The ongoing circulation of SARS‐CoV‐2 variants of concern (VOCs) has caused global concerns, because VOCs could escape current vaccines, antiviral drugs, and diagnosis. Analyzing mutations and intra‐host diversities in different and widespread VOCs can provide important insights to virus adaptive evolution and validity of vaccines, antiviral drugs, and diagnosis. In this study, by analyzing 1744 high‐throughput sequencing data for intra‐host single‐nucleotide variations (iSNVs) and 3,668,205 genome sequences for mutations in different VOCs, it was found that Omicron variant is still evolving at high speed, especially having high iSNVs frequency in its S and N genes. The efficacies of antibodies or detection primers targeting these two genes are at high risks to be invalid. Instead, highly conserved regions such as NSP8 gene could be better therapeutic and detection targets. Furthermore, mutations in later VOCs could be traced to the minor alleles in the previous variant samples such as Alpha and Delta in different countries. Finally, it was found that mutations C14408T in RdRp and A18163G in NSP14 gene might be associated with the higher genetic diversity in Omicron. Our findings not only contribute to understanding the adaptive evolution of SARS‐CoV‐2 VOCs, but also provide useful information for both drugs and diagnostic kits development. The distributions of mutations and iSNVs in the genomes of SARS‐CoV‐2 VOCs were characterized. The potential correlation between mutations and iSNVs of SARS‐CoV‐2 VOCs was evaluated. Analysis results of both mutations and iSNVs provided some helpful implications to SARS‐CoV‐2 detection and therapeutics. As severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) rapidly and continuously evolves, more than 50,000 lineages had been classified  of the samples from COVID‐19 patients were collected and used for iSNVs analysis. These data included eight independent SARS‐CoV‐2 genomics sequencing datasets, two of which belonged to our previous work,Public high‐throughput sequencing raw data n = 744 of thBesides, another 3,668,205 high‐quality genomes  of SARS‐CoV‐2 from GISAID were used to analyze the distribution of mutations and unique mutations specific to each SARS‐CoV‐2 VOC Table .2.2The well‐sequenced 258 NGS raw data were selected for iSNVs screening. In summary, we calculated and sorted the frequencies of four bases at each base site of the genome. The base with the second highest frequency is seen as the potential iSNV  at the site. Only the qualified minor alleles with the minor allele frequency (MAF) value larger than 0.02, which is about 20 times larger than the sequencing error rate, was considered as the iSNVs. The detailed identification of iSNVs screening can be seen in the method selection.Ka/Ks analysis revealed that most iSNVs in the coding regions of SARS‐CoV‐2 variants were under the purifying selection  of each SARS‐CoV‐2 variant were identified based on the large‐scale genomes data. As shown in Figure 2.3During analysis of the distributions of iSNVs and common mutations in different VOCs, we found that many common mutations of the SARS‐CoV‐2 variants were present as iSNVs  in previous sequencing datasets. For example, two unique mutations C22686T (S375F) and T23075C (Y505H) were in the receptor binding domain (RBD) of Omicron variant and reported to be associated with decreased protein stability and increased risks of infectivity.2.4p‐values <0.0001). Omicron variants have the highest diversity among the analyzed VOCs, irrespective of the individual strain level or lineage level.We evaluated the intra‐host diversities of the SARS‐CoV‐2 strains by calculating the root‐mean‐square‐deviation (RMSD) value at each site across the genome. Each column in Figure x or B.x, but with P316L mutation in RdRp and a few mutations in other regions, were included to calculate intra‐host diversities . One thing to be noticed is that B.1.x or B.x lineages with P316L mutation only contain a few mutations (Table Previous studies reported that RNA‐dependent RNA polymerase (RdRp) and NSP14 are two major contributors to the intra‐host diversities of coronaviruses,es Table . As showns Table  comparedns Table . Owing tns Table .2.5The current gold‐standard approach for SARS‐CoV‐2 detection is RT‐PCR. One of the obvious factors to keep the effectiveness of these assays is that the oligonucleotide primers and probes can accurately match the target sequence of different variants. Mutations, especially the mutations in the probe or at 3′ end of the primers, may lead to invalid PCR assays. We further analyzed whether mutations and iSNVs in N gene, which was chosen mostly as the target gene for detection because of its high abundance lead by the complex numerous discontinuous transcription events,2.6The S protein of SARS‐CoV‐2 majorly interacts with the host angiotensin‐converting enzyme 2 (ACE2) receptor before entering a cell to initiate the infection, so it was mainly used as the target for vaccine development. With the circulation of the SARS‐CoV‐2 variants, most monoclonal antibodies approved by FDA such as bamlanivimab, etesevimab, casirivimab, imdevimab, tixagevimab, and cilgavimab were reported to have reduced efficacies against Omicron variants because of the heavy mutations in RBD region.p‐values <0.0001 by Kruskal–Wallis test). Therefore, it is highly possible that efficacy of vaccines targeting S gene is variable to different VOCs. For example, two mRNA vaccines named BNT162b2 and Spikevax approved by FDA, another mRNA vaccine named ARCoV under phase III clinical trials, and two other subunit vaccines, NVSI‐06‐08 in clinical trials and NVX‐COV2373 for Emergency Use Authorization (EUA) by FDA (https://www.fda.gov/emergency‐preparedness‐and‐response/coronavirus‐disease‐2019‐covid‐19/covid‐19‐vaccines),As shown in Figure 2.7Most of the antiviral drugs target the products of ORF1ab region, where few mutations in SARS‐CoV‐2 VOCs are included Figure  sheet B.3Previous studies on other RNA viruses, such as yellow fever virus and influenza virus, revealed that the intra‐host diversity could provide useful information on virus adaptive evolution and host‐to‐host transmission during the epidemic.Overall, the iSNV results indicated that the VOCs have adapted to humans easier than the ancestral strain during spread and the higher iSNVs of Omicron variants may contribute to their much higher transmission rates. We also compared our results to the deep‐sequencing NGS data of the B.1/A lineage submitted by UK scientists in the early stage of the pandemic.Ka/Ks values of iSNVs and mutations in RdRp were smaller than 1, which means the deleterious mutations in this gene might be eliminated .4.2https://genome.sph.umich.edu/wiki/BamUtil:_bam2FastQ). The de novo assembly was performed by the megahit software (version 1.2.9) based on the viral reads.https://pangolin.cog‐uk.io/ to calculate the lineages. Based on the lineage information, the taxonomy of SARS‐CoV‐2 variant could be accurately determined.After mapping the clean reads to the SARS‐CoV‐2 reference genome, the viral reads were extracted by the bam2fastq software  at the same position within the same sample. The identification of iSNVs was basically executed by our established pipelines and referred to Nicole's work.4.4As mentioned in Rozen‐Gagnon's publication, the root mean square deviation (RMSD) values of the variance at each nucleotide position were used to estimate the viral genetic diversity within a deep sequencing sample.4.5https://www.gisaid.org/) covering all isolates that emerged from 2019 to April 2022. These genomes were selected based on the criteria: (a) genome length is longer than 29 kbp, (b) the unknown base(s) are less than 30, and (c) the degenerate base(s) are less than 70. These large‐scale genomics data were used to construct the mutations landscapes and identify unique mutations specific to SARS‐CoV‐2 VOCs according to our established pipelines.A total of 3,668,205 SARS‐CoV‐2 completed high‐quality genomes were downloaded from the GISAID database  from Protein Data Bank (PDB) database was used for amino acid substitution prediction. The Chimera software4.7The selection pressures of iSNVs and mutations were calculated by the software KaKs_calculator (version: 3.0).4.8https://cran.r‐project.org/bin/windows/base/old/3.6.3/). Before statistical analysis, the distribution feature of each data was judged. Collectively, in this study, the independent groups were analyzed by Kruskal–Wallis test and the correlation coefficient was calculated by Pearson correlation analysis. Data were showed as mean ± SEM. Statistical differences were considered significant at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.All the statistical analyses were performed in the GraphPad Prism 7 software  or the R program language (version: 3.6.3, Dongyan Xiong designed experiments, performed all bioinformatics analysis, analyzed the experiment results, and drafted the manuscript. Xiaoxu Zhang attended the results discussion. Junping Yu designed experiments, attended the results discussion, and revised the manuscript. Hongping Wei generated the idea, designed experiments, finalized the manuscript, and provided funding. All authors have read and approved the final manuscript.The authors declare that they have no conflicts of interest.No research activity for this manuscript requires an ethics approval.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.


The field of robotic surgery has seen significant advancements in the past few years and it has been adopted in many large hospitals in the United States and worldwide as a standard for various procedures in recent years. However, the location of many hospitals in urban areas and a lack of surgical expertise in the rural areas could lead to increased travel time and treatment delays for patients in need of robotic surgical management, including cancer patients. The fifth generation (5G) networks have been deployed by various telecom companies in multiple countries worldwide. Our aim is to update the readers about the novel technology and the current scenario of surgical procedures performed using 5G technology. In this article, we also discuss how the technology could aid cancer patients requiring surgical management, the future perspectives, the potential challenges, and the limitations, which would need to overcome prior to widespread real‐life use of the technology for cancer care.The expansion of 5G technology has enabled some countries to conduct remote surgical procedures, tele‐mentored and real‐time interactive procedures on animal models, cadavers, and humans, demonstrating that 5G networks could offer a potential solution to previously experienced latency and reliability hurdles during the remote surgeries performed in the 2000s.New technological advancements could serve as a ground for emerging novel therapeutic applications. While limitations and challenges related to the 5G infrastructure, cost, compatibility, and security exist; researching to overcome the limitations and comprehend the potential benefits of integrating the technology into practice would be imminent before widespread clinical use. Remote and tele‐mentored 5G‐powered procedures could offer a new tool in improving the care of patients requiring robotic surgical management such as prostate cancer patients. The expansion of the novel technology has enabled clinicians to conduct remote procedures, tele‐mentored surgeries, and real‐time interactive surgeries on animal models, cadavers, and humans. In the following article, the authors discuss detail the history of robotic surgical systems, a summary of the internet and the 5G networks, and the current scenario of remote procedures using the 5G network with an aim to update the readers about the novel technology, illustrate the efficacy and feasibility of the use the novel technology and potential benefits for the cancer patients requiring surgical interventions.2Robotic surgical systems have been in development for over 40 years. Arthrobot, developed and used in Vancouver in 1983, was the world's first surgical robot.The PUMA 200 was a robotic system used initially for industrial purposes. In 1985, researchers released a better and improved version of PUMA 200 called the PUMA 560, both made by Unimation Limited.PROBOT was developed in England and utilized in prostate reconstruction and transurethral prostate resection surgeries.Through the late 1980s and early 1990s, Computer Motion developed their robotic system named AESOP, Automatic Endoscopic System for Optimal Positioning. AESOP assisted surgeons by providing a steady operating field without the risk of a fatigued or inexperienced scope holder. AESOP controlled the orientation of the laparoscope initially using a foot pedal. Later the orientation was controlled using voice commands. AESOP received FDA approval for intra‐abdominal surgeries in 1994 and became the first FDA‐approved robotic device for intra‐abdominal procedures.While ZEUS gained significant popularity, the da Vinci robotic system, developed by American company Intuitive Surgical, Inc., was shown to have a shorter learning curve than ZEUS and more intuitive technical movements. The da Vinci robotic system was approved by the FDA in 2000 for general laparoscopic procedures.The robotic‐assisted surgical device space is growing rapidly with the development and expansion of various devices, including Ion , Mako (Stryker), NAVIO (Smith & Nephew), and Monarch 3A simplified visual representation of how devices transfer data via wired or broadband Figure  and wireIt is essential to understand latency. Latency is the response delay between a device and the hosting server or target device, which is affected by the data transfer rate of a network and the amount of data needed to be transferred. In remote surgery, these characteristics are essential for the surgeon to respond adequately to changing circumstances and the quality of the video stream to provide appropriate information. Inadequate time or poor video quality could lead to surgical trauma and complications for the patient. The maximum resolution possible for Dr. Marescaux and colleagues, in their surgery conducted in early 2000s, was 1024 pixels by 768 pixels and a data transfer rate of 10 Mbps.Similarly, data transfer speed increases can help the surgeon reduce the latency and allow the surgeon to respond more quickly. The DaVinci robot is capable of at least 1920‐pixel by 1080‐pixel resolution (about 2.6 times larger than Dr. Marescaux), and 5G networks can reduce both the latency and improve video stream quality through their maximum transfer rate of 10Gb/s. Concerning surgeons, Zheng and colleagues found the average endoscopic surgeon's response time to their most complex scenario was 397 ms ± 19 s.4Many countries are slowly adopting 5G networks.In December 2018, a remote hepatectomy in a porcine model was performed at the Fujian, People's Republic of China (PRC) using the Kangduo robotic surgery system and 5G network by Huawei Technologies Company and China Unicom. The equipment control was located in the Fujian Branch of China Unicom, and the operating theater was located in Mengchao Hepatobiliary Hospital in Fujian, Fuzhou, PRC, approximately 48 km 30 miles) away. Two robotic arms (bipolar coagulation and electrocoagulation hooks) and lens were controlled remotely by the surgeon to resect a 2 cm × 2 cm × 3 cm portion of the liver. The surgery lasted for around 60 min, with a total blood loss of approximately 5 ml. In addition, average latency was reported at less than 150 ms0 miles aIn 2019, a study to evaluate the performance of 5G in two different medical applications was conducted in Munich, Germany, and involved camera positioning. The video streaming rate was 900 KB–1 Mbps (7.2–8 Mbps), and the robotic control command rate was 2.4–7.2 KB/s (19.2–57.6 KB/s). The latency was 2–60 ms, and it depended on the transmitted data packet length. A Delphi study was also conducted, which showed that the participants agreed that 5G has a great potential in the healthcare domain and needs to be further exploredIn March 2019, clinicians remotely controlled implants for deep brain stimulation (DBS) through 5G to treat Parkinson's disease and brain ailments. Three patients  were recruited and operated on‐site to install the headframe, craniotomy, and microelectrode puncture in Beijing. The remote‐control group controlled the microelectrode recording and imaging to confirm the implantation of the electrode. The remote‐control group was located approximately 2400 km away  from the operating site . The 5G network utilized in this study was through Huawei and China Mobile. During the procedures, peak download speed was reported at 119 Mbps with an average latency of 76 ms. At the 3‐month follow‐up, it was recorded that the patients improved by 43.6%, 84.9%, and 90.5%, respectively, according to the Unified Parkinson's Disease Rating Scale (MDS)In February 2019, the first tele‐mentored surgery over 5GIn June 2019, remote tele‐assisted surgery was done between MWC Shanghai 2019, Shanghai New International Expo Center (SNIEC) in Shanghai, PRC, and Shanghai East Hospital, approximately 6 km away. Surgeon 1 provided live guidance to Surgeon 2 via the 5G connection provided by China Mobile. The surgery lasted around 138 min, with an average latency of 148 ms. The mean data upload speed was 101 Mbps with no observable signal loss. Image quality was graded as a 9.5/10, and the audio exchange was again 10 out of 10In June and August 2019, two surgeries with a total of 12 orthopedic telerobotic spinal placement on patients  were conducted. The first procedure, the “one‐to‐two” simultaneous remote orthopedic pedicle screw placement, was conducted in June 2020 between Beijing Jishuitan Hospital, Shandong Yantaishan Hospital, and Zhejiang Jiaxing Second Hospital.2 laser (SmartXid2 C60 DEKA), Panda robot , and a VITOM 3D stereo exoscope (Karl Storz). Support staff set up the cadaver and managed the laser settings. The 5G network utilized in this study was the 5G Radio Access Network with a 1 Gbps bandwidth by Vodafone. Throughout the lecture, the average latency observed for 3D high‐definition video was 102 ± 9 ms, with a maximum latency of 280 ms and a mean round trip delay between devices estimated at 40 ms. The surgical evaluation was deemed highly precise after procedures were conductedIn October 2019, transoral laser microsurgery procedures  were conducted on an adult human cadaver. The otolaryngologist was located at Vodafone Village, Milan, Italy, while the cadaver was in the San Raffaele Hospital, Milan, Italy, approximately 15 km away. The computer‐assisted laser microsurgery system (CALM) was used to control a medical COIn November 2019, a live interaction surgery using 5G was performed from a distant location. Surgeon 1, who was located in Rome, Italy, was able to view laparoscopic gastrectomy in Santa Maria Hospital in Terni, Italy performed by Surgeon 2 and his team via the visual‐reality visor. The patients' biometrics via visor connected to three cameras, including one super high‐definition 360° camera, in the operating room. In addition, the laparoscopic camera allowed viewing organs. The event was presented live at the 30th International Congress of Digestive Surgery and was watched by over surgeons present at the Congress and over 30 000 surgeons worldwide via online video streaming platformsFurthermore, four laparoscopic procedures: left nephrectomy, partial hepatectomy, cholecystectomy, and cystectomy, were performed on 25 kg porcine models in 2020. Surgeons were located in Qingdao, PRC, and porcine models were in Anshun, PRC, approximately 3000 km away.Stable 5G networks have made robotic surgeries and telementoring via 5G networks a reality in some countries. Surgeons in PRC, Germany, Spain, and Italy have demonstrated that robotic procedures using 5G can safely and effectively be conducted. The time to complete the surgeries could be similar to non‐5G robotic surgeries.5The challenges created by the COVID‐19 pandemic have increased the importance and need for telemedicine. Integrating 5G into healthcare could provide various benefits, including virtual check‐ins, mobile waiting rooms, and encrypted high‐definition medical imagery transfers; creating 5G enabled virtual reality scenarios to reduce pain and anxiety for terminally ill, immunocompromised, or elderly patients.It is also essential to conduct a cost–benefit analysis before any potential implementation of 5G‐powered robotic surgery for rural areas needing surgeons. From 1981 to 2005, the number of surgeons per 100 000 in rural counties has declined from 7.7 to 5.7 per 100 0006et al.et al.The procedures discussed demonstrate that the latency and reliability difficulties experienced by Marescaux et al.,In the early 2000s, with the development of improved robotic surgical devices, the prospective trial comparing the results of RARP and open retropubic radical prostatectomy by Menon In the initial year of implementation, the performance of 5G networks would vary depending on the user's location because 5G uses various frequency bands to operate, namely: high band 5G (transmitting on millimeter‐wave/24–39 GHz frequency), mid‐band 5G (transmitting on 2.5/3.5 GHz frequency), and low band 5G (transmitting on 600–700 MHz frequency).7Remote tele‐mentored collaboration could help expand knowledge between clinicians in real‐time, increasing medical aid where 5G networks have been established. The high‐speed data transfer could allow large volumes of research and data collection to be shared in real‐time. New technological advancements could serve as a ground for emerging novel therapeutic applications. While limitations and challenges related to the 5G infrastructure, learning curve, cost analysis and device compatibility exist, conducting further research to overcome these limitations and comprehend the potential benefits of integrating the technology before widespread use for cancer management is imminent. Remote and tele‐mentored 5G‐powered procedures could offer a new tool in improving care of patients requiring robotic surgical management such as PCa patients.Ashutosh K. Tewari has served as a site‐PI on pharma/industry‐sponsored clinical trials from Kite Pharma, Lumicell Inc, Dendreon, and Oncovir Inc. Ashutosh K. Tewari has received research funding (grants) to his institution from the United States Department of Defense, National Institutes of Health, Axogen, Intuitive Surgical, the Arthur M. Blank Family Foundation, and other philanthropy. Ashutosh K. Tewari has served as an unpaid consultant to Roivant Biosciences and advisor to Promaxo. Ashutosh K. Tewari owns equity in Promaxo. Krunal Pandav, Austen G. Te, Nir Tomer, Sujit S. Nair declare no conflicts.Not applicable.Krunal Pandav: Conceptualization ; data curation (lead); formal analysis (lead); investigation (lead); methodology (lead); validation ; visualization ; writing‐original draft (lead); writing‐review & editing (lead). Austen G Te: Data curation ; formal analysis ; investigation ; validation ; visualization ; writing‐original draft ; writing‐review & editing . Nir Tomer: writing‐original draft (supporting); writing‐review & editing (supporting); Sujit S Nair: Conceptualization ; supervision ; visualization ; writing‐review & editing . Ashutosh Tewari: Conceptualization ; funding acquisition (lead); project administration (lead); supervision ; writing‐review & editing .


This population‐based follow‐up study investigated the comorbidities, medication use, and healthcare services among women with polycystic ovary syndrome (PCOS) at age 46 years.n = 246) and controls without PCOS symptoms or diagnosis (n = 1573), referred to as non‐PCOS women. The main outcome measures were self‐reported data on symptoms, diagnosed diseases, and medication and healthcare service use at the age of 46 years.The study population derived from the Northern Finland Birth Cohort 1966 and consisted of women reporting oligo/amenorrhea and hirsutism at age 31 years and/or a PCOS diagnosis by age 46 years  and medication use by 27% [RR 1.27, 95% CI 1.08–1.50) compared with non‐PCOS women, and the risk remained after adjusting for body mass index. Diagnoses with increased prevalence in women with PCOS were migraine, hypertension, tendinitis, osteoarthritis, fractures, and endometriosis. PCOS was also associated with autoimmune diseases and recurrent upper respiratory tract infections and symptoms. Interestingly, healthcare service use did not differ between the study groups after adjusting for body mass index.Women with PCOS are burdened with multimorbidity and higher medication use, independent of body mass index. BMIbody mass indexHAhirsutismMETmetabolic equivalent of taskNFBC1966Northern Finland Birth Cohort 1966NWnormal weightOW/OBoverweight/obesePCOMpolycystic ovary morphologyPCOSpolycystic ovary syndromeWomen with polycystic ovary syndrome are burdened with multimorbidity and higher medication use, independent of body mass index.1Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of reproductive age, with a prevalence of 5%–18%.Surprisingly, only a few studies have systematically assessed the overall comorbidity in women with PCOS.22.1n = 280, 4.8% of the population) consisted of women who reported both hirsutism and oligo/amenorrhea at age 31 (4.1%) and/or PCOM/PCOS at age 46 (3.1%), of which 246 replied to the 46‐year questionnaire. The non‐PCOS control population consisted of all the remaining women . Women who were pregnant or using hormonal contraceptives at age 31 (n = 1488) were excluded because of possible bias towards oligo/amenorrhea. Moreover, women who did not permit the use of their data were excluded (n31 = 41 and n46 = 14) ,2.2A questionnaire was sent to participants at the age of 46. Morbidity was assessed via the following question: “Do you currently have, or have you ever had, any of the following symptoms, diseases, or injuries diagnosed or treated by a doctor?” Table . The diaRegarding respiratory/allergic symptoms, the questionnaire included the question: “Have you had the following respiratory and/or allergic symptoms or diseases?” The answer options were Never; Yes, within the last 12 months; and Yes, but more than a year ago. Affirmative answers were combined to form a new variable, which was used for the analysis. The questionnaire also included two questions about symptoms of infection and autoimmune diseases, with no diagnostic data. The items are listed in Table 2.3n = 68) was calculated. Perceived health status at age 46 years was assessed by the following question: “How would you estimate your current state of health?”. Answer options were very good, good, moderate, poor, and very poor. Answer options 1 and 2 and options 4 and 5 were merged for the analysis.To create an overall morbidity score, a sum score of all the Yes answers from the questions regarding morbidities diagnosed by a doctor  classification. The data were analyzed as the main ATC groups, and if the main group was significantly different between the PCOS cases and the controls, the medication subgroups were also analyzed. The risk for medication use was calculated based on the sum score of the medication main groups used. The medication data are reported in Figure 2.62, 25–30 kg/m2, and >30 kg/m2.All the confounding variables included in the study represent factors commonly considered to relate to exposure or overall strongly to morbidity  and R (version 4.0.3) software. A value of p less than 0.05 was considered statistically significant. Differences in the categorical variables between the study groups were analyzed using Pearson's chi‐squared test. The specific group differences were deciphered by adjusted standardized residual counts. The continuous variables were tested for normal distribution using the Kolmogorov–Smirnov test. The independent samples t test or the Mann–Whitney U test were used to analyze the differences in continuous variables. The associations between self‐reported diagnoses as well as symptoms and medication use among the PCOS group vs the control group were analyzed with binary logistic regression models. The adjusted logistic regression models were performed by including the following variables in the models: BMI, physical activity, alcohol consumption, smoking, marital status, and education. To control for type I errors due to multiple‐hypothesis testing, values of P below the false discovery rate calculated by the Benjamini–Hochberg method were considered as statistically significant study‐wide. The risk ratios for morbidity, medication use, and health service visits were calculated using negative binomial regression models.Statistical analyses were conducted using IBM‐SPSS Statistics (version 24) for Windows software, and figures were produced using 2.8The ethical committee of the Northern Ostrobothnia Hospital District approved the study (EETTMK 94/2011) on December 14, 2011. All the participants of NFBC1966 gave informed consent for the use of their collected data for scientific purposes.33.1After correction for multiple testing, the women with PCOS had a higher prevalence of hypertension, DMT2, depression, migraine, tendinitis, osteoarthritis in the knee, back or shoulder, fractures, gestational diabetes, pre‐eclampsia, and endometriosis compared with the non‐PCOS controls by age 46 years. After adjusting for the confounding factors , significance remained for hypertension, migraine, tendinitis, arthroses, fractures, and endometriosis. Table .3.2P < 0.001). In line with this, the women with PCOS presented with multimorbidity at age 46. The morbidity score distributions at age 46 in the PCOS cases and controls are presented in Figure 2) and the overweight/obese  women with PCOS had higher median morbidity scores than controls in the same weight group. Most importantly, the morbidity scores of NW vs OW/OB women with PCOS did not differ, whereas NW controls had lower morbidity scores than OW/OB controls  Figure , which dr = 0.338, p < 0.001), indicating that those with poorer perceived health also reported a higher number of morbidities.To validate if the morbidity score, specific for this study, truly reflected the health status of an individual, we conducted a correlation analysis with the self‐reported health status, a measure previously described to be associated with mortality and morbidity, and to predict survival better than medical records.3.3At age 46, women with PCOS had more “cough with wheezing”, “recurrent respiratory infections”, and “atopic, infantile, or allergic eczema” than controls. PCOS cases also reported having “two or more pneumonias”, “recurrent otitis in adulthood”, “other health‐endangering recurrent infections”, and “common colds”, and being “more susceptible to infections than other people”. PCOS cases also reported more symptoms related to autoimmune diseases, such as “joint pain”, “joint swelling”, and “pain in the heel” than the controls. Adjustments did not affect the results Table .3.4Regarding self‐reported medication use at the age of 46 years, the main medication groups that were more commonly used by women with PCOS than the controls were “alimentary tract and metabolism”, “dermatological”, “systemic hormonal preparations excluding sex hormones and insulins”, and “nervous system” after adjustment for confounding factors. As for the subgroup analysis, “drugs for functional gastrointestinal disorders”, “drugs used in diabetes”, “beta‐blocking agents”, “agents acting on the renin‐angiotensin system”, “antibiotics and chemotherapeutics for dermatological use”, “thyroid therapy”, and “analgesics” were more commonly used among women with PCOS compared with controls before and after adjustments, except for “beta‐blocking agents” Table . Overall3.5p = 0.010), adjustments for confounding factors rendered the risk nonsignificant .Even though the median number of visits to any healthcare provider was higher in women with PCOS  than in controls , fractures, endometriosis, gestational diabetes, and pre‐eclampsia, although after adjustments the risks for DMT2, depression, gestational diabetes, and pre‐eclampsia were no longer significantly increased. Indeed, it has been established that normal‐weight women with PCOS are not at risk for developing DMT2, especially in Nordic populations.Our study also revealed an increased risk for migraine in PCOS, in line with the existing literature.Respiratory tract problems were more prevalent in women with PCOS. The women reported having cough with wheezing, recurrent respiratory infections, and atopic, infantile, or allergic eczema more often than the controls. The evidence of a higher prevalence of various respiratory infections and diseases in women with PCOS is increasing.This is the first study to assess self‐reported symptoms related to infections and autoimmune diseases among women with PCOS. The affected women more often reported recurrent infections, including pneumonias, otitis and common colds, and a higher susceptibility to infections than the controls at age 46 Table . Also, sOnly one previous population‐based study on medication use among women with PCOS exists.The women with PCOS reported morbidities, symptoms, and higher medication use more often than the controls. Moreover, women with PCOS estimated their health as poor or very poor almost three times more often compared with controls, in line with our previous finding.The present study has many strengths, including the possibility to adjust for various confounding factors, which was possible because of the unique cohort data. The study screened symptoms for the first time, thus identifying conditions impossible to discern by International Classification of Diseases codes from the hospital registers. The self‐awareness bias was low because the birth cohort data collection did not target PCOS; rather, it focused on lifelong health and work ability. Indeed, the data represent participants from the same general community, with low variation in terms of ethnicity, education, or access to healthcare services, in comparison to selected PCOS populations from infertility clinics. Additionally, in Finland, there is very little variation in healthcare access, as the healthcare system is public and accessible to everyone regardless of area of residence. Although PCOS diagnosis might be considered a limitation, as mentioned above, the validity of the diagnosis has been well established in several previously published studies.5Our study emphasizes the multimorbidity and poor self‐rated health among women with PCOS. Further studies are warranted to explore the disease mechanisms in more detail.The authors have stated explicitly that there are no conflicts of interest in connection with this article.LK and TTP planned the study. LK, TTP, and RKA wrote the manuscript. LK, RKA, EK, and PP conducted the statistical analyses. RKA created the figures. SK, M‐RJ, SF, LM‐P, and JT provided help with the interpretation of the results. All the authors reviewed the manuscript.This study was supported by grants from The Finnish Medical Foundation, The Academy of Finland , The Sigrid Juselius Foundation, The Finnish Cultural Foundation, The Jalmari and Rauha Ahokas Foundation, The Päivikki and Sakari Sohlberg Foundation, Genesis Research Trust (UK), The Medical Research Council, UK (G0802782), University of Oulu , Oulu University Hospital , Ministry of Health and Social Affairs , National Institute for Health and Welfare (54121), Regional Institute of Occupational Health, , and ERDF European Regional Development Fund (539/2010 A31592).Figure S1Click here for additional data file.Table S1Table S2.Table S3.Table S4.Click here for additional data file.


To determine the association between dynamic neutrophil-to-lymphocyte ratio (NLR) during hospitalization and mortality 1 month after ischemia reperfusion in patients undergoing endovascular treatment (EVT) with successful revascularization for acute large vessel occlusion stroke.This retrospective study included patients who had undergone successful EVT. Information was collected regarding patients’ clinical characteristics, imaging data, and mortality at 1 month. Univariate and multivariate logistic regression models were applied to assess the association between NLR and mortality. We used a generalized additive model and a generalized additive mixed model to compare trends in NLR over time between survivors and nonsurvivors.P = 0.008), 24 to 48 hours , and 48 to 72 hours  after EVT were independently associated with mortality at 1 month. In addition, there was a trend for NLR to decrease gradually over time for both survivors and nonsurvivors; however, NLR in survivors decreased by an average of 0.29 daily than in nonsurvivors.A total of 237 patients were included. During the 1-month follow-up, 42 of these patients (17.7%) died. The multivariate analysis demonstrated that NLR obtained within 12 to 24 hours (odds ratio [OR] = 1.18; 95% confidence interval [CI]: 1.04, 1.33; Increased NLR in the early period after EVT was associated with an increased risk of mortality, and a continued trend toward higher NLR over time was also linked with a higher mortality risk. Endovascular treatment (EVT) has become the gold standard for the treatment of large vessel occlusion in patients with acute ischemic stroke ; howeverPrevious studies have shown that the neutrophil-to-lymphocyte ratio (NLR) in the early poststroke period may reflect the state of inflammation and which plays an important role in ischemia-reperfusion injury –15, thusTo address these uncertainties, we collected data on stroke patients undergone successful revascularization for anterior circulation large vessel occlusion stroke, and sought to assess the potential relationship between dynamic NLR during hospitalization and mortality at 1 month, in the hope that a proven correlation would help physicians make treatment decisions.This case series was approved by the Ethics Committees of Changzhou Second People’s Hospital Affiliated to Nanjing Medical University . Because this study involved an observational retrospective cohort, written consent from patients was not required.Consecutive patients aged ≥18 years who underwent EVT for large vessel occlusion stroke at Changzhou Second People’s Hospital Affiliated to Nanjing Medical University between May 2017 and December 2021 were considered for study inclusion. Eligible patients were those who had experienced an anterior circulation large vessel occlusion stroke, confirmed by head computed tomography (CT) angiography or digital subtraction angiography (DSA); those with a modified Rankin Scale score ≤ 1; those with a National Institutes of Health Stroke Scale (NIHSS) score > 5 at admission; and those in whom treatment was initiated within 12 hours after stroke onset .Patients were excluded from the study if > 6 hours after stroke onset and CT perfusion evaluation did not meet established criteria guidelines , 22; if We collected information regarding patient demographics, clinical history, past medical history and history of drug use, imaging data, laboratory results, NIHSS score, Alberta Stroke Program Early CT Score (ASPECTS), complications during hospitalization，medication during hospitalization and mortality at 1 month after EVT. DSA was used to assess collateral compensation; grades 3 and 4 were defined as favorable collateral compensation based on guidelines from the American Society of Interventional and Therapeutic Neuroradiology/Society of Interventional Radiology .symptomaAll leukocyte and lymphocyte counts obtained during hospitalization were collected, and NLR for each sample were calculated by dividing the neutrophil count by the lymphocyte count. NLR were categorized into values obtained at admission, immediately after EVT, within 12 to 24 hours after EVT, within 24 to 48 hours after EVT, and within 48 to 72 hours after EVT.Mortality at 1 month after EVT was recorded by clinicians either in the hospital or via telephone consultation after the patient had been discharged. All clinical and imaging data were assessed by 2 experienced cerebrovascular physicians who were not familiar with the clinical information and the imaging data. In case of disagreements, a third senior cerebrovascular physician was consulted.t test or a Kruskal–Wallis rank-sum test for continuous variables and a chi-square test for categorical variables. Univariate and multivariate logistic regression models were applied to evaluate the association between NLR at different time points and mortality at 1 month after successful revascularization, with adjustments made for multiple confounders in multivariate models. For the multivariate analysis, the covariates were included as potential confounders in the final models if they changed the estimates of NLR on mortality at 1 month by more than 10% or were significantly associated with mortality. One such confounder was the occurrence of pneumonia during hospitalization, as poststroke infections such as pneumonia are common, and the presence of infection will affect the NLR ). Categorical variables were described as rate and composition ratio. Baseline characteristics were analyzed using a  the NLR –27.The smoothing plots made by generalized additive model were used to assess NLR and mortality at different points after successful revascularization. The difference in NLR between survivors and nonsurvivors at 1 month after EVT was derived from a generalized additive mixed model. The model is used to analyze repeated measurement data, especially when some data are missing and the time interval for repeated measurements is irregular , 29. and EmpowerStats . A P value < 0.05 (two-sided) was considered statistically significant.All analyses were performed using the R package P = 0.088) and symptomatic intracerebral hemorrhage (P = 0.400) among the categorical variable (tertile) groups of NLR.Of the 370 patients who were treated with EVT during the study period, 237 64.1%) were included in the analysis . In addition, 30 cases (71.4%) died in hospital.The sample included 195 patients who were alive at 1 month after EVT (82.3%) and 42 patients who had died at 1 month after EVT (17.7%). Among nonsurvivors, the cause of death was cerebral hemorrhage in 13 patients (31.0%), malignant brain swelling in 8 patients (19.0%), respiratory failure in 12patients (28.6%), heart failure in 5 patients (11.9%), septic shock in 2 patients (4.8%), and “other cause” in 2 patient (4.8%). Among them, 21 cases (50%) died of cerebral causes and 21 cases (50%) died of extra-cerebral causes. The median NLR of the two groups was 6.69 (2.40–7.88) and 3.95 (2.62–5.48) respectively, with no significant difference between them (P = 0.003), baseline NIHSS score (P < 0.001), history of diabetes (P = 0.001), history of chronic kidney disease (P = 0.031), glucose at admission(P = 0.017), bridging intravenous thrombolysis (P = 0.040), cardioembolism (P = 0.040), internal carotid artery occlusion (P = 0.049), Pneumonia during hospitalization (P = 0.024), anticoagulation during hospitalization (P = 0.004), use of invasive mechanical ventilation and vasopressors during hospitalization (p < 0.001), sepsis during hospitalization (P = 0.001), any intracerebral hemorrhage and symptomatic intracerebral hemorrhage during hospitalization (p < 0.001), NLR within 12 to 24 hours after EVT (p < 0.001), NLR within 24 to 48 hours after EVT (P = 0.002), and NLR within 48 to 72 hours after EVT (p < 0.001) were associated with mortality at 1 month , within 24 to 48 hours after EVT , and within 48 to 72 hours after EVT  were independently associated with mortality at 1 month after treatment  [P = 0.002) and greater temporal change in NLR (2.4 (IQR 1.1–7.6) vs 1.3 (IQR 0.1–3.7); P = 0.05) within 3 to 7 days after EVT were also associated with mortality at 90 days [P = 0.971), but NLR obtained within 12 to 24 hours after EVT were associated with mortality at 1 month . For every unit increase in NLR in the early stage, the risk of mortality increased by approximately 18%. Differences in findings between our study and previous research may be partially explained by differences in the enrolled populations  and the stress reaction associated with undergoing EVT.Previous studies have demonstrated a relationship between mortality and NLR at admission  . Higher  90 days . In the P = 0.002) [P = 0.422). Additionally, we found that NLR in patients still alive at 1 month decreased by an average of 0.29 daily than in nonsurvivors. To our knowledge, this is the first study to examine the association between persistent dynamic changes in NLR during hospitalization and mortality, thus extending previous research in several ways. First, we demonstrated the trends in dynamic NLR over time during hospitalization after successful revascularization. Second, we compared these trends in survivors and nonsurvivors to assess the association between dynamic NLR and mortality at 1 month after EVT. The NLR might be a biological marker of prognosis in patients undergoing EVT and could also sensitively reflect changes in the severity of a patient’s condition [Our previous research has shown that increased NLR immediately after EVT is associated with an unfavorable prognosis  at 3 months for anterior circulation large vessel occlusion stroke  , but in ondition , 42. Addondition . HoweverOur study had some limitations. First, this study was limited to patients who had experienced an anterior circulation stroke, and so the results cannot be extrapolated to the posterior circulation stroke population. Second, in order to study the mechanism of ischemia reperfusion injury, the enrolled patients had all undergone successful recanalization after thrombectomy; patients in whom revascularization was not successful were not included in the analysis. Third, some repeated measurements of NLR were missing for study patients. However, we used a generalized additive model and a generalized additive mixed model to analyze all collected data, and these models have been found to be useful when some data are missing. Finally, although we adjusted for multiple confounding factors, other potential confounding factors may have been missed in these analyses.We demonstrated the pathophysiological changes of dynamic NLR on stroke and the persistent effect of inflammation on ischemia-reperfusion injury. Because NLR can be easily obtained repeatedly, these measurements can be used to assess the changes in disease progress over time. These results suggest that physicians should be aware of the potential harm associated with persistently high NLR in patients who have undergone successful revascularization for anterior circulation large vessel occlusion stroke.


Amaranthus species are appealing betalain sources given their extensive pigmentation patterns and recognized food status around the world. An advantage of amaranths as natural food colorants is that, when grown as leafy vegetables, water extracts would be compliant with U.S. Food and Drug Administration guidelines as “vegetable juice” colorants. Thus, we developed a methodology based on U.S. FDA guidelines to investigate betalain diversity among forty-eight amaranth accessions grown as leafy vegetables. Total betacyanin concentrations ranged from 4.7 to 478.8 mg/100 g dry weight, with amaranthin and isoamaranthin identified as major constituents. Our findings will guide future research on amaranths to determine economic viability and suitability for growing natural colorant markets.Growing consumer demands for healthier foods have evoked trends in the food industry to replace synthetically produced colorants with naturally derived alternatives. Anthocyanins currently comprise the bulk of the natural colorant market, but betalains offer advantages where anthocyanins have limits.  The use of naturally derived food and beverage colorants has grown substantially in recent years. By 2024, the global natural colors market is estimated to surpass 2 billion USD . DespiteAnthocyanins and betalains are the most commonly used natural alternatives to synthetic reds, with anthocyanins typically performing best in high acidity applications (pH < 4) and betalains performing best at pH levels above 4. Anthocyanins are commercially sourced from many crops such as grapes, carrots, and corn, while betalains are exclusively sourced from red beets . Other cA. caudatus, A. cruentus, and A. hypochondriacus. Two additional amaranths, A. tricolor and A. blitum, are also grown as leafy vegetables mainly in Africa and Asia.Interest in amaranths as resilient and healthy alternative crops continues to gain traction . AmarantGluconacetobacter diazotrophicus , whereas betanin (betanidin 5-O-β-glucoside) is the major betacyanin in beets .Formal studies of amaranth as an alternative to beet-derived colorants began in the 1980s . Amarantin beets . BetalaiAmaranthus germplasm to determine the diversity of betacyanin content and composition were conducted two decades ago  for which little or nothing of betalains are known.Our selection of plants was guided in part by FDA natural colorant guidelines (CFR Title 21 73.260) and practical considerations for identifying top pigment producers in greenhouse screens as a first step for eventual field trials. Although some amaranths have densely pigmented inflorescences, this part of the plant typically is not consumed (seeds excluded) and therefore would require FDA approval as a colorant source. Alternatively, when grown as a leafy vegetable, amaranth could be harvested multiple times throughout the growing season and extracts would be FDA compliant as a “vegetable juice colorant.” For this study, we use the term ‘vegetable amaranth’ to represent all amaranth species we analyzed at the same vegetative growth stage. We selected forty-eight uppliers . The majuppliers . Our sel2. Plants were manually watered daily and fertilized with an organic 4–1-1 fertilizer  weekly starting at week 2. All above-ground biomass was harvested 7 weeks after planting and immediately frozen at-20°C. This frozen plant material was then lyophilized, ground into a fine powder, and stored at-20°C until analysis.Plants were grown in the greenhouse facilities at the University of Illinois at Urbana-Champaign in four replicates, planted in February 2018, August 2018, September 2018, and May 2019. In each replicate, five individual plants were grown for each accession. Seeds were sown directly into 5-inch pots filled with Sunshine® LC1  professional soil mix and thinned to 1 plant per pot upon germination. Plants were grown under a 31/23°C, 15/9 h day/night temperature regime with high-pressure sodium (HPS) lighting provided from 06:00 to 21:00 h if light intensities fell below 900 watts/m20 min, 30 min), temperature  and plant mass  so that betacyanin pigment yields were maximized. Bolded conditions were selected after optimization trials and used for all experimental samples. Optimization trials were conducted on five randomly selected amaranth accessions, coded as a categorical variable during analysis, to better represent the extractability of the broad range of accessions surveyed. After extraction, tubes were centrifuged at a relative centrifugal force (RCF) of 3,000× g for 5 min. Supernatants were then filtered through a 30 μm CellTrics cell strainer  followed by a 0.45 μm hydrophilic PFTE syringe filter  and chilled on ice until analysis. To limit protein precipitation under acidic HPLC conditions, samples for HPLC analysis were acidified with a small amount of formic acid after straining , briefly vortexed, and centrifuged in a 2 ml tube for 10 min at 21100 RCF prior to syringe filtration. This additional step was used rather than simply acidifying the starting extraction solvent to avoid the accelerated degradation observed for betalains at high temperatures and low pH values  was used to optimize extraction variables so that total betacyanin yield from a single extract was maximized. This method is commonly used to optimize complex biotechnological processes in food research and consists of plotting multiple independent factors at different levels  in a three-dimensional space so that their interaction with a response variable can be precisely monitored as a response surface plane . This suH values .Spectral readings for ultraviolet–visible (UV–Vis) well plate assays were collected using a Synergy 2 multi-well plate reader . A final volume of 200 μl was added to each well and any bubbles were removed by blowing ethanol vapor from a squeeze bottle across the sample(s). Data were corrected to a pathlength of 1 cm by analyzing select samples for each extraction system in a reference 1 cm pathlength cuvette using a UV–Vis spectrophotometer . These cuvette-derived data were divided by data from the plate reader to obtain the correction factor for each extraction system.* a* b*L (CIELAB) color space. Parameters were calculated with ColorBySpectra software  and hue angle (*h) were calculated according to the equations below:Extract color parameters were calculated using the CIE  software  using 38a* and b* correspond to the CIELAB color space values in which L* measures lightness, a* redness/greenness, and b* blueness/yellowness. Samples were buffered with a 0.1 M pH 6.5 phosphate buffer prior to analysis. For each accession, samples were analyzed independently from three separate extractions prior to being averaged.where Total betacyanin concentrations were determined as described by A536 = absorbance at a 536 nm, MW = molecular weight, DF = dilution factor, V = extraction volume (ml), ε = molar coefficient, M = mass (g) of material being extracted, and L = pathlength (cm). Coefficients for amaranthin  were used  and a SecurityGuard C18 guard column . The mobile phase used 1.5% (v/v) phosphoric acid as solvent A and 1.5% phosphoric acid, 20% acetic acid, and 25% acetonitrile for solvent B. A 30-min linear gradient from 10 to 55% solvent B in solvent A was used, with an added 1-min 100% B wash followed by a 5-min equilibration at 10% B after each sample. An injection volume of 20 μl was used with monitoring at 535 nm. The column was kept at ambient temperature at a constant flow rate of 1 ml/min and samples were injected via a 20 μl sample loop. Purified betanin  was used as a standard for compound identification with each set of samples analyzed, and identified compounds were quantified as a percentage of the total betacyanin content determined previously using a UV–Vis spectrophotometer. Spectral DAD data were simultaneously collected between 250 and 600 nm.Betalain composition analyses were carried out as described by Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS–MS) was performed on selected samples with diverse pigment profiles  to confirm the identity of peaks observed using HPLC. Analyses were performed at the School of Chemical Sciences Mass Spectrometry Lab at the University of Illinois with a Poroshell 120 SB-C18  at ambient temperature using 1% formic acid in water as solvent A and 1% formic acid in acetonitrile as solvent B. A gradient of 100% solvent A to 80% solvent B over 10 min with a flow rate of 0.7 ml/min was used. Mass spectra were acquired in positive mode scanning in a mass to charge ratio (m/z) range of 100–1,000 at a cone voltage of 35 V. Spectral data were collected at 535 nm. Compound identities were confirmed based on published literature of MS and MS–MS measurements and standard compounds.0: total betacyanin content is the same across all tested accessions) using a generalized linear model with replication as a blocking factor, followed by Tukey’s HSD procedure to perform pairwise mean comparisons (α = 0.05). Response surface methodology experiments for optimization of betalain extractions were analyzed using Minitab 19 . Statistical correlations were made using the non-parametric Spearman rank correlation test.Results in via grinding and to maximize stability of pigments in storage prior to analysis. Water was chosen as the solvent for betalain extractions to maximize the direct applications of our results to the food industry. FDA guidelines (CFR Title 21 73.260) state that all natural colorants labeled as ‘vegetable juice’ must either be expressed from fresh tissue or extracted from dry tissue with a simple water infusion—any specialized extraction or purification protocol would likely require a significant investment of time and money to gain explicit regulatory approval in the US and beyond.Harvested plant material was lyophilized to facilitate homogenization of tissue p < 0.01) with an adjusted R2 value of 83.1%. The interactions between time*temperature, temperature*mass, and temperature*accession were also significant (p < 0.05). Accession or genotype was the most influential factor, followed by mass, temperature*accession, time, time*temperature, temperature, and lastly temperature*mass. Using the optimization tool to maximize betalain content, the optimal parameters selected for further experimental analyses were a temperature of 30°C, incubation for 20 min, and the use of 0.15 g of ground lyophilized tissue in 40 ml of deionized water. The efficiency of this first extraction was found to be 96.2% (SD = 0.0062), which was determined from 10 separate successive extractions of the same starting material.The temperature, time, and mass of extraction material for betalain extractions were optimized using response surface methodology with a Box–Behnken design. Example contour plots showing the effects of extraction parameters on betacyanin content are shown in A. cruentus and suspected hybrids thereof. Accession PI 566897 (A. cruentus) had the highest betacyanin yield at 478.8 mg BCY/100 g DT when averaged across all four replicates. The single highest betacyanin yield within a single replicate was in A. cruentus ‘Hopi Red Dye’  with 655.6 mg betacyanins (BCY)/100 g dry tissue , betanin (x̄ = 1.4%), isobetanin (x̄ = 0.1%), and celosianin II (x̄ = 0.5%). An example of the HPLC-MS–MS method used is shown in Five different betacyanin compounds were identified from the water extracts and summarized as percentages of total betacyanin concentration . Peak anAmax = 1 to allow for direct comparisons at equal tinctorial strengths. Only lines with considerable betacyanin concentrations (>70 mg/100 g DT) were analyzed (A. cruentus (PI 689689) had a uniquely low b* value, with a vivid magenta appearance  and positively with chroma  to yielding bluer and more saturated colors with increasing proportions of amaranthin. These correlations, however, appear to be confounded by total betacyanin content— regression analysis (not shown) indicated that betacyanin composition did not significantly affect any color parameters when total betacyanin content was accounted for. Given this information, the variability seen in b* is likely due to the presence of unquantified dehydrogenated or decarboxylated betacyanins, betalamic acid, and other non-betalain compounds selectively absorbing blue light.Color parameters were measured from the same samples used for determining total betalain content, buffered to a pH of 6.5 and normalized to analyzed . The visanalyzed . One USDpearance , #1. The535.7 nm . AccessiAmaranthus cruentus generally showed the most visually intense pigmentation in leaves and stems among all amaranth types examined , were listed to be of hybrid origin, presumably involving A. cruentus, and both ranked in the top-third of all tested types based on total betacyanin content ; A. tricolor PI 419057  and A. tricolor containing significantly higher proportions of isoamaranthin (x̄ = 12.1%) and betanin (x̄ = 3.1%). These trends between species are seen in other published work on Amaranthus betacyanins  determined to be betalamic acid was identified with LC–MS–MS in very minor concentrations in most accessions. Betalamic acid is a key precursor to both betacyanins and betaxanthins but is also a common degradation product of betacyanins. Given the spontaneous formation of betaxanthins upon conjugation of betalamic acid and an amino acid, extracts allowed enough time to undergo this reaction would not be representative of the plant’s in vivo betalain profile. The general absence or low abundance of betaxanthins among our test panel is intriguing and worthy of more research, especially compared to similar-colored carotenoids, although this finding might be partly explained by the tissues we tested.Full spectral data from HPLC analyses were collected from 300 to 650 nm using a photodiode array detector and visualized with an isoabsorbance plot to further verify the absence of betaxanthins, which would be observed with a Betacyanin composition is often explored from application-based perspectives due to the implications it is thought to have on color stability and performance in food and beverage products. Work done by Amaranthus extracts that are substantially darker and less vivid colors that appear dark plum to gray when visualized after conversion to RGB coordinates using the Colorspace package in R  extract colors also show close similarity to amaranth colors , with 478.8 mg BCY/100 g DT. Another finding was that total betacyanin yield is hard to predict based on phenotype alone, as several intensely dark-pigmented types had lower yields compared to other types with less intense pigmentation (Amaranthus species.Our investigation yields new findings on betalain pigments among a diverse panel of amaranth accessions from the USDA National Plant Germplasm collection and genotypes from commercial sources. In addition, our focus on betalains as potential sources of natural food colorants provided the context for optimizing high-throughput extraction and quantification methods in compliance with current US FDA food colorant standards. Simple water extractions of dried edible biomass yielded vivid magenta extracts appearing bluer than color values reported for red beet extracts, which highlights the potential of betacyanin-rich amaranth extracts as an alternative to beet-sourced food colorants. The major betacyanin of all accessions surveyed was amaranthin, but yields of betacyanins were quite variable and mainly dependent on genotype. The highest average total betacyanin yield was from accession PI 566897 (entation . FurtherThe original contributions presented in the study are included in the article/This manuscript represents partial fulfilment of a graduate thesis project by JH under the supervision of CR, both of whom conceived the study. JH performed the experiments, analysed and interpreted the data, and drafted the manuscript with contributions and suggestions from CR and MV. All authors contributed to the article and approved the submitted version.This project was funded in part by a University of Illinois ACES-OIP Seed Grant and USDA-NIFA-AFRI grant 2019-67013-29355. Fellowship support for JH was provided by a USDA-NNF Grant 2015-38420-23707 administered through the MINDS-in-Ag Program at the University of Illinois.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study evaluated the effects of less invasive surfactant administration (LISA) and intubation-surfactant-extubation (InSurE) on bronchopulmonary dysplasia (BPD) in preterm infants with respiratory distress syndrome (RDS).Neonates with respiratory distress syndrome requiring surfactant, with gestational age < 32 weeks and birth weight < 1500 g admitted to our neonatal intensive care unit from January 2018 to December 2019, were retrospectively analyzed. LISA and InSurE were used independently. The incidence of BPD at 36 weeks postmenstrual age, pre-discharge mortality, and need for mechanical ventilation (MV) within 72 h of birth were compared between LISA and InSurE group. Secondary outcomes including necrotizing enterocolitis requiring surgery, retinopathy of prematurity ≥ stage 3, patent ductus arteriosus requiring medical therapy or surgery, and length of hospitalization were analyzed.P > 0.05, respectively). The incidences of necrotizing enterocolitis requiring surgery, retinopathy of prematurity ≥ stage 3, patent ductus arteriosus requiring medical therapy or surgery, and length of hospitalization did not differ significantly between the two groups .Among the 148 included neonates, there were 46 and 102 infants in LISA group and InSurE group, respectively. There were no significant differences in BPD incidence, the severity of BPD at 36 weeks postmenstrual age, and the rate of MV within the first 72 h after birth between the two groups (For surfactant administration among preterm infants with respiratory distress syndrome, LISA did not decrease bronchopulmonary dysplasia and severity of BPD at 36 weeks postmenstrual age. The benefits of LISA would require further evaluations. Neonatal respiratory distress syndrome (RDS) is a common disease in premature infants, specifically in early preterm infants, due to surfactant deficiency. Surfactant replacement therapy has been widely used for infants with RDS, because it reduces mortality and the incidence of bronchopulmonary dysplasia (BPD) . IntubatThis study analyzed LISA’s effects on the incidence of BPD and mortality in preterm infants with RDS, compared with the traditional InSurE method.This retrospective observational study was performed in the tertiary neonatal intensive care unit (NICU) of the Women’s Hospital, School of Medicine, Zhejiang University, between January 2018 and December 2019. The requirement for informed consent was waived due to the study's retrospective nature. The participants were classified into two groups based on the method of surfactant administration during the study period: (1) LISA group, in which infants were treated with surfactants by the LISA method, and (2) InSurE group, in which infants were treated with surfactants by the traditional method.2O; (3) requiring fraction of inspired oxygen (FiO2) ≥ 0.3; and (4) needing surfactant administration within 2 h after birth.The inclusion criteria were as follows: (1) having RDS with the gestational age < 32 weeks and the birth weight < 1500 g; (2) having spontaneous breathing and being stable on nasal continuous positive airway pressure (nCPAP) with a pressure of 5–8 cmHThe exclusion criteria were as follows: (1) requiring intubation in the delivery room or before the application of surfactant; (2) having congenital diseases affecting respiratory function; (3) having severe congenital birth defects; and (4) having inherited metabolic disease.In the retrospective study, porcine surfactant  was used. Based on literature concerned on the efficacy of surfactant and our experience, 200 mg/kg of porcine surfactant was used as the first dose. A second or third dose of 100 mg/kg of porcine surfactant was used, if the infant’s condition is deteriorated and RDS was still the primary consideration.2 before surfactant administration.The following demographic data were collected in the two groups: gestational age, birth weight, sex, mode of delivery, Apgar score at 5 min, multiple fetuses, small for gestational age (SGA), maternal complications, complete course of antenatal corticosteroids, time between birth and surfactant administration, positive end-expiratory pressure (PEEP) prior to surfactant administration, and FiO2 ≥ 0.6 with PEEP ≥ 7cmH2O; (2) respiratory acidosis (pH ≤ 7.2); and (3) frequent apnea. The secondary outcomes included using a second or further dose of surfactants; the incidence of pulmonary hemorrhage, pneumothorax, intraventricular hemorrhage (IVH) ≥ grade III, surgical necrotizing enterocolitis (NEC), or retinopathy of prematurity (ROP) ≥ stage 3; hemodynamically significant patent ductus arteriosus (hsPDA); adverse events during surfactant administration; duration of respiratory support mode in hospitalization; and length of hospitalization.The primary outcomes included the occurrence and severity of BPD at 36 weeks PMA or discharge, mortality before discharge, combined incidence of mortality and BPD, and mechanical ventilation (MV) rate within the first 72 h after birth. BPD was defined by the 2001 NICHD definition  as an oxP-values < 0.05 were considered statistically significant.All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) software, version 20.0 . Continuous variables are presented as mean with standard deviation and median with interquartile range (25th–75th percentile) and were assessed using t-tests and Mann–Whitney U-tests for normally and non-normally distributed data, respectively. Categorical variables are described using frequency and were assessed using the chi-square test or Fisher’s exact test for between-group comparison. +5 weeks and 31+6 weeks and of birth weights between 745 and 1490 g were diagnosed with RDS by both clinic presentations and/or chest X-ray and require surfactant replacement. Of these, 46 infants were in the LISA group; with gestational age ranged from 25+5 to 31+6 weeks and birth weight ranged from 790 to 1490 g. The remaining 102 infants were in the InSurE group; with gestational age ranged from 26+2 to 31+6 weeks and birth weight ranged from 745 to 1490 g. Other demographic and baseline clinical characteristics were similar in both groups (P > 0.05). The details were shown in Table A total of 148 preterm infants of gestation age between 25P = 0.85). There were two infants with severe BPD in LISA group, compared with four in the InSurE group (4.35 vs. 3.93% P = 0.903). No mortality was observed in each group. There was no significant difference in the MV rate within the first 72 h after birth, there were eight infants in the LISA group, compared with 19 in the InSurE group . The details were shown in Table Ten infants had BPD in the LISA group, compared with 28 infants in the InSurE group . No infant had pneumothorax or intraventricular hemorrhage ≥ stage 3 during hospitalization in each group. There were also no significant differences in other neonatal morbidities, such as pulmonary hemorrhage, surgical NEC, hsPDA, and ROP ≥ stage3 between the groups (P > 0.05). There was no significant difference in the length of hospitalization between the groups . The average durations of MV, nCPAP/high-flow nasal catheter, and oxygen supplementation during hospitalization were 5, 15, and 23 days in the LISA group, compared with 7, 13, and 25 days in the InSurE group, respectively (P > 0.05). The details were shown in Table Three infants required second or further dose of surfactant in LISA group, while 6 infants did in InSurE group. There were no significant differences between the two groups , who were relatively older than those included in two previous multi-center studies conducted by Kribs et al.  and DargNo mortality in either group was observed in our study. The result was consistent with that of Kribs et al. , who fou+0–28+6 weeks, while no effect was observed among neonates with gestational ages 29+0–31+6 weeks.We did not find significant differences in the incidence of BPD between the two groups. The result was consistent with the result of our previous study . This coIt is well known that infants with severe BPD are at a higher risk of mortality after discharge , and theMV is a significant risk factor for BPD in preterm infants, specifically those with very low birth weights , 20. We 2, PEEP and the number of additional surfactant doses. Thus, further studies should focus on the issue of the optimal surfactant dose for LISA.Several adverse events have been observed during surfactant administration . HoweverIn our retrospective study, the number of infants in the InSurE group was relatively higher than that in the LISA group. As shown in previous surveys, the doctors’ attitudes may affect the clinical application of LISA , 26. HalFurther studies may focus on two issues. One was which population could take most advantage from the method of LISA, although it had been proven to be safe for neonates with a gestational age < 26 weeks . The othThere were some limitations in our study. Firstly, our study was a single-center retrospective study, and the cohort was relatively small. Secondly, the mean gestational age in our study population was about 29 weeks, which is not the population at the highest risk for mortality and BPD. Finally, we did not perform follow-up in survivors. Our further stratified comparison studies would focus on these issues.In our study, compared with the traditional application of surfactant administration, the LISA technique was an alternative method to delivery surfactant, but did not significantly reduce the mortality and incidence of BPD. Further researches are required to explore the potential effects of LISA on late pulmonary outcomes or neurodevelopment in survivors.