We find a strongly growing, multidisciplinary LC research domain. That domain appears tightly connected based on shared research knowledge. However, we also observe notable concentrations of research activity in different disciplines. Data trends over 3 years of LC research suggest heightened attention to psychological and neurodegenerative symptoms, fatigue, and pulmonary involvement.While the COVID-19 pandemic morphs into less malignant forms, the virus has spawned a series of poorly understood, post-infection symptoms with staggering ramifications, i. e., long COVID (LC). This bibliometric study profiles the rapidly growing LC research domain  to make its knowledge content more accessible. The article addresses What? Where? Who? and When? questions. A 13-topic Concept Grid presents bottom-up topic clusters. We break out those topics with other data fields, including disciplinary concentrations, topical details, and information on research “players”  engaging in those topics. We provide access to results  As the pandemic wanes, the outpouring  of research on COVID-19 slows. However, what is the research pattern for long COVID (LC), the “pandemic after the pandemic”? The aim of this study was to profile LC research to generate actionable research intelligence for researchers, clinicians, and policymakers. That should serve to accelerate the resolution of LC medical and other effects.We cast this article as a “research profile”  Where is the work being done? [countries].(2) Who is doing it? .(3) When? [trends].(4) Of particular interest is to provide usable intelligence on combinations of those “4 W's,” e.g., to identify who is researching what sub-themes recently?“Research profiling” . This is shorthand for text analyses of Science, Technology & Innovation (ST&I) information resources to inform R&D management in various guises. Tech mining applies bibliometrics and text analyses of various sorts to gain usable research intelligence. A sampling of tech mining applications gives some feel for how this approach can help perceive research concentrations on given topics: nanotechnology systems of innovation  approaches extract intelligence on research concentrations within a domain to, then, identify pertinent research beyond the domain .Offering even greater potential for ST&I management would be forecasts of topics that are “emerging” , and search queries. Zhang et al. addressed some 5,000 articles; we  through December 2021. Our current study analyzes 5,243 articles from PubMed  through November 2022. Therefore, our dataset is strikingly larger (some 7X) in less than one extra year of data. However, LC research is tiny compared to COVID-19 research, as noted. A few comparisons between Jin et al. .➢ Top countries for articles published, i.e., Jin et al. vs. this studyCureus.➢ Top journals, i.e., their top five remain in our top six (refer to Section 4.5.1) given the ~seven-fold increase in our dataset, which shows surprising consistency. The one added by us is ➢ Top cited authors vary from our current results interestingly . Nalbandian et al. is #1 for them with 396 citations, up to 594, and #3 for us . GreenhaMost interestingly, our #1 is Huang et al. , with 78via Concept Grid (based on PCA), i.e., different perspectives.➢ Their explorations of topical emphases focus on keyword frequency, whereas ours offer topical categorization Jin et al.  also anaOur search in PubMed on 15 November 2022, used the National Library of Medicine (NLM) standard LC search:→ (covid) AND LitCLONGCOVID[filter].https://pubmed.ncbi.nlm.nih.gov/?term=%28covid%29$+$AND$+$LitCLONGCOVID%5Bfilter%5D.→ at: That search yielded 5,243 records with abstracts . We downloaded these and conducted our analyses on them. The 5,243 PubMed IDs were available, so we entered those in a PubMed search and retrieved the record set for further analyses:https://sites.google.com/searchtech.com/covidproject/home, or at.➢ COVID http://bit.ly/3iJ5OAt.➢ Someone seeking to extend these analyses could start with the query operations noted; then, adjust for record additions from the time of our search, as warranted. The data treatments described in Section 3.2 indicate how we refined the record content.On 16 November 2022, we searched for those 5,243 records in WoS using PubMed ID, thereby retrieving 4,292 (some 82% of the PubMed set). For topical analyses, we used the PubMed data. The main purposes for also getting the WoS version of the same articles were to use Web of Science Categories (WoSCs) to study disciplinary involvement and to use WoS Cited Reference content to enable citation analyses. Essentially, all the 4,292 records have Cited References.VantagePoint tools to extract, clean, and consolidate the fielded data of the record sets. These computer operations, plus certain manual refinements, included:We utilized various ➢ Processed date information to generate publication month for each article over the period of January 2020, through December 2022  for 4,393 records. For example, some date variants were: “2021 Apr”; “2021 Apr 1”; and “2021 Apr–Jun 01.”➢ Combined institution name variations (and consolidated department levels within an organization) to reduce 15,010 names to 10,497, of which 2,356 were associated with more than a single article. PubMed Affiliation names are dependent upon submitted data and therefore inconsistent. Manual curation was completed on institutions, generally on all institutions with more than five records in the dataset. Individual department or campus location was not considered, with the exception of the University of California system.VantagePoint fuzzy matching routine tailored to person names. To illustrate, the second most active author was Patrizia Rovere-Querini; the “List Cleanup” routine combined 18 instances of her name in that format with six lacking the hyphen, one showing as “Querini, Patrizia Rovere,” and one as “Rovere Querini, P.”➢ Consolidated author name variations using a VantagePoint's “RefineNLP” set of routines to consolidate closely related term variations . We further tuned the abstract NLP phrases by removing terms closely related to LC search terms . These processes yielded a set of 97,996 noun phrases. To illustrate, two of the resultant leading phrases were “sequelae,” appearing 1,101 times in 949 records after treatment (vs. 586 times in 505 records prior), and “persistent symptoms,” appearing 410 times in 303 records after treatment (vs. 383 in 288 records beforehand).➢ Extracted, using Natural Language Processing (NLP), 126,740 abstract noun phrases; applied We considered various topical data resources, i.e., Medical Subject Headings (MeSH); keywords ; and title or abstract NLP-derived noun phrases. MeSH terms are pre-established and then applied to the set of articles under study. Here, we used them for some purposes but prefer a more adaptable set of terms deriving from the articles in our fast-evolving domain for “bottom-up” topic identification.We also used the WoSCs for disciplinary characterizations; those are based on the journal (or conference) in which the articles appear, not on the article's content. We drew on abstract phrases, on the rationale that these were most prevalent and most fluid to pick up new research matter at the article level. Alternative topical data resources included Title NLP-derived phrases. We explored using these in conjunction with the Abstract NLP-derived phrases but went with the abstract NLP noun phrases in investigating this very new LC research domain.Likewise, we treated the WoS 4,292-record dataset. Of particular note, we sought to mine the Cited Reference (“CR” field) information therein. As an illustration, here are a few references cited by J. Calvo-Paniagua et al.  in an article titled “A tele-health primary care rehabilitation program improves self-perceived exertion in COVID-19 survivors experiencing Post-COVID fatigue and dyspnea: A quasi-experimental study”:10.1371/journal.pone.0243882.➢ Jacobs L. G., 2020, PLOS ONE, V15, doi: ➢ Kendrick K. R., 2000, J Emerg Nurs, V26, P216.➢ Lee K., 2009, GLOB INST, P1.Note the sparsity of the cited record information. We obtained information of interest for our analyses on first author  of the cited article; year of cited article publication; cited journal (or conference) name (abbreviated); and DOI, where available. We did not use the volume and page information. For our purposes, we elicited the cited (first) author, cited year, cited journal, and cited DOI. We used a thesaurus to associate the cited journal (or conference) names with their corresponding WoSCs. This gave us a field of Cited WoSCs for further analyses (leading to strong indications of connectedness in the LC research domain).Grouping-related variations of terms and phrases to determine meaningful topics are vital to comprehending latent themes in a body of text. There are a number of diverse methods to achieve such ends, including factor analyses, cluster analyses, and hierarchical analyses. Our colleagues have applied three such approaches to COVID-19 research to compare results and identify four promising research topics . In the past, we have compared topic modeling approaches to generate effective topic clusters on WoS abstract records . A key feature is the application of an optimization routine in conjunction with PCA to cover a maximum number of records with a minimum number of groups (factors). The resulting set contains fewer factors than our PCA solution, but those cover more of the records. The 13 Concept Grid factors have their constituent high-loading terms (above threshold) associated with 4,358 of the 5,243 records. In contrast, our favored PCA solution of 23 factors only covers 3,124 of the records.We ran the Principal Components Analysis (PCA) routine provided in Concept Grid gives a reproducible solutionWe decided to apply the Concept Grid approach here. A key advantage is that it offers the Concept Grid (We ran Concept Grid on the record-term matrix (for abstract NLP phrases); it yielded 13 factors. We proceed to use these for our topical analyses to follow.VantagePoint (www.theVantagePoint.com), to perform a number of text cleaning and consolidation steps, as well as analytical operations. Its RefineNLP routine consolidated abstract NLP phrases in preparation for topical analyses. Other software  can do many of the analyses. The open-access Dashboard [https://searchtechnology.github.io/LongCovidDashboard/] provides many “what, where, who, and when” results in a form suitable for zooming in on particulars. Some analyses requiring VantagePoint are:We used proprietary software, ➢ NLP routine has been tailored to scientific text; generally speaking, differences from other NLP routines should not be excessive. Note that we process noun phrases, including single-word ones.➢ Emergence indicators ,➢ Broader issues .With such disciplinary breadth of the LC research domain, it is surprising to see that research shows a high degree of citation “connectedness.” While citation concentrations certainly vary by discipline , they doWe also analyze specific topical phrases that appear as “emerging”. We duly note important limitations. We used the “official” LC query devised by NLM to identify relevant research. For an emerging research domain with especially amorphous boundaries, there are many conceivable alternative formulations that would yield different datasets. LC diagnosis is not clear-cut; the definition of what research on diverse organ systems and symptoms belongs “in” the domain is hard. LC research invites tools, such as LBD, to scout out to identify pertinent research outside a given set of research bounds.ClinicalTrials.gov) offer additional information resources worthy of exploration. They would surely present different perspectives on LC.Our analyses are of a “snapshot in time,” the 5,243 PubMed articles as of 15 November 2022. We note that the prior NLM query applied a month or so earlier yielded a dataset less than half the size due to rapidly advancing research and, more so, adjustment of the query to adapt to evolving research patterns. We also analyzed the corresponding 4,292 WoS records. Special COVID-19 compilations ; other databases, such as Scopus; and other data forms  and use that to retrieve a suitable set of records that are highly related. Those could be presented via Dashboard.We welcome inquiries about particular interests that we might help you pursue. Future research, above all, warrants tracking how LC evolves. Conducting literature-based discovery (LBD) holds particular appeal. We explored the use of a knowledge model for vaccination regarding COVID-19 (Wu et al., The original contributions presented in the study are included in the article/AP coordinated the analyses and led the article drafting. MM developed and executed many of the core analyses. NN contributed insights and analytical guidance throughout the study. All authors contributed to the article and approved the submitted version.


People Who Inject Drugs (PWID) are at a higher risk of acquiring bloodborne infections. We aimed to estimate the seroprevalence of the Hepatitis C Virus (HCV) in PWID and identify correlates and risk factors using data from the Puerto Rico National HIV Behavioral Surveillance System, PWID cycle 5, conducted in 2018.A total of 502 San Juan Metropolitan Statistical Area participants were recruited through the Respondent Driven Sampling method. Sociodemographic, health-related, and behavioral characteristics were assessed. Testing for HCV antibodies was completed after the face-to-face survey. Descriptive and logistic regression analyses were performed.a = 2.23; 95% CI: 1.06–4.69; ORa = 2.14; 95% CI: 1.06–4.30, respectively).Overall seroprevalence of HCV was 76.5% (95% CI: 70.8-81.4%). A significantly (p < 0.05) higher HCV seroprevalence was observed among PWID with the following characteristics: heterosexuals (78.5%), high school graduates (81.3%), tested for sexually transmitted infections (STI) in the past 12 months (86.1%), frequent speedball injection (79.4%), and knowing the HCV serostatus of the last sharing partner (95.4%). Adjusted logistic regression models showed that having completed high school and reported STI testing in the past 12 months were significantly associated with HCV infection (ORWe report a high seroprevalence of HCV infection in PWID. Social health disparities and potential missed opportunities validate the continuing call for local action for public health and prevention strategies. It is well documented that People Who Inject Drugs (PWID) are at a higher risk of acquiring various infections through blood, such as Human Immunodeficiency Virus (HIV) and Hepatitis C Virus (HCV) –3. ThereLike HIV, HCV in PWID is a public health concern with potential chronic severe sequelae if left unattended. Worldwide, around 5.5 million PWID live with Hepatitis C . In PR, The National HIV Behavioral Surveillance System (NHBS) has identified an increase in age among cohorts recruited as part of this surveillance system . HCV canAlthough the epidemiology of HIV and HCV among PWID has been extensively evaluated across different studies in PR , 15–19, NHBS is a serial cross-sectional survey that monitors the prevalence of HIV, sexual and drug-use behaviors, and HIV testing. The methodology used in the NHBS study has been described elsewhere , 21. We Participants signed informed consent before beginning the survey, which was read by an interviewer who also offered a copy to the participants for their record. Recruited participants completed an anonymous standardized face-to-face survey that lasted approximately 25 min with trained interviewers who noted responses using a handheld computer device with the Questionnaire Development System software . Both HIV and HCV tests were offered to those who completed the interview. The interview included questions about demographics, sexual practices, drug use, and HIV and HCV testing. The participant’s responses and test results were labeled with a survey number, removing identifying information.We used the following variables from the NHBS core questionnaire for our analysis : age, geTesting for HCV was done using the OraQuick® HCV Rapid Antibody Test  by following the manufacturer’s instructions. The test is an immunoassay for the qualitative detection of antibodies to the HCV (anti-HCV) and was performed through fingerstick and venipuncture of whole blood specimens from individuals 18 years or older. Antibodies in the blood reacting with these peptides and antigens are visualized by colloidal gold-labeled, generating a visible line in the test zone for a reactive sample. The PR-NHBS staff interpreted the result according to the manufacturer’s instructions 20 min later. The test has a sensitivity and specificity of 97.9% and 98.5%, respectively. In addition, for participants who tested positive on the rapid test, the RT-PCR Qualitative HCV RNA test was used as a confirmatory test with a sensitivity of 94.1%, positive predictive value of 72.7%, negative predictive value of 99.9% ; and speParticipants samples were tested using Alere Determine HIV-1/2 Ag/Ab combo assay , an immunochromatographic fingerstick test for the simultaneous and separate qualitative detection of free Human Immunodeficiency Virus Type 1 (HIV-1), p24 antigen, and antibodies to HIV-1 and HIV-2 in whole blood or plasma samples. The viral capsid protein p24 (core) present in HIV-1 and HIV-2 is detected in the blood before the HIV antibody during acute infection. The result was interpreted according to the manufacturer’s instructions between 20 and 30 min after the test. If the preliminary result was positive, we did the confirmatory test by another rapid test, INSTI® HIV-1/HIV-2 Antibody Test . INSTI® HIV-1/HIV-2 Antibody Test [p < 0.10) in the bivariate analysis. Variables such as speedball (mixture of cocaine and heroin) injection frequency and the participant’s knowledge of the HCV serostatus of the last person with whom they shared injection equipment were significant in the bivariate analysis but were excluded in the final model because both are conditioned variables. We used behavioral and clinical data from the core PR-NHBS interview to describe drug use by HCV serostatus. Statistical significance was set at p < 0.05. All analyses were conducted using STATA 17™.Participants identified as transgender (n = 4) were excluded from the sample due to the small sample size. Therefore, a total of 502 participants were included in our analysis. The data were weighted for HCV serostatus using the CDC software Respondent-driven Sampling Analysis Tool (RDSAT 8.1.45™). Descriptive analyses were stratified by HCV serostatus. HCV seroprevalence was calculated for sociodemographic, behavioral, and clinical characteristics. Chi-square and Fisher exact tests were used to assess statistical differences in the distribution of different characteristics and behaviors. A logistic regression analysis was used to assess the associations of selected variables that were statistically significant (p < 0.05) or tended to statistical significance (0.05 ≤ (data not shown).The sociodemographic, health-related, and drug consumption characteristics by HCV serostatus of the PWID are shown in Table The PWID weighted seroprevalence of HCV was 76.5%. Those PWID who reported being heterosexuals had a significantly higher HCV seroprevalence (78.5%). Those PWID who reported either being a high school graduate or having less than high school had a higher seroprevalence of HCV (81.3%), and 77.9%, respectively). The PWID who tested for STIs in the past 12 months had a significantly higher HCV seroprevalence (86.1%).Of those PWID who started injecting at or before 20 years of age, 80.9% were HCV positive. Among PWID who reported injecting speedball daily or more than daily, 79.4% were HCV positive Table . No stata = 2.23; 95% CI: 1.06–4.69). PWID tested for a sexually transmitted infection, such as gonorrhea, chlamydia, or syphilis, had higher odds of having HCV when compared to those that were not tested . Moreover, those PWID who started injecting at or before 20 years of age had higher odds of being HCV positive when compared to those that started injecting at 21 years or more ; however, the relative excess was not statistically significant (p > 0.05)  to a study of PWID in rural PR between April and June 2015 .4% to a . In addiPunto Fijo in the San Juan Metropolitan Area [Intercambios Puerto Rico in the Eastern region [El Punto en la Montaña in the Central Eastern region [We also report that despite HCV high-risk behavioral practices identified, the practice of sharing needles was lower compared to other injection equipment use, which suggests harm reduction awareness within this group. Harm reduction programs have helped reduce the negative effects or consequences of certain behaviors, particularly drug use . Locallytan Area , Intercan region  and El Pn region . These nn region .In addition to sharing injection equipment, the lack of accessible HCV treatment in PR during the study period may have contributed to the high HCV prevalence. Most participants reported a household income of less than $5,000, which is lower than the estimated median household income . Social After adjusting for behavioral and hepatitis-related factors, lower education was associated with a high risk of HCV compared with the highest educational attainment group, which was also reported in a population-based study in PR ; and a DWe report that testing for STIs in the past 12 months is positively associated with HCV which suggests that STI testing is a proxy for access to care, leading to HCV testing opportunities. Interventions by community-based organizations have helped identify HCV-positive cases since HIV/STI, and HCV testing opportunities or referrals to testing are made available to PWID , 44, 45.p < 0.001), heroin (p = 0.051), and painkillers (p = 0.064) and have lower education (p = 0.083) , which have been documented in other studies [Frequent speedball injectors had a higher HCV prevalence. Studies have found that injection of speedball was associated with HCV infection , 47. The studies , 50.We report that those who knew the HCV serostatus of the last sharing person had a higher HCV prevalence which could be explained by selecting higher risk partners based on concordant HCV serostatus . AwareneOur study does have several limitations. Firstly, our participants of PWID in PR is mostly adults, which is similar to national data , it is iWe have shown that the burden of HCV in the PWID population in PR is high which demonstrates a challenge in health management due to pressing problems related to socioeconomic determinants of health. Efforts to enhance HCV treatment availability and accessibility for PWID are needed. Furthermore, linkage to care, follow-up, and harm reduction programs are critical to reducing HCV infection among PWID which is an ongoing challenge in Puerto Rico.


The objective of this study was to develop a model combining ultrasound (US) and clinicopathological characteristics to predict the pathologic response to neoadjuvant chemotherapy (NACT) in human epidermal growth factor receptor 2 (HER2)-positive breast cancer.p < 0.1 were subjected to multivariate analysis and then the conventional US and clinicopathological characteristics independently associated with pathologic complete response (pCR) from the analysis were used to develop US models, clinicopathological models, and their combined models by the area under the receiver operating characteristic (ROC) curve (AUC), accuracy, sensitivity, and specificity to assess their predictive efficacy.This is a retrospective study that included 248 patients with HER2-positive breast cancer who underwent NACT from March 2018 to March 2022. US and clinicopathological characteristics were collected from all patients in this study, and characteristics obtained using univariate analysis at The combined model had an AUC of 0.808, a sensitivity of 88.72%, a specificity of 60.87%, and an accuracy of 75.81% in predicting pCR of HER2-positive breast cancer after NACT, which was significantly better than the clinicopathological model (AUC = 0.656) and the US model (AUC = 0.769). In addition, six characteristics were screened as independent predictors, namely the Clinical T stage, Clinical N stage, PR status, posterior acoustic, margin, and calcification.The conventional US combined with clinicopathological characteristics to construct a combined model has a good diagnostic effect in predicting pCR in HER2-positive breast cancer and is expected to be a useful tool to assist clinicians in effectively determining the efficacy of NACT in HER2-positive breast cancer patients. Breast cancer is the most prevalent cancer among women and is a heterogeneous solid tumor with complex genetic and molecular variations –3. ThereNACT is a long-term treatment process during which tumor changes occur owing to fibrosis, fragmentation, and/or necrosis, so imaging allows for non-invasive monitoring of treatment response during this time and serves to predict which patients will achieve pCR early in the course of treatment , 14. Thin = 156). ②Distant metastasis (n = 287). ③Multiple breast cancer of the unilateral and bilateral breast (n = 149). ④HER2 negative breast cancer (n = 488). Finally, a total of 248 HER2-positive patients were included in this study  with a high-resolution linear probe (ML6-15) and a Philips iU22 ultrasonic instrument  with a high-frequency linear probe (L12-5). In this study, considering that different doctors’ US reports may have different interpretations of the same characteristics, three doctors with more than 5 years of experience were selected to interpret the corresponding US maps stored in the database. If there were differences, an agreement was reached after discussion. ①  Routine For all patients, an US-guided puncture biopsy of the target breast tumor was performed and IHC indices were measured before NACT. We collected the estrogen receptor (ER), progesterone receptor (PR), HER2, and Ki67 index status of the pathology reports. ① The critical level of Ki67 is 30% . ② ER- aThe NACT regimen follows the National Comprehensive Cancer Network (NCCN) and China Anti-Cancer Association (CACA) guidelines. HER2-targeted therapy (trastuzumab in combination with or without pertuzumab) is added to chemotherapy for patients with HER2-positive breast cancer. Treatment cycles of 6 or 8 course of 21 days each.Clinicopathological information was obtained from the patient’s medical records. Clinical data included age, menopausal status, clinical TNM stage, NACT regimen, NACT cycle, and surgical approach. Histopathological findings included tumor type, ER status, PR status, HER2 status, Ki67 index, and pCR status.U test for comparisons between two groups. Categorical information is expressed as instances (%) and comparisons between two groups were made using the χ2 test or Fisher’s exact test. Statistical significance was defined as a two-tailed p < 0.05.For all statistical analyses, SPSS (version 25.0) and R (version 4.2.1) were used. Continuous variables were expressed as mean ± standard deviation (SD) or median M (interquartile range IQR), t test, or Mann–Whitney p < 0.1 in the univariate analysis were entered into a multivariate logistic regression analysis to determine independent correlations between the above characteristics and pCR. Subsequently, a combined model was developed by integrating conventional US and clinicopathological characteristics using multivariate logistic regression analysis. The area under a receiver operating characteristic (ROC) curve (AUC) was utilized to compare the predictive effect of the conventional US model, the clinicopathology model, and the combined model on pCR status. An AUC value greater than 0.8 is considered acceptable [All the clinicopathological and conventional US characteristics associated with pCR were first assessed using univariate analysis and then variables with ceptable .A total of 248 patients with HER2-positive breast cancer  who received the full NACT regimen were included in this study, 146 postmenopausal patients (58.9%) and 102 premenopausal patients (41.1%). All patients had pathologically confirmed invasive carcinoma, including 232 (93.5%) invasive ductal carcinoma (IDC) and 16 (6.5%) non-IDC. All patients underwent breast and axillary surgery after NACT, with 20 patients undergoing breast-conserving surgery and 228 mastectomies . Post-operative pathology showed a breast pCR rate of 53.6% (133/248) and a non-pCR rate of 46.4% (115/248) (Table p = 0.013), lower clinical N stage (p = 0.020), and negative PR compared to non-pCR (p = 0.010). In addition, the NACT regimen and Anti-HER2 therapy were significantly correlated with pCR status . There were no statistically significant differences between the two groups in terms of age, menopausal status, location of the tumor, histological type, and molecular subtype.For the univariate analysis of Clinicopathological characteristics Table , HER2-pop < 0.001), posterior acoustic (p < 0.001), calcification (p < 0.001), and elasticity score (p = 0.001) were significantly correlated with pCR. There was no significant difference between the two groups in terms of maximum diameter, echogenicity, shape, boundary, lateral shadow, and blood flow score.For the univariate analysis of breast US characteristics Table , tumor mFigure p < 0.05). Increasing Clinical T stage grade decreased the pCR rate ; increasing Clinical N stage grade decreased the pCR rate ; and PR-positive pCR rate decreased relative to PR negative (OR 0.45. 95% CI 0.23 to 0.87). Compared with non-spiculation, the pCR rate was lower for spiculation at the margin of the breast mass ; lower for posterior acoustic attenuation compared with non-attenuation ; and lower for the presence of calcification compared with absence of calcification . All of the above factors were hindering factors for pCR.Variables with p-values less than 0.1 in the univariate analysis were selectively included in the binary logistic regression for multifactorial analysis and the results were found Fig. . Clinicap < 0.001, DeLong’s test)  and the clinicopathological model (AUC of 0.656), respectively. To improve the predictive performance of the model, we integrated the US and clinicopathological characteristics to construct a regression model with an AUC of 0.808 for the combined model, which further demonstrated the great clinical application of the combined model we constructed in predicting the early outcome of NACT in HER2-positive breast cancer patients.HER2 is a transmembrane glycoprotein with receptor tyrosinase activity . In normIn the present study, the independent predictors of pCR in terms of clinicopathological characteristics were the Clinical T stage, Clinical N stage, and PR status, in line with many previous studies –34. The There are several limitations to this study. Firstly, the interpretation of breast US characteristics is largely influenced by the sonographer’s personal experience, and the US section images stored in the database may cause the characteristics of other US sections to be ignored; secondly, this study excluded masses that could not be accurately measured by the US, which may result in selective bias; finally, this study was a retrospective, single-center study with a small total sample size. Therefore, a multicenter prospective study with a larger sample size will be conducted in future to further improve and validate.In conclusion, US characteristics combined with clinicopathological indices have higher predictive performance than conventional US or clinicopathological indices alone for predicting the early efficacy of HER2-positive breast cancer after NACT. The combined model has improved AUC, sensitivity, specificity, and accuracy, suggesting that our model may provide important decision support for the clinical formulation of HER2-positive breast cancer treatment strategies.


Acidobacteria and Planctomycetaceae bacteria, respectively , and those in β-glucosidase, sucrase, and urease activities mainly controlled by fungi. The β-glucosidase and sucrase were both positively correlated with Herpotrichiellaceae, and urease was positively correlated with Sebacinaceae (p < 0.05). These findings contribute to the conservation and management of mountain ecosystems in the face of changing environmental conditions. Further research can delve into the specific interactions between microbial communities, soil properties, and vegetation to gain deeper insights into the intricate ecological dynamics within earthquake-prone mountain ecosystems.Patterns of microbial diversity on elevational gradients have been extensively studied, but little is known about those patterns during the restoration of earthquake-fractured alpine ecosystems. In this study, soil properties, soil enzyme activities, abundance and diversity of soil bacterial and fungal communities at four positions along a 2.6-km elevational gradient in the Snow Treasure Summit National Nature Reserve, located in Pingwu County, Southwest China. Although there were no significant changes in the soil chemical environment, bacterial and fungal communities were significantly different at different elevations. The overall fungal community presented an N-shaped diversity pattern with increasing elevation, while bacterial diversity decreased significantly with elevation. Changes in microbial diversity were associated with soil phosphorus, plant litter, and variations in dominant microbial taxa. Differences in enzyme activities among elevations were regulated by microbial communities, with changes in catalase and acid phosphatase activities mainly controlled by  Based on above advantage, extensive studies on biodiversity of higher organisms  demonstrate different patterns of richness with increasing elevation . HoweverAt present, climate, soil properties, vegetation, and historical impacts are the four main determinants of composition and diversity of soil microbial communities along elevational gradients . ClimatiThe Snow Treasure Summit National Nature Reserve in the Longmen Mountain seismic zone in Northwest Sichuan, China, is a nature reserve of wild creatures that mainly protects giant pandas and their habitats. The Longmen Mountain seismic zone is one of the strongest seismic zones in Sichuan. Since 1169 AD, there have been 26 destructive earthquakes, 20 of which measured 6 or higher on the Richter scale. The 8.0 magnitude Wenchuan earthquake and the 7.0 magnitude Ya’-an earthquake occurred in the Longmen Mountain seismic zone. Years of effects of geological activities on the soils and vegetation in the Snow Treasure Summit National Nature Reserve have seriously affected the survival of wild creatures. In this study, soil bacterial and fungal communities were measured at four positions along a 1.0-km elevational gradient in the Snow Treasure Summit National Nature Reserve. The gradient was used to test the following hypotheses: (i) soil bacterial and fungal communities will have unique elevational patterns of diversity, but different environmental drivers will be correlated with those patterns; and (ii) differences in bacterial communities will be the dominant factor regulating microbial enzyme activity along the elevational gradient. Testing those hypotheses will generate information to help to maintain the sustainability of ecosystems in the Snow Treasure Summit National Nature Reserve and will also provide a relevant theoretical basis for the restoration and reconstruction of damaged soil ecosystems in the reserve.2.2.1.The study was conducted in the Snow Treasure Summit National Nature Reserve , which is responsible for protecting the giant panda, golden monkey, and other rare wild animals and their habitats. The nature reserve, originally established in 1993, is in Sichuan Province, China, with a total area of 63,615 ha. Because the area is in the heart of the east slope of the Minshan Mountain system and has been protected by national and local governments for over 30 years, it is characterized by natural forest ecosystems with little human disturbance but disturbance from natural hazards. The study area is in the transition from subtropical mountain humid monsoon to cold climate zones in Northwest Sichuan. According to records of the Pingwu County meteorological station , mean annual maximum and minimum air temperatures are 37°C in July and −6.6°C in January, respectively. Mean annual precipitation is 1,300 mm. The dominant soil types are yellow and yellow-brown earths .Taxus baccata Linn (Taxaceae), T. chinensis (Taxaceae), Davidia involucrata Baill (Nyssaceae), and Metasequoia glyptostroboides Hu & W. C. Cheng (Taxodiaceae) are some rare tree species in the forest. Along the elevational gradient, the vegetation types range from evergreen broad-leaved forest , to mixed coniferous broad-leaved forest , to coniferous forests , to alpine irrigation meadow , and to the vegetation of limestone beach and snow covers   . The foc2.2.Soil samples were collected in July 2019. Four 20 m × 20 m permanent plots were systematically set up. Plots were roughly evenly distributed by elevation and collectively spanned 1026.19 m, from 1627.19 m to 2653.38 m a.s.l. Within each plot, six evenly distributed sampling points were set up, and six soil cores within each sampling point  were collected randomly and composited as a single sample. Visible plant roots and residues were removed before mixing. Soil samples were stored in plastic bags on ice. In the laboratory, samples were divided into two portions: one was air-dried and sieved through a 2-mm mesh screen for physicochemical analyses, and the other was stored at −80°C for DNA extraction and soil enzyme activity.2.3.Soil pH, soil organic carbon (SOC), total nitrogen (TN), total phosphorus (TP), total potassium (TK), available nitrogen (AN), available phosphorus (AP), and available potassium (AK) were measured using standard protocols following 4+-N produced per unit time and per unit dry soil. Sucrase was determined by a 3,5-dinitrosalicylic acid colorimetric method, and activity was expressed in terms of glucose production per unit time and per unit dry soil mass. Catalase was determined by potassium permanganate titration, and activity was indicated by the volume of 0.02 mol/L KMnO4 consumed by 1 g of dry soil filtrate. β-glucosidase was determined by nitrophenol colorimetry, and activity was expressed by the content of p-nitrophenol produced per unit time and per unit dry soil. Activity of soil extracellular acid phosphatase (ACP) was estimated by measuring the release of p-nitrophenol from p-nitrophenyl phosphate . The PCR was performed in a reaction mixture containing 12.5 μL of ABI Power SybrGreen qPCR Master Mix (Applied Biosystems), 0.5 μM each primer, 1 μL of 20 ng μL−1 template DNA, and sterile distilled water to make up a final volume of 25 μL. The qPCR program consisted of an initial denaturation at 95°C for 1 min and 30 cycles of 95°C for 15 s, 55°C for 30 s, and 72°C for 30 s. The PCR products were extracted from 2% agarose gel, further purified using an AxyPrep DNA Gel Extraction Kit , and quantified using QuantiFluor™-ST . Purified PCR products were paired-end sequenced (2 × 300 bp) on an Illumina MiSeq platform by Origingene Biotechnology Co., Ltd., Shanghai, China.The bacterial 16S rRNA gene was amplified using primers 338F/806R , and theRaw sequence data were processed using the QIIME v1.9 pipeline, where sequences were quality filtered, chimera-checked, OTU-clustered, and taxonomically annotated . Operati2.5.Differences in soil properties and enzyme activities were tested using a random permutation test . Principp < 0.1) between two contiguous sites along the elevational gradient. Partial Mantel tests were used to test the correlations between environmental variables and bacterial and fungal taxa.An OTU table was used for downstream alpha and beta analyses. Taxonomic alpha diversity of soil bacteria and fungi was estimated using OTU richness, Pielou’s evenness, and the Shannon–Wiener diversity index. To calculate taxonomic beta diversity, the Bray–Curtis index for abundance-weighted dissimilarity was used. Phylogenetic beta diversity was calculated using both weighted and unweighted UniFrac distance. To examine the elevational differences in compositional dissimilarities, principal coordinates analysis (PCoA) and permutational multivariate analysis of variance (PERMANOVA) were generated using “vegan” package in the R statistical software platform (v2.15.0). Differences in taxa among elevations were analyzed using linear discriminant analysis (LDA) effect size (LEfSe) analysisFor the predicted functional groups, a linear model of Spearman’s rank correlations was used to examine the relations between enzyme activities and relative abundances of bacterial and fungal groups, with relations visualized using the Cytoscape package v3.2.1 .3.3.1.p < 0.05; p < 0.05), and activity of ACP was negatively correlated with pH and positively correlated with SOC, TN, AP, and AK  and negatively correlated with TK and AK , and the Chao1 index was positively correlated with SOC  and AK (p < 0.05) were positively correlated with elevation, whereas pH and AP were negatively correlated with elevation . By contrast, differences in soil fungi were not significantly affected by elevation and soil properties (Pearson correlations indicated that TK (operties .3.4.Acidobacteria and Tepidisphaeraceae and the fungi Thelephoraceae and Chaetomiaceae (p < 0.001). The enzyme ACP was significantly positively correlated with the bacteria Planctomycetaceae and Isosphaera and the fungi Agaricales and Leotiomycetes (p < 0.01). Urease was significantly positively correlated with only the fungal family Sebacinaceae (p < 0.05). In addition, sucrase and β-glucosidase were both positively correlated with the fungal family Herpotrichiellaceae. Although catalase and ACP shared some functional genera, the functional genera did not have the same effects. For example, Gemmatimonadaceae, a family of bacteria, was significantly positively correlated with catalase and negatively correlated with ACP.Network analysis was used to determine the correlations between the top 50 genera of bacteria and fungi and activities of different enzymes . The net4.4.1.The change in SOC along the elevational gradient in the Snow Treasure Summit National Nature Reserve was not consistent with increases in SOC with elevation in previous study , which m4.2.Along the elevational gradient in the Snow Treasure Summit National Nature Reserve, fungal communities showed N-shaped changes in diversity indices. By contrast, in bacterial communities, the richness index increased with elevation and evenness and Shannon indices decreased. The different responses of soil bacterial and fungal community diversity are consistent with results in study , suggestNitrospirae with increasing elevation  can indicate soil nutrient status, with higher values indicating more nutrient-rich soils. Therefore, changes in relative abundances of bacterial phyla with elevation may be attributed to differences in ecological strategies  can be found in the article/TH proposed the study, designed the experiments, and participated in supervision. YW analyzed data and wrote the manuscript. XW and LM conducted experiments. XW and XY participated in field investigation. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (No. 32001232 and 32101363) and Open Fund Project of Key Laboratory of Ecological Safety and Protection of Sichuan Province (No. ESP1701).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.


During the COVID-19 pandemic, a great number of patients required Mechanical Ventilation (MV). Tracheostomy is the preferred procedure when difficult weaning is presented. Surgical techniques available for performing tracheostomy are open and percutaneous, with contradictory reports on the right choice. This paper aims to describe the clinical results after performing a tracheostomy in patients with COVID-19, regarding both surgical techniques.An observational, analytical study of a retrospective cohort was designed. All patients admitted to the Hospital Universitario Mayor Méderi, between March 2020 and April 2021 who presented COVID-19 requiring MV and who underwent tracheostomy were reviewed. Open versus percutaneous tracheostomy groups were compared and the primary outcome evaluated was in-hospital mortality.n = 73) of the patients and percutaneous tracheostomy in 35.4% (n = 40) with an in-hospital mortality of 65.7% (n = 48) and 25% (n = 10), respectively (p < 0.001). In a multivariate analysis, open tracheostomy technique [OR 9.45 (95% CI 3.20–27.92)], older age [OR 1.05 (95% CI 1.01–1.09)] and APACHE II score [OR 1.10 (95% CI 1.02–1.19)] were identified as independent risk factors for in-hospital mortality. Late tracheostomy (after 14 days) [OR 0.31 (95% CI 0.09–1.02)] and tracheostomy day PaO2/FiO2 [OR 1.10 (95% CI 1.02–1.19)] were not associated to in-hospital mortality.A total of 113 patients were included in the final analysis. The median age was 66.0 (IQR: 57.2 – 72.0) years old and 77 (68.14%) were male. Open tracheostomy was performed in 64.6% (Percutaneous tracheostomy was independently associated with lower in-hospital mortality and should be considered the first option to perform this type of surgery in patients with COVID-19 in extended MV or difficulty weaning. During the COVID-19 pandemic, before massive vaccination, up to 32% of hospitalized patients required Mechanical Ventilation (MV) . MV remoTracheostomy is recommended in stable patients with extended MV, as it reduces laryngotracheal stenosis, ventilator-associated pneumonia, and hospital stay length . Its advSurgical techniques for tracheostomy are open and percutaneous, with contradictory reports about the preferred choice –14. ThisAn observational, analytical study in a retrospective cohort was designed. All the electronic records of patients who were admitted to the Hospital Universitario Mayor Méderi, located in Bogotá, Colombia, between March 2020 and April 2021 who required MV, had COVID-19 confirmed by a polymerase chain reaction (PCR) positive result in a nasopharyngeal swab and who underwent tracheostomy were reviewed. This study was reviewed and approved by the Universidad del Rosario's Ethics Committee under number DVO005 2172-CV1656. We followed STROBE guidelines to report this study .All patients with PCR-confirmed COVID-19 on a nasopharyngeal swab who were managed with MV and underwent tracheostomy were included in the final analysis. All patients were managed under the institutional protocol based on the evidence available at each of the different moments of the pandemic. Patients under 18 years old and ventilated for a cause other than respiratory failure secondary to COVID-19 were excluded. All included patients received steroids during their hospital stay and protective mechanical ventilation guidelines were followed.2/FiO2  with which the patient was intubated and with which the tracheostomy was conducted; SOFA and APACHE II scores; number of intubation days before tracheostomy (considering early tracheostomy as the ones performed in under 14 days of MV); type of tracheostomy (open and percutaneous); days of hospital stay and days of intensive care unit (ICU) stay; tracheostomy-associated complications; reintervention and in-hospital mortality; pulmonary mechanics were assessed on the day of mechanical ventilation initiation, considered as day 0 of mechanical ventilation. The first measurement was taken within the first 6 h after orotracheal intubation, and the initial reported measurement occurred on day 4 of mechanical ventilation. These evaluations were conducted in patients receiving volume-controlled ventilation.The following variables were analyzed: patients’ demographics; body mass index (BMI); presence of arterial hypertension, diabetes mellitus, chronic obstructive pulmonary disease, chronic kidney disease, cardiovascular disease and malignancy; PaOThe decision of performing tracheostomy was made jointly by the intensive care physician and the surgeon, in which both clinical course and prognosis was evaluated previous to informed consent signing by the patient’s family. The technique of choice depended on the surgeon's expertise, resources, and the patient’s features such as short neck, obesity, and cervical extension capacity.Percutaneous tracheostomy was conducted next to the patient's bed in the ICU. In the case of open tracheostomy, this was performed in the operating room. Conscious sedation and neuromuscular relaxation were used in all cases. The staff involved in the procedure used the recommended personal protective equipment. The use of a fiberoptic bronchoscope is not routinely used in our institution during tracheostomy, however all procedures were successful. Early tracheostomy was considered when it was conducted during the first 13 days of MV and late tracheostomy when the time exceeded 14 days.A horizontal incision was made 2 fingers length distance above the sternal notch, and the subcutaneous tissue and platysma muscle were incised. Both the sternohyoid and sternothyroid muscles were separated from the midline. Dissection of the pre-tracheal fascia was continued, and the thyroid isthmus was superiorly displaced, in cases where this was not possible the isthmus was divided. Two lateral silk sutures were placed over the second or third tracheal ring, between which the trachea was opened. A tracheostomy tube was then inserted and connected to the ventilator. A thorough review of hemostasis was performed during the procedure .Performed using a Tracoe percutaneous tracheostomy device®. A 14-fr needle was inserted with its helix 2-finger lengths above the sternal notch to cannulate the trachea between the second and third tracheal rings. Appropriate placement was verified by aspirating air. The needle was then withdrawn, and the catheter was left in place, through which the guide was passed using the Seldinger technique. The catheter was removed, and a series of dilators were advanced over the guidewire until an appropriate dilatation size was achieved that allowed for passage of the tracheostomy cannula. After placing the tracheostomy cannula, the guidewire was removed, the balloon was inflated, and appropriate positioning was verified using capnography and respiratory noises. Once proper positioning was verified, the orotracheal tube was removed .The primary outcome evaluated was in-hospital mortality according to the surgical approach used. Secondary outcomes were ICU stay, hospital stay, decannulation , and complications according to tracheostomy technique.p value r < 0.05. A multivariate analysis was conducted concerning the outcome of in-hospital mortality, variables with a p < 0.25 in the marginal analysis were included in the model. Finally, using automatic selection, a backward stepwise regression model was applied, for which variables with a p < 0.1 were considered as independent variables associated to mortality. Potential interactions between the included variables in the final model were tested individually, one by one. Collinearity was evaluated by calculus of the variance inflation factor VIF (Variance Inflation Factor). For variables included in the final model, ORs with their respective 95% confidence intervals were reported. Univariate and bivariate analyses were conducted using SPSS®28, considering a statistically significant p < 0.05, the multiple regression model was conducted using RStudio version 4.1.A description of demographic, clinical, surgical, and outcome variables was made. The distribution was evaluated using the Shapiro–Wilk and Kolgomorov-Smirnov tests. Categorical variables were described as rates and continuous variables as medians with their corresponding interquartile range (IQR) or means and standard deviations according to normality. Bivariate analyses were conducted related to the primary outcome , technique used (open vs. percutaneous tracheostomy), and tracheostomy time (early versus late). The Chi-squared test was used in the case of categorical variables and the t-test or Mann–Whitney U-test  in the case of continuous variables to evaluate differences between the groups, considering a statistically significant difference as a A total of 113 patients were included in the study, the following flowchart . Table The median age was 66.0 (IQR: 57.2– 72.0) years and 77 patients 68.14%) were male. In-hospital mortality was 51.3%  in Table Regarding the employed surgical technique, patients who underwent percutaneous tracheostomy had higher decannulation and lower mortality rates; although they presented a greater hospital stay median time and no differences in ICU stay.Demographical and clinical characteristics and outcomes are shown according to moment of tracheostomy (open vs. percutaneous) in Table Patients who underwent early tracheostomy had a significantly shorter median hospital stay and ICU stay than patients who underwent tracheostomy after 14 days of MV. However, decannulation rates were lower in patients who underwent early tracheostomy and presented higher mortality rates.Figure 2/FiO2 [OR 1.10 (95% CI 1.02–1.19)] were not associated to in-hospital mortality ], age [OR 1.05 (95% CI 1.01–1.09)] and APACHE II score [OR 1.10 (95% CI 1.02–1.19)] were identified as independent risk factors for in-hospital mortality. Late tracheostomy (after 14 days of MV) [OR 0.31 (95% CI 0.09–1.02)] and tracheostomy day PaO.20–27.92One hundred thirteen patients were included in our study, where we found that overall mortality in patients who underwent tracheostomy was 51.3%. Factors associated with mortality were age, APACHE II score, and surgical approach. Age and APACHE II scores seem logically related to mortality as they evidence higher patient vulnerability, their relation to mortality has also been evidenced in multiple other pathologies.When comparing the mortality in this study to results reported in different systematic reviews on tracheostomy, ours presented higher rates than average. In a systemic review and the meta-analysis by Ferro, et al., a cumulative mortality of 19.23% (5% CI 15.2% – 23.6%) was reported ; in a syWhen comparing moment of tracheostomy (early versus late), a statistically significant difference was found in our study in favor of early tracheostomy in terms of shorter hospital stays and shorter ICU stays. In terms of mortality, the bivariate analysis showed a higher mortality for early tracheostomy, however, there were no statistically significant differences in the multivariate analysis. In a systematic review and meta-analysis performed by Ji, et al., a shorter ICU stay was evidenced in early tracheostomies, with no statistically significant differences between early and late tracheostomy regarding mortality . The dec2/FiO2 ratio with which procedures were conducted. Patients who underwent percutaneous tracheostomy had a higher PaO2/FiO2 ratio (162.0 IQR: 131.5 – 203.5) than patients who underwent open tracheostomy (147.0 IQR: 113.0 – 180.0), however this variable showed no statistically significant differences in the multivariate analysis. Another factor that could explain this finding is that patients selected for open tracheostomy had unfavorable anatomical structures, such as short neck and obesity considering the latter is also a risk factor for mortality due to Sars-CoV2 infection [When evaluating percutaneous versus open tracheostomy, open tracheostomy was found to be a factor associated with mortality; the reason for this difference could be a lower PaOnfection ; howevernfection . Furthernfection , suggestIn a pre-pandemic systematic review and meta-analysis by Iftikhar, et al., three different percutaneous approaches and open tracheostomy were compared reporting no statistically significant differences in major complications . AnotherIn a systematic review and meta-analysis by Ferro, et al., mortality between both surgical techniques was compared (percutaneous versus open) in patients with Sars-CoV2 infection, reporting no statistically significant differences (RR 1.96 95% CI 0.19 – 20.37); however, this study only included 4 studies for a total of 250 percutaneous tracheostomies and 172 open tracheostomies [This study recognizes some limitations such as using data from a single high complexity center exclusively, which limits the generalization of the results; its retrospective nature which may influence selection bias; being conducted on patients with COVID-19 only, which also does not allow to generalize these findings in patients using MV suffering from other etiologies; in addition, despite the use of steroids in all patients and the use of protective mechanical ventilation, the therapies used on all patients were not specifically analyzed (including the position of the patient).Percutaneous tracheostomy was independently associated with lower in-hospital mortality and should be considered as the first option for performing tracheostomy on COVID-19 positive patients in extended MV or with difficult weaning. The non-mobilization of the patient to the operating room, its performance at the patient's bedside, speed, and low rate of complications may explain these findings.


The indicators for metabolic syndrome also had significant coefficients of 0.20 (p = 0.673), 0.36 (p = 0.645) and 0.15 (p = 0.576), but they were not statistically significant. There were no significant relationships observed between the SNPs, dyslipidemia and metabolic syndrome. The SEM produced an acceptable model according to the fit indices. Genome-wide association studies (GWASs) have been used to discover genetic polymorphisms that affect cardiovascular diseases (CVDs). Structural equation modelling (SEM) has been identified as a robust multivariate analysis tool. However, there is a paucity of research that has conducted SEM in African populations. The purpose of this study was to create a model that may be used to examine the relationships between genetic polymorphisms and their respective cardiovascular risk (CVR) factors. The procedure involved three steps. Firstly, the creation of latent variables and the hypothesis model. Next, confirmatory factor analysis (CFA) to examine the relationships between the latent variables, SNPs, dyslipidemia and metabolic syndrome, with their respective indicators. Then finally, model fitting using JASP statistical software v.0.16.4.0. The indicators for the SNPs and dyslipidemia all indicated significant factor loadings, −0.96 to 0.91 ( Cardiovascular diseases (CVDs) are still the primary cause of premature mortality worldwide ,2. For tVarious factors have been reported to contribute to the development of CVDs. Evidence has shown that some of these factors are unique to an individual. Genetics has been considered as a factor that impacts the pathogenesis of CVDs because mutations alter the structure and quantity of the gene products, ultimately affecting the function of the gene products such as cardiovascular risk factors (CVR) . For exaAt molecular a level, GWASs have also been effectively applied to discover genetic polymorphisms that affect CVDs. The limitations of some of the techniques in GWASs are that only important SNPs that are related to specific diseases are reported, and there are concerns that these techniques might ultimately link the entire genome to disease susceptibilities . For thiSEM has been successfully used in a variety of fields such as psychiatry research  and geneThis study used a convenience sampling method of the elderly (n = 61) who attend a day care in a black peri-urban population of Sharpeville, Vaal region, South Africa. Fasting blood samples were collected on the same day (within 2 h) from the elderly participants who met the inclusion criteria of age 60 years or older and voluntary attendees of the Sharpeville day care centre, as well as signing the informed consent form for participation. Participants who were not able to provide substantial information to complete the consent process due to conditions such as dementia were excluded from this study.g for 1 min. The collection tube with the flow through was discarded. A total of 400 μL of the DNA pre-wash buffer was added to the same spin column in a new collection tube and centrifuged at ≥12,000× g for 1 min. The collection tube was then emptied. The mixture was washed in two steps by adding 700 μL and 200 μL g-DNA wash buffers to the spin column, respectively, with subsequent centrifugation at ≥12,000× g for 1 min. The collection tube was then emptied again after each wash. The spin column was transferred to a clean microcentrifuge tube and 50 μL DNA elution buffer was added directly on the matrix. This was followed by incubation for 5 min at room temperature, then centrifugation at maximum speed for 1 min to elute the DNA. The DNA concentration was measured by spectrophotometry using the NanoDrop® 2000  and DNA purity was determined by 1% agarose gel analysis using the A260/A280 ratio. A ratio of ~1.8 is generally accepted as “pure” for DNA.Genomic DNA was extracted from peripheral blood samples using the Quick-DNA™ Miniprep DNA purification kit  following the manufacturer’s instructions. Microtubes were placed onto a rack using a cool block. A total of 200 μL of each sample was added to a 1.5 mL microcentrifuge tube. Next, 200 μL of BioFluid & Cell Buffer (Red) and 20 μL of Proteinase K was then added to the same 1.5 mL microcentrifuge tube. The contents were mixed using a vortex for 15 s and then incubated at 55 °C for 10 min. One volume of the genomic binding buffer was added to the digested sample. The mixture was then mixed for another 15 s. The mixture was transferred to a Zymo-Spin™ IIC-XL Column in a collection tube. This was then followed by centrifugation at ≥12,000× ®Pre-designed SNP Genotyping Assay Kit . The 20 µL reaction mix consisted of: 1 µL template DNA (15 ng/µL), 10 µL TaqMan®Genotyping Master Mix (Cat. # 4371355), 1 µL probe (TaqMan®Pre-designed SNP Genotyping Assay), and 8 µL deionized water. The probe was diluted in Tris EDTA buffer (10 mM Tris–HCl (pH 8.0), 0.1 mM EDTA) (1:1) before the reaction. Polymerase Chain Reaction (PCR) was performed according to the manufacturer’s specifications using the Bio-Rad CFX Real-Time PCR System . The primers and probes were purchased form Inqaba Biotech  at concentrations of 10 μM. PCR amplification of the target loci involved amplifying each specific fragment of genomic DNA, which was then genotyped on the Agena MassARRAY  platform. A Multiplex PCR cocktail was then prepared according to the manufacturer’s instructions. The products were then measured using the MassARRAY Compact mass spectrometer and Agena real-time detection software . A no-template control was included in every PCR reaction to detect false positive reactions. Detection of rs675, rs699, rs247616, rs1968905, rs1801278, rs1805087, rs28362286 and rs67608943 was successful in the participants. Genotypes of the SNPs were also detected.Genotyping was outsourced to Inqaba Biotechnical Industries (Pty) Ltd., Pretoria, South Africa, which uses the Agena Bioscience, MassARRAY genotyping system, based on single base-extension or cleavage chemistry in conjunction with matrix-assisted laser desorption/ionization–time of flight (MALDI–TOF) mass spectrometry. Current advances in GWASs have enabled the identification of several SNPs that have been linked to human diseases including CVDs . The SNP® duo control monitor was wrapped firmly around the right arm wrist consistently to measure systolic and diastolic BP readings in duplicate on two different days at the same time by a Health Professions Council of South Africa (HPCSA)-registered nurse. For accuracy, the mean systolic and mean diastolic readings were then determined and recorded. Hypertension was specified according to the South African Hypertension Society (SAHS) guidelines at ≥140/90 mmHg.Blood pressure (BP) measurements were conducted using a previously reported method . The par® . The kit consisted of assay plates with micro-titre wells pre-coated with antibodies specific to PCSK9 . Reagent preparation included reconstitution of a wash buffer with distilled water, reconstituting a standard with sample diluent, and preparation of a detection reagent A and B working solution with their corresponding assay diluents . Washing steps were conducted with the W206—Microplate Washer . The final absorbance readings were conducted using a microplate reader . The plate was immediately read using the M201—ELISA microplate reader . These calibration standards were run in duplicate from which a standard curve was generated to determine serum PCSK9 levels.Serum was prepared from the vacutainer blood collection tubes by centrifugation of whole blood for 15 min at 4500 rpm. Aliquots of the serum were then stored in the laboratory at −20 °C until analysis. Serum apolipoprotein A-IV (Apo A-IV), apolipoprotein B (ApoB), high-density lipoprotein cholesterol (HDL-C), homocysteine (hcy), insulin, lipoprotein (a) (Lipo (a)), low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC) and triglyceride (TG) were measured quantitatively using standardized commercial kits  on a Konelab 20i Thermo Scientific autoanalyzer . The Konelab 20i Thermo Scientific autoanalyzer is a clinical chemistry analyzer that works on colorimetric and immunoturbidimetric principles. Lyophilised calibrator and quality control samples from the manufacturer were reconstituted before use and run before the serum samples to validate the tests. Proprotein convertase subtilisin/kexin type 9 (PCSK9) was assessed using an internationally standardized kit from EIAabIn this analysis, the SNP names rs1801278 (SP1), rs1805087 (SP2), rs1968905 (SP3), rs247616 (SP4), rs28362286 (SP5), rs675 (SP6) and rs699 (SP7) were denoted to enable shorter identification in the JASP statistical software v.0.16.4.0 for Windows . The preliminary analysis using a covariance matrix assay  showed that in our population only SP1 (IRS1), SP2 (MTHFR), SP3 (CETP), SP4 (CETP) and SP5 (PCSK9) were strongly correlated; for this reason, only these five were used as indicators for SNPs. Initially, we attempted to use the usual CVD risk indicators for an extended lipid profile )  and metaAll the raw data was cleaned and captured in Microsoft Office Excel . A conceptual framework was developed to indicate the possible relationships between the latent variables . SEM wasThe procedure involved three steps. Firstly, we created the latent variables and the hypothesis model. The variables were derived from the SNPs and CVR factors in our population. The SNPs for this study were selected based on their association to CVR in the literature; namely, rs675 (ApoA-IV), rs699 (AGT), rs247616 and rs1968905 (CETP), rs1801278 (IRS-1), rs1805087 (MTHFR), rs28362286 and rs67608943 (PCSK9). The CVR factors that were investigated are glucose, blood pressure (BP), apolipoprotein A-1 (ApoA1), apolipoprotein B (ApoB), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), insulin, lipoprotein (a) (Lipo(a)), low-density lipoprotein cholesterol (LDL-C), homocysteine (hcy), proprotein convertase subtilisin/kexin type 9 (PCSK9) and triglyceride (TG). This was then followed by confirmatory factor analysis (CFA) which was used to examine the relationship between the latent variables: SNPs, dyslipidemia and metabolic syndrome and their respective indicators (CVR). Once this was performed, the model was then fit using the JASP statistical software v.0.16.4.0 for Windows  using the lavaan (v. 0.6–1) package for R [Following the SEM, we estimated and validated the model using the chi-square test (χ2), root mean square error of approximation (RMSEA), the standardized root mean square residual (SRMR), comparative fit index (CFI), goodness-of-fit index (GFI) and Tucker–Lewis index (TLI). The chi-square test (χ2) measures a relationship between two categorical variables. Hence, a non-significant difference is ideal ,26,27. Tp = <0.001) and 0.92 to 0.96 (p ≤ 0.001), respectively. The indicators for metabolic syndrome also had significant coefficients of 0.20 (p = 0.673), 0.36 (p = 0.645) and 0.15 (p = 0.576), but they were not statistically significant.The factor loadings and standardized coefficients as shown in There were no significant relationships observed between the SNPs , dyslipidemia ) and metabolic syndrome . The findings of the SEM analysis that explored the relationships between the SNPs, dyslipidemia and metabolic syndrome are shown in p ≤ 0.001). Regarding the goodness-of-fit indices, RMSEA was 0.164 (90% CI: 0.126–0.203) suggesting a marginal fit. This is because we had a small population and RMSEA has been reported to be affected by sample size [The chi-square statistic for the model was 118.483 with 51 degrees of freedom . Several studies have shown a high prevalence of dyslipidemia in South Africa [The goal of our study was to develop and explore the relationships between genetic polymorphisms and cardiovascular risk factors in an elderly population from a peri-urban community. Studies have reported that rs1801278 , rs18050h Africa ,43,44. Fh Africa  reportedh Africa . In our h Africa ,47,48 haAccording to our hypothesized model, we expected to see an effect of the SNPs on dyslipidemia ,49 and mThere are, however, some conflicting results on the effect of genetic polymorphisms on the different markers of CVDs. This might be the other reason why there is inadequate information on the mechanisms and inheritance of these complex conditions. For example, there is a body of evidence that suggests that some genetic mutations or polymorphisms that occur in the genes of the lipid profile parameters ,52,53 anCVDs are a growing challenge with devastating outcomes. Studies have been conducted to try and understand these conditions but there is an urgent need to further investigate them at a molecular genetic level ,62. The We propose a model that may provide an opportunity to better understand the complex interaction of genetic polymorphisms in the CETP, IRS1, MTHFR and PCSK9 genes with the ApoB, BP, glucose, TC, LDL-C, Lipo (a) and PCSK9 levels in a black elderly population from Sharpeville in South Africa. Models such as this may enable early detection of CVDs, especially in asymptomatic individuals, allow precise treatment and lessen the mortality efforts by the department of health or policy makers . From published reports, it appears that the use of indices to assess the fitness of a model is still adaptable. While there is no benchmark, it is evident that the more fit indices are applied to a model, the better for its acceptance. Hu and Bentler  recommenThe novelty of the present study is that we developed a model to explain some of the mechanisms that link genetic polymorphisms with the risk factors of CVD (dyslipidemia and metabolic syndrome). Prior to this study, there were no reports that measured the pathways that relate the variables of dyslipidemia and metabolic syndrome in this elderly population. This study used SEM which enabled simultaneous analysis of a combination of different SNPs from different genes with an extended number of different CVR factors. Additionally, our findings supplement insights into an area with a paucity of information known as nutrigenomics. We recommend further studies with such models in diverse populations with larger samples to clarify and directly highlight the exact roles of genetic polymorphisms on the CVR factors. Additionally, we recommend potential interventions by the ministry of health and policy makers such as awareness of dyslipidemia and metabolic syndrome, regular screening of such conditions, promotion of physical activity and facilitation of access to treatment.


A total of 47 and 51 participants completed the Oxycodone Study and the Ketamine Study, respectively (Completer Population). In both studies, esmethadone doses ranging from therapeutic (25 mg) to 6 times therapeutic (150 mg) had a meaningful and statistically significantly (p < 0.001) lower Drug Liking VAS Emax compared with the positive control. Results were consistent for all secondary endpoints in both studies. In both studies, all doses of esmethadone were statistically equivalent to placebo on Drug Liking VAS Emax (p < 0.05). In the Ketamine Study, Drug Liking VAS Emax scores for esmethadone at all tested doses were significantly lower vs. dextromethorphan (p < 0.05) (exploratory endpoint). These studies indicate no meaningful abuse potential for esmethadone at all tested doses.Esmethadone (REL-1017) is the opioid-inactive dextro-isomer of methadone and a low-affinity, low-potency uncompetitive NMDA receptor antagonist. In a Phase 2, randomized, double-blind, placebo-controlled trial, esmethadone showed rapid, robust, and sustained antidepressant effects. Two studies were conducted to evaluate the abuse potential of esmethadone. Each study utilized a randomized, double-blind, active-, and placebo-controlled crossover design to assess esmethadone compared with oxycodone (Oxycodone Study) or ketamine (Ketamine Study) in healthy recreational drug users. Esmethadone 25 mg (proposed therapeutic daily dose), 75 mg (loading dose), and 150 mg (Maximum Tolerated Dose) were evaluated in each study. Positive controls were oral oxycodone 40 mg and intravenous ketamine 0.5 mg/kg infused over 40 min. The Ketamine study included oral dextromethorphan 300 mg as an exploratory comparator. The primary endpoint was maximum effect (E Major depressive disorder (MDD) is the second leading cause of disability and chronic disease burden in the United States, among all medical conditions . AccordiEsmethadone (REL-1017) is the opioid-inactive dextro-isomer of methadone and a low affinity, low potency uncompetitive NMDAR antagonist , 23. EsmPhase 1 and Phase 2 results with esmethadone provided safety, tolerability, and pharmacokinetic (PK) results across a range of doses sufficient to inform the design and conduct of the human abuse potential (HAP) studies , 35. PhaThese studies were conducted in accordance with relevant federal regulations of the Declaration of Helsinki, in compliance with the International Council for Harmonisation good clinical practice guidelines, and according to the appropriate regulatory requirements in the United States. Study protocols were reviewed and approved by a qualified Institutional Review Board. All participants signed the written informed consent prior to study procedures.max) of ≥65 points for the positive control and ≥15 point difference compared with placebo). Participants had to show an appropriate response following placebo administration, which was defined as scoring within the neutral range  and also acceptable neutral responses on other scales. If a participants showed a positive response on Drug Liking following placebo (>60), they were excluded. In the Treatment Phase, eligible participants were randomized to receive each of the planned study drugs in a crossover manner. Study drug administration in each treatment period was separated by a minimum washout interval of 11 days. While there is always the potential for functional unblinding, double-and triple-dummy procedures and multiple treatment sequences were put in place to mitigate this risk. The ketamine study included two comparators, further decreasing the potential for functional unblinding.Each study utilized a single-dose, randomized, double-blind, active- and placebo-controlled crossover design to assess the abuse potential of esmethadone compared with oxycodone (Study 1—Oxycodone Study) or ketamine (Study 2—Ketamine Study) in healthy recreational drug users. All study drug administration and assessments were conducted in an inpatient setting. The overall design was consistent with FDA guidelines for assessing HAP . Each st2, were healthy according to physical examination, medical history, vital signs, clinical laboratory assessments, and 12-lead electrocardiogram (ECG); and had negative urine drug screens and pregnancy tests  at each visit. Participants with a history/presence of drug or alcohol dependence or psychiatric disorder, according to the Diagnostic and Statistical Manual of Mental Disorders  or who had ever participated in a substance or alcohol rehabilitation program were excluded. This was defined as any psychiatric disease that was anticipated in the Investigator’s or Medical Monitor’s opinion to be clinically significant and to potentially compromise safety or adversely affect the evaluation of the study data. No participant was excluded in either study for failing to satisfy this inclusion/exclusion criteria. Other exclusion criteria included corrected QT interval by Fridericia (QTcF) > 450 ms, pregnancy or breastfeeding , and allergy to NMDAR antagonists or related drugs or allergy to opioids. Concomitant medications  were prohibited during the study.Participants were healthy individuals 18 to 55 years of age, inclusive, who were experienced with nontherapeutic  drug use. In the Oxycodone Study (Study 1), participants had prior experience with recreational opioid use (defined as ≥10 lifetime occasions of use and ≥1 use in the 12 weeks prior to Screening). In the Ketamine Study (Study 2), participants had prior experience with NMDAR antagonists , and had specifically used ketamine ≥1 in their lifetime and had ≥1 use of drugs for nonmedical purposes by either the intranasal or IV route in the past year. Participants were recruited separately for each study and were compensated according to IRB-approved parameters. In addition, participants had a body mass index (BMI) ranging from 18 to 35 kg/mEsmethadone doses of 25 mg, 75 mg  , adminisOxycodone, a Schedule II opioid, was selected as the positive control in the Oxycodone Study (Study 1). The dose of oxycodone  was consistent with those previously evaluated in HAP studies .Ketamine, a Schedule III NMDAR antagonist, was selected as the positive control in the Ketamine Study (Study 2). The dose and infusion duration of IV ketamine (0.5 mg/kg over 40 min) was consistent with a previous HAP study (CDER review of NDA 211243 [esketamine]). DXM, an unscheduled, over-the-counter, NMDAR antagonist, and antitussive drug, was selected as an exploratory comparator in the Ketamine Study. The oral dose of DXM (300 mg capsule) was based on a prior single ascending dose study showing that this dose had detectable subjective effects but was not associated with prominent emesis that was more commonly observed at higher supratherapeutic doses of 400 to 800 mg .To ensure blinding, study drugs were administered in a double- (Oxycodone Study) or triple- (Ketamine Study) dummy fashion, where participants received the same number of tablets and capsules (and a 40-min IV infusion in the Ketamine Study) administered as a combination of active or placebo product in each treatment period.max of “at-the-moment” Drug Liking VAS was defined as the primary endpoint. Unipolar “at-the-moment” 100-point VAS were used to measure positive , negative (Bad Effects), and other  subjective drug effects. In the Ketamine Study, perceptual/dissociative effects were assessed using “at-the-moment” 100-point Hallucinations and Bowdle VAS  ) ) , 53–55. tamine]) . The tesmax), area under/over the effect curve) were initially analyzed using a linear mixed effects model containing treatment, period, sequence, and first-order carryover effect as fixed effects . The participant nested-within-treatment sequence was included as a random effect. Baseline was also included as a covariate where applicable . The first-order carryover effect was the previous treatment received in the Treatment Phase. If the carryover effect was found to be nonsignificant at the 25% level, then the term was dropped from the model [p value of the test was ≥0.01. If the p value was <0.01 for the Shapiro–Wilk W test on the residuals from the mixed model, a test of skewness was conducted on each paired difference. If the distribution of the paired differences was not skewed , then the endpoint was analyzed using a paired t-test. If the distribution of the paired differences was skewed , then the endpoint was analyzed nonparametrically using the Sign Test. Based on assumptions, derived from prior studies [Pharmacodynamic endpoints  for each analyte were calculated using non-compartmental analysis  for the PK Population, which included all participants who received at least 1 dose of active study drug and had at least 1 measurable PK sample for the respective treatment. Derived parameters were summarized descriptively. All safety analyses were summarized descriptively using the Safety Population, which included all participants who received at least 1 dose of study drug in the Treatment Phase.Pharmacokinetic (PK) parameters  to the Treatment Phase, and 47 completed all treatment periods and were included in the Completer Population. Six participants discontinued early: 5 were lost to follow-up , and 1 was discontinued due to noncompliance or major protocol violation (repeated visit cancellation/no show). In the Ketamine Study (Study 2), 54 participants were randomized at two clinical sites  to the Treatment Phase, and 51 completed all treatment periods and were included in the Completer Population. Three participants discontinued early: 1 withdrew consent, 1 discontinued for safety reasons , and 1 was discontinued for administrative reasons.In the Oxycodone Study (Study 1), 50 participants were randomized at two clinical sites  are shown in Fig. max between the positive control, oxycodone 40 mg, and placebo. The median  difference was 35.0  for the Completer Population, indicating that oxycodone had a meaningful and statistically significantly higher Drug Liking VAS Emax compared with placebo, using a prespecified margin of 15  (Tables max of each dose with placebo. The median difference from placebo was 0 for esmethadone 25 mg and 75 mg (p < 0.001) and 7.0 for the 150 mg dose (p = 0.036), indicating that esmethadone was not meaningfully different from placebo and was statistically equivalent to placebo at doses up to 6 times the planned therapeutic dose and MTD (Tables Effects on Drug Liking VAS E5 Tables  and 3. T) Tables  and 3. Tp < 0.001).Consistent with the primary endpoint, oxycodone had greater effects compared with placebo on all secondary endpoints, including global effects , positive effects , and other effects  and all doses of esmethadone had lower effects on all secondary endpoints compared with oxycodone Table . Based omax (primary endpoint) are shown in Fig. max between the positive control, ketamine 0.5 mg/kg administered IV over 40 min, and placebo (Table p < 0.001) for the Completer Population, indicating that ketamine had a meaningful and statistically significantly higher Drug Liking VAS Emax compared with placebo, using a prespecified margin of 15. The abuse potential of esmethadone relative to ketamine was determined from the comparison of Drug Liking VAS Emax of each esmethadone dose with ketamine. Drug Liking VAS Emax for each esmethadone dose was meaningfully and statistically significantly lower than that of ketamine at a prespecified margin of 0  , indicating that esmethadone was equivalent to placebo at doses up to 6 times the planned therapeutic dose. In the exploratory comparisons with DXM, Drug Liking VAS Emax for DXM 300 mg was meaningfully and significantly lower than the Drug Liking VAS Emax for ketamine . DXM was found not to be equivalent to placebo in Drug Liking VAS Emax using the margin of 11  (Table Effects on Drug Liking VAS E1) Table . The abumax scores for Hallucinations VAS were 0.2 (0.55); 0.3 (0.77); 0.6 (0.24) points for esmethadone 25 mg; esmethadone 75 mg; esmethadone 150 mg, respectively; 23.1 (38.5) points for ketamine; 7.5 (19.21) points for DXM; and 0.2 (0.45) points for placebo. Based on Drug Similarity VAS, ketamine was rated to be most similar to the category of Ketamine  and less so with Opioids . Participants did not perceive esmethadone as similar to Ketamine or Opioids, with mean scores <20 and median scores of 0.Ketamine had greater effects compared with placebo on all secondary endpoints Table . All dosPharmacodynamic analyses were also performed for the Modified Completer Population. The results for the Modified Completer Population for the Oxycodone Study and for the Ketamine Study are presented in Supplemental Tables max and AUC0-last) increased with increasing dose of esmethadone. Median Tmax ranged from approximately 2 to 3 h post dose for all esmethadone doses in both studies, whereas Tmax for oxycodone occurred at approximately 1 h post dose  of treatment-emergent AEs (TEAEs) was observed with oxycodone 40 mg (52.1%), followed by esmethadone 150 mg 28.6%) , while a similar incidence of TEAEs occurred with esmethadone 75 mg (41.5%) and 150 mg (39.5%) . In both HAP studies, all three esmethadone doses  had meaningful and statistically significant lower scores compared with positive control (oxycodone or ketamine) on the primary endpoint of Drug Liking VAS E effects , indicat effects . While p effects .We presented results for the Completer Population as indicated in the FDA 2017 Guidance  Tables  to 6 andIn summary, the results of these two, randomized, double-blind, active- and placebo-controlled, crossover studies testing the abuse potential of esmethadone in recreational drug users are consistent with the profile of a drug without meaningful abuse potential and are similar to results observed for unscheduled drugs without abuse potential such as eslicarbazepine and difelikefalin , 63 and Considering that MDD is a highly prevalent and life-threatening condition and available treatments have limited and delayed efficacy and metabolic and neurological side effects, an unmet need exists for safe, well-tolerated, and rapidly effective antidepressants. Patients in need should not have restricted or delayed access to potentially safe, well-tolerated, and life-saving drugs .In conclusion, the results of these two HAP studies, in accordance with results in preclinical models , 59, indSupplemental Material


Currently, over 200 research publications have examined different aspects of cytoglobin structure, redox chemistry and potential roles in cell signalling pathways. This research is wide ranging, but common themes have emerged throughout the research. This review examines the current structural, biochemical and in vivo knowledge of cytoglobin published over the past two decades. Radical scavenging, nitric oxide homeostasis, lipid binding and oxidation and the role of an intramolecular disulfide bond on the redox chemistry are examined, together with aspects and roles for Cygb in cancer progression and liver fibrosis.Since its discovery in 2001, the function of cytoglobin has remained elusive. Through extensive  Cytoglobin (Cygb) is the fourth member of the globin family found in humans after erythrocyte haemoglobin (Hb), myoglobin (Mb) and neuroglobin (Ngb). Discovered in 2001 and initially named stellate cell activating protein (STAP) . The namPhylogenetic analysis of Cygb suggests a common ancestor with vertebrate Mb ∼500 Mya  and is ain vitro depending on the redox state of two cysteines, Cys38(B2) and Cys83(E9). Various crystal structures show the protein in either a monomeric state with the cysteines reduced (monomerSH) or mutated (e.g. PDB:1VH5 [S–S) (e.g. PDB: 2DC3 [S–S).The structure of cytoglobin shows the typical 3-over-3 alpha helical fold of non-truncated globins . The hemPDB:1VH5  Figure 1DB: 2DC3 , Figure 2DC3 [S–S conformation. The Cygb-1 from teleost fish are typically truncated, having lost the C-terminal intron. The N-terminal extension is absent from zebrafish Cygb-2, possibly by a mutation in the lineage of D. rerio [Cygb has extended N- and C-terminal extensions beyond the typical alpha helical fold of the globular part of the protein with each extension ∼20 amino acids long, making the human protein 190 amino acids in length and a molecular weight of ∼21 kDa . Crystal2DC3 ). Trunca2DC3 . TruncatD. rerio . Globin D. rerio . HoweverD. rerio .in vivo. This is a critical question to be answered as the biochemical and biophysical properties of the monomerS–S form behaves very differently from the other two states. The protein monomerS–S form has a distal histidine off rate over 600-fold faster than the monomerSH or dimerS–S, with only slight changes to the distal histidine on rate [S–S state the protein has more pentacoordinate-like features, as exhibited by faster rates of exogenous ligand binding and higher affinities [in vivo.There has been considerable debate over the oligomeric state of the protein  on rate . Therefofinities . Allostefinities ,33. Initfinities . Howeverfinities . It is ifinities . It has finities . Analysifinities . Therefo2 homeostasis, with disruption of O2 homeostasis activating HSCs in liver tissues inducing hepatic fibrogenesis [Cygb shows several activities that could have potential physiological or pathological roles. Cygb binds oxygen with an affinity of 0.2–3 Torr, depending on the conformation of the protein and the oxidation state of the disulfide bond ,38. Thisogenesis . Howeverogenesis . Additioogenesis .7 M−1 s−1 for this reactivity [in vitro and in vivo studies suggest a physiological or pathological role for this redox chemistry (currently 39 articles). Such studies show that an enhanced expression of Cygb in fibroblasts resulted in decreased NO consumption and intracellular nitrate production [As with all globins, oxyferrous Cygb shows rapid nitric oxide dioxygenase (NOD) activity, converting NO to nitrate with the oxidation of the Cygb from ferrous to ferric. A rate constant of 3 × 10activity  is only activity . Numerouoduction . Furtheroduction . The cysoduction . In Cygboduction .−1 s−1 for Hb (T state) and Ngb [−1 s−1 for globin X [−1 for the monomerS-H and dimerS–S, respectively, to 32.3 M−1 s−1 for the monomerS–S [Under hypoxic conditions, Cygb also shows a high nitrite reductase (NiR) activity, generating NO from nitrite. Typical NiR rate constants for globins range from 0.12 M and Ngb ,49 to 26globin X . Cygb shnomerS–S . Thus winomerS–S . Ligand nomerS–S . It shounomerS–S . SeveralnomerS–S , attenuanomerS–S  and corrnomerS–S .in vivo. In vitro ferric Cygb reacts rapidly with ascorbate, two orders of magnitude faster than pentacoordinate globins such as Mb and Hb [in vivo reduction systems of Cygb endorses redox activity as a physiological role, such NO metabolism [For such redox activities to be physiologically relevant there needs to be an effective reducing mechanism to return the high oxidation states of Cygb to ferrous b and Hb . In addib and Hb . The raptabolism .The link between Cygb and cancer progression, particularly with hypoxic tumours, is well established from numerous studies on a variety of cancer types. The cellular pathways involving Cygb in cancer suppression or promotion are still unclear. However, up-regulation of the NFkappaB/iNOS signal pathway and NO production has been observed in human cardiac stem/progenitor cells with up-regulated Cygb, suggesting Cygb functions as a pro-survival factor in response to oxidative stress . FurtherCygb, originally discovered in hepatocytes as STAP, is up-regulated in hepatic fibrosis. Cygb overexpression reduces tissue fibrosis in both toxic and cholestatic models of liver injury and promotes recovery after the onset of injury . Carbon S–S form exhibits this conformational change and only in the ferric oxidation state [sn-glycero-3-phosphate) and PIP3  predicts the aliphatic chain extending into the hydrophobic core of the protein, with phosphate groups interacting with surface amino acids such as Lys111 and Lys116 and the ester groups interacting with Arg84 and Thr91 [Unusual amongst vertebrate globins, Cygb interacts with lipids and fatty acids resulting in marked changes to the heme geometry and redox activity . Only thon state . Interacon state  with sucon state . Similaron state . Currentnd Thr91 . Mutationd Thr91 .in vitro, with the monomerS–S form having significantly higher levels of activity compared with the monomerSH and dimerS–S states [It has been demonstrated that Cygb is also an effective lipid peroxidase S states . AnionicS states . These lS states . Both MbS states  or acuteS states . The mecS states ,111 or aS states . For morS states .in vitro observations of prevalent lipid oxidation with cell responses may be explained by the observation that lipid peroxidation, either through enzymatic or non-enzymatic pathways, results in a conversion of the saturated fatty acid to an oxylipidome. Many members of this oxylipidome are electrophilic and can modulate antioxidant and anti-inflammatory protective pathways [in vivo [It has been observed that Cygb overexpression in rat hepatic stellate cells decreases lipid radicals as shown by decreased levels of lipid peroxidation biomarkers malondialdehyde and 4-hydroxy-2-nonenal . In a THpathways . Hence, pathways ,119. The[in vivo ,102. How[in vivo . The datin vivo. Cygb-induced protection against cancer proliferation and hepatic inflammation and fibrinolysis gives us the clearest indication of the role of Cygb in cells, however, the biochemical mechanisms of Cygb in these conditions does not yet have consensus. Increasing bodies of evidence support the redox role of Cygb, be it related to NO homeostasis, ROS scavenging, inhibition of DNA damage or lipid oxidation, it is likely to play a role in the activation of pathways that facilitate cell protection  of cell protection.in vivo are crucial for elucidating the mechanisms and pathways that Cygb induces cell protection. Furthermore, a clearer understanding of the molecular mechanisms of Cygb and pathways that Cygb induces would clarify the potential for Cygb as a biomarker or novel target for therapeutic intervention for cancer and other diseases.Understanding the roles of the redox chemistry of Cygb and the effect of the disulfide bond as a potential molecular redox switch