck Units per milliliter (mMU/mL), while those vaccinated against HPV exhibit levels greater than 800 mMU/mL 4)et al.To evaluate the ability of the PiReader to accurately analyze HPV serology tests from patient samples, we used the reader to re-read tests obtained in a previously reported clinical evaluation by Grant III.A.The lightweight CNN model was able to successfully transfer learn the binary output of the existing HRME image analysis algorithm with high accuracy. Initial model accuracy of the MobileNetV2 on the first training/validation batches were 57% and 50% respectively. Upon completion of 4,000 training iterations, the training and validation of the final batches improved to 93% and 92% respectively. Final accuracy for the 405 images in the test set was 90%. Whereas the raw numerical output of the pre-existing algorithm is a metric ranging from 0 to 417 with a positivity threshold of 120, the output of the retrained CNN ranged from 0 to 1 with a binary threshold for positivity of 0.50. In deployment, the CNN required an average of 5.2 seconds to perform inference on a single frame. The frame rate of the PiHRME system was much slower with real-time inference activated (30 FPS vs 0.19 FPS). Although the image processing time was not ideal for real-time inference, it was still faster than the pre-existing algorithm running on a PC tablet which requires ~8 seconds per frame.1)2)3)B.1)2)IV.The work described here demonstrates the utility of single-board computers for developing low-cost, point-of-care technologies for cervical cancer prevention. Using a Raspberry Pi computer, a low-cost HRME system (PiHRME) was developed  at half the cost of the current tablet HRME system , while still being able to accurately classify and provide high-resolution imaging of the cervix. In addition, a low-cost, portable lateral flow assay reader (PiReader) was developed to read a point-of-care HPV serology test with the same accuracy as a computer and high-resolution scanner.The value of using single-board computers, such as the Raspberry Pi, to create biomedical devices for point-of-care diagnostics includes their low cost as well as a number of other advantages. Because of their small size and low-power requirements, they allow devices to be portable and battery powered. They can also be programmed with easy-to-use interfaces to automate analysis of results. These advantages are particularly important when developing medical devices for use in low-resource settings, where electrical power is not always available, where devices often must be operated by local medical personnel with limited clinical laboratory expertise, and where there is limited availability of support for preventive or reparative maintenance. Larger scale clinical field studies are needed to verify these advantages for the PiHRME and PiReader; however, the results presented here indicate that these technologies have the potential to meet the cost, usability, and effectiveness constraints associated with low-resource settings.The PiHRME was able to provide real-time visualization of cervical nuclei and accurately classify images, which was confirmed by the final histopathology diagnosis. Real-time image analysis when examining the cervix can help clinicians either reject or confirm suspected cases of high-grade precancer or cancer, especially in low-resource areas where expert physicians may not always be available. In order to accomplish real-time image classification for the PiHRME system we leveraged recently developed TensorFlow libraries for the Raspberry Pi (MobileNetV2). Future work will explore using more efficient processors for accelerated neural network computation on embedded systems in addition to other state of the art deep learning models for semantic segmentation and classification using nuclear features. Additionally, the usability and frame-rate could be improved by automatically rejecting low-quality frames (frames with motion blur or not in contact with tissue), and only running inference on high-quality frames.Ongoing development of TensorFlow Lite can potentially extend this approach even more broadly to microcontrollers and edge computing devices The PiReader demonstrates how real-time imaging can help make lateral flow test interpretation easier to perform at the point-of-care. The PiReader was designed using a low-cost USB microscope with lower resolution (96 dpi) compared to a commercial flatbed scanner (800 dpi). However, even with lower resolution, there was enough detail within the images to accurately determine the presence and absence of positive signals. We demonstrated the accuracy of the PiReader by interpreting the results of an HPV serology test. The algorithms used here can be applied to read other lateral flow tests that rely on colorimetric changes to distinguish positive and negative results.Further clinical work is ongoing to validate the utility of these technologies for cervical cancer prevention in low-resource settings. Taken together this work demonstrates the great promise single-board computers have for the development of low-cost, point-of-care medical technologies.