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"The clinical data for all patients were summarized in Additional file 1: Table S1. Relative BANCR expression in NSCLC tissues and its clinical significance. (A) Relative expression of BANCR in NSCLC tissues (n?=?113) compared with corresponding non-tumor tissues (n?=?113). BANCR expression was examined by qPCR and normalized to GAPDH expression. Results were presented as the fold-change in tumor tissues relative to normal tissues. (B) BANCR expression was classified into two groups. (C D) KaplanMeier disease-free survival and overall survival curves according to BANCR expression levels. *P?<?0.05 **P?<?0.01. Correlation between BANCR expression and clinicopathological characteristics of NSCLC patients (n?=?113) Characteristics BANCR P High no. cases (%) Low no. cases (%) Chi-squared test P-value Age(years) 0.616 ?65 29(54.7) 30(50.0) >65 24(45.3) 30(50.0) Gender 0.232 Male 35(66.0) 33(55.0) Female 18(34.0) 27(45.0) Histological subtype 0.466 Squamous cell carcinoma 30(56.6) 38(63.3) Adenocarcinoma 23(43.4) 22(36.7) TNM Stage <0.001* Ia + Ib 25(47.2) 9(15.0) IIa + IIb 17(32.1) 21(35.0) IIIa 11(20.7) 30(50.0) Tumor size 0.001* ?5cm 35(66.0) 21(35.0) >5cm 18(34.0) 39(65.0) Lymph node metastasis 0.001* Negative 34(64.2) 20(33.3) Positive 19(35.8) 40(66.7) Smoking History 0.127 Smokers 39(64.2) 36(60.0) Never Smokers 14(35.8) 24(40.0) * Overall P?<?0.05. Association of BANCR expression with patients survival Kaplan-Meier survival analysis was conducted to investigate the correlation between BANCR expression and NSCLC patient prognosis. According to relative BANCR expression in tumor tissues the 113 NSCLC patients were classified into two groups: the high BANCR group (n?=?53 fold-change???4); and the low BANCR group (n?=?60 fold-change ?4) (B). With respect to progression-free survival (PFS) this was 35.3% for the high BANCR group and 17.2% for the low BANCR group. Median survival time for the high BANCR group was 31 months and 16 months for the low BANCR group (C). The overall survival rate over 3 years for the high BANCR group was 46% and 27.5% for the low BANCR group. Median survival time for the high BANCR group was 32 months and 18 months for the low BANCR group (D). Univariate analysis identified three prognostic factors: lymph node metastasis; TNM stage; and BANCR expression level. Other clinicopathological features such as gender and age were not statistically significant prognosis factors (). Multivariate analysis of the three prognosis factors confirmed that a low BANCR expression level was an independent predictor of poor survival for NSCLC (p?=?0.031) in addition to TNM stage (p?=?0.038) (). Univariate and multivariate analysis of overall survival in NSCLC patients (n?=?113) Variables Univariate analysis Multivariate analysis HR 95% CI p value HR 95% CI p value Age 1.257 0.712-2.219 0.431 Gender 1.185 0.670-2.098 0.559 Smoker 1.120 0.842-1.491 0.436 Histological subtype 0.982 0.738-1.307 0.902 Chemotherapy 0.787 0.587-1.055 0.110 Tumor size 1.233 0.926-1.640 0.151 Lymph node metastasis 0.424 0.235-0.764 0.004* 0.577 0.311-1.071 0.081 TNM stage (I vs. II or IIIa) 0.320 0.149-0.685 0. 003* 0.431 0.195-0.954 0.038* BANCR expression 0.367 0.201-0.669 0. 001* 0.496 0.262-0.938 0.031* HR hazard ratio; 95 % CI 95 % confidence interval * Overall P?<?0.05. Histone deacetylation is involved in the downregulation of BANCR Expression levels of BANCR in NSCLC cell lines were determined by qPCR. Compared with that in 16HBE cells relative expression levels of BANCR were reduced in NSCLC cells (A). Because of the different expression patterns for BANCR in NSCLC and melanomas we investigated the mechanisms controlling tissue-specific expression of BANCR. We analyzed the promoter region of BANCR and found there were no CpG islands (data not shown). Histone protein modification was thought to play an important role in the transcription of lncRNAs; however knockdown of two core subunits of polycomb repressive complex 2 (SUZ12 and EZH2) had no influence on BANCR expression (Additional file 2: Figure S1A). Histone deacetylation is involved in BANCR downregulation. (A) BANCR expression levels of NSCLC cell lines (A549 SPC-A1 H1299 H1650 H1975 and SK-MES-1) compared with that in normal human bronchial epithelial cells (16HBE). (B) qPCR analysis of BANCR expression levels following the treatment of SPC-A1 and A549 cells with TSA. (C) qPCR analysis of HDAC2 and HDAC3 expression levels following the treatment of SPC-A1 and A549 cells with si-HDAC2 or si-HDAC3.(D E) qPCR analysis of BANCR expression levels following the treatment of SPC-A1 and A549 cells with si-HDAC1 and si-HDAC3. We observed that BANCR expression was upregulated in SPC-A1 and A549 cells following treatment with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) (B). We sought to determine whether repression of BANCR was mediated by HDACs. Specific anti-HDAC1 and HDAC3 siRNAs were transfected into NSCLC cells and HDAC1 and HDAC3 expression was significantly decreased (C). Expression levels of BANCR were significantly upregulated in cells transfected with si-HDAC3. Transfection with the scrambled siRNA or si-HDAC1 did not induce BANCR expression (D and E). Moreover the HDAC3 expression was upregulated in NSCLC cells and negatively correlated with BANCR expression (Additional file 2: Figure S1B). Furthermore NSCLC cells were treated with RGFP966 which is an seletive inhibitor for HDAC3 with an IC50 of 0.08?M and no effective inhibition of other HDACs at concentrations up to 15?M. The results of qPCR showed that the expression of BANCR was upregulated in NSCLC cells after treated with RGFP966 when compared with control cells (Additional file 2: Figure S1C). " | 1 |
"objectives to describe the benefits and limitations of using individual and combinations of linked english electronic health data to identify incident cancersdesign and setting our descriptive study uses linked english clinical practice research datalink primary care cancer registration hospitalisation and death registration dataparticipants and measures we implemented case definitions to identify first site specific cancers at the most common sites based on the first ever cancer diagnosis recorded in each individual or commonly used combination of data sources between and we calculated positive predictive values and sensitivities of each definition compared with a gold standard algorithm that used information from all linked data sets to identify first cancers we described completeness of grade and stage information in the cancer registration data setresults gold standard cancers were identified positive predictive values of all case definitions were ¥ and ¥ for the four most common cancers breast lung colorectal and prostate sensitivity for case definitions that used cancer registration alone or in combination was ¥ for the four most common cancers and ¥ across all cancer sites except bladder cancer using cancer registration alone for case definitions using linked primary care hospitalisation and death registration data sensitivity was ¥ for the four most common cancers and ¥ for all cancer sites except kidney oral cavity and ovarian cancer when primary care or hospitalisation data were used alone sensitivities were generally lower and diagnosis dates were delayed completeness of staging data in cancer registration data was high from minimum in and in for the four most common cancerss ascertainment of incident cancers was good when using cancer registration data alone or in combination with other data sets and for the majority strengths and limitations of this study º this is the first study to present comprehensive information on the implications of using different individual and combinations of linked electronic health data sources in england to identify cases of the most common incident cancers º using a gold standard algorithm that combined all available data from multiple sources as a comparator we were able to estimate both positive predictive values and sensitivity values for a range of pragmatic case definitions º we described similarities and differences in values between age groups sexes and calendar years the impact of choice of sources on diagnosis dates and mortality rates and completeness of stage and grade in cancer registration data º a key limitation was that our gold standard algorithm is not validated and may be affected by differences in clinical diagnosis and coding of invasive cancers between data sourcesof cancers when using a combination of primary care hospitalisation and death registration dataintroductionthe clinical practice research datalink cprd provides de identified primary care data linked to additional secondary health data sources under a well governed framework1 use of linked data helps researchers to answer more epidemiological questions and increase study quality through improved exposure outcome and covariate classification2 in the field of cancer epidemiology cprd primary care data linked to hospital episode statistics admitted patient care strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access data hes apc office of national statistics ons mortality and national cancer registration and analysis service ncras cancer registration data are used to analyse factors contributing to the risk of cancer and the consequences of cancer and its treatment use of linked data reduces the sample to the common source population and data coverage period for each included data set and has cost and logistical implications which are greatest for ncras data research teams therefore commonly choose not to use all available linked data3 cancer epidemiology studies can also be conducted using ncras and hes apc data provided by national health service nhs digital and public health england phe without linkage to cprd primary care data4 this provides national coverage at the expense of the detailed health data that are available in primary care recordsvalidation studies assessing concordance between cprd gold hes apc and ncras data have estimated high positive predictive values ppvs for cprd gold data and varying proportions of registered cancers that are not captured in cprd gold and hes apc5 the most up to date analysis by arhi et al included the five most common cancers and all papers focussed on concordance between cprd gold only and ncras existing evidence therefore does not provide a complete assessment of the benefits and limitations of using different combinations of data sources within the context of practical study designs national data are available describing completeness of data fields within the cancer registry data in each collection year9 and over time for all cancers combined4 missingness for individual years has been associated with age comorbidities and clinical commissioning groups10 we aim to describe and compare the benefits and limitations of using different combinations of linked cprd primary care data hes apc ons mortality and ncras cancer registration data for conducting cancer epidemiology studies our analyses focus on incident cancer ascertainment as it is a common and important outcome in cancer epidemiology and it is more difficult to distinguish between secondary recurrent and primary cancers at a second site in these data sets we have compared definitions of the most common cancers based on the first ever cancer recorded in individual or combinations of data sets with a gold standard definition comparing information from all four data sets we also describe the availability of stage grade and treatment variables over time in the cancer registration data for the cprd linked cohort this reflects real life study design and will help researchers to decide which combination of data sources to use for future studiesmethodsstudy design and settingwe completed a concordance study using linked2 english cprd gold hes apc ons mortality and ncras data cprd gold data were extracted from the january monthly release and the 13th update to cprds linked data the study period was from january to december with december matching the end of the ncras coverage periodthe cprd gold database includes de identified records from participating general practices in the uk who use vision software1 general practice staff can record cancer diagnoses using read codes or in free text comments boxes though the latter are not collected by cprd diagnoses will typically be entered duringfollowing a consultation or from written information that is returned to the practice from secondary care cprd gold data are linked to hes apc ons mortality and ncras through a trusted third party for english practices that have agreed to participate in the linkage programme2 hes apc data are collected by nhs digital to co ordinate clinical care in england and calculate hospital payments12 admissions for and related to cancer diagnoses are recorded using international classification of diseases version icd10 codes national cancer registration data are collected by ncras which is part of phe in accordance with the cancer outcomes and services data set13 which has been the national standard for reporting of cancer in england since january data include icd10 codes to identify the cancer site and more detailed information such as stage and grade ons mortality data includes dates and causes of deaths registered in england recorded using icd10 codesparticipants exposures and outcomesour underlying study population included male and female patients registered in cprd gold practices who were eligible for linkage to hes apc ncras and ons mortality data and had at least days of follow up between january and december start of follow up was defined as the latest of the current registration date within the practice and the cprd estimated start of continuous data collection for the practice up to standard date end of follow up was determined as the date the patient left the practice ons mortality date of death or practice last collection dateidentification and classification of cancer codeswe used code lists to classify cancer records in each of cprd gold hes apc and ons mortality data as one of the most common sites other specified cancers history of cancer secondary cancers benign tumours administrative cancer codes unspecified and incompletely specified cancer codes https org data incompletely specified cancer codes could be mapped to cancer site eg icd10 code c689 malignant neoplasms of urinary organ unspecified was considered consistent with both bladder and kidney cancer for ncras we accessed coded records for the most common cancers we included cancers recorded in the clinical or referral file for cprd gold cancers recorded in any diagnosis field for hes apc and the underlying or strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure gold standard algorithm to identify incident site specific cancers using all data sources hes hospital episode statistics ncras national cancer registration and analysis service ons office of national statisticsmost immediate cancer cause of death in ons mortality datacancer case definitions based on individual sources and combinations of sourceswe developed alternative cancer case definitions mirroring those commonly used in epidemiology studies based on identifying the first malignant cancer excluding administrative codes and benign tumours recorded in various combinations of data sources ncras alone ncras and hes apc all sources cprd gold hes apc and ons mortality cprd gold alone hes apc alone multiple malignant cancers recorded on the index date in cprd gold or hes apc were reclassified as multiple site cancer and were not considered as individual site cancer records for positive predictive value and sensitivity calculations multiple codes recorded in different sources on the same date were reclassified as the site identified in the ncras data if available and as multiple site cancer if not for each case definition we only examined the first malignant cancer per individual where this occurred within the study period and at least year after the start of follow upgold standard cancer case definitionwe developed a gold standard algorithm that classifies incident records of the most common cancers by comparing the first malignant cancer identified in each individual source figure cancers recorded in ncras alone with no contradictions ie records for first cancers at different sites were considered true cases whereas cancers recorded in hes apc alone or gold alone required internal confirmation within that source in the form of another code for cancer consistent with the same site or with site unspecified within months and no contradictory codes eg for cancers at other sites in this period where cancer records were present in data source we considered a site specific cancer to be a true case a if it was recorded as the first cancer in ncras and the total number of data sources with records for cancer at that site was equal to or greater than the number of data sources with contradictory records ie records for first cancers at different sites or b where the cancer was not present in ncras if there were more data sources in total with records for cancer at that site than data sources with contradictory recordswe used ncras data to identify stage grade and treatment where available in the cancer registry only cohort binary surgery chemotherapy and radiotherapy variables were derived using individual records of treatment from the first year after diagnosisstatistical analysisfor each cancer site and each individual or combined data source we combined our applied study definitions strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access with our gold standard definition to classify each applied study definition as a true positive false positive or false negative recordwe used these categories to calculate sensitivity and positive predictive value overall and stratified by age categories to and calendar year and sex we calculated differences in diagnosis dates for true positives by subtracting the gold standard index date from the index date for each source and combination of sourceswe used kaplan meier methods to describe mortality over time for cancers identified using each definition the ons mortality death date was used for these analyseswe used the ncras only definition to calculate proportions of patients with complete stage and grade and recorded cancer treatment modalities over timepatient public involvementpatients and the public were not involved in conceiving designing or conducting this study and will not be consulted regarding the dissemination of study resultsresultsof research quality patients in the cprd gold january build were eligible for linkage to hes ons mortality and ncras data in set were excluded due to unknown sex of the remainder and had at least year of follow up between january and december and were included in the study population using the gold standard algorithm incident cases of cancer were identified the number of patients identified with each cancer is presented in online supplementary appendix table half n82 of these patients were male aged to aged to and aged or olderfigure presents ppvs for each case definition comparing the first recorded cancer in each combination of data sources with the gold standard algorithm when using ncras data alone to of cancers were confirmed by the algorithm for out of cancer sites the ncras only case definition gave the highest ppv case definitions using data sources not including ncras generally had lower ppvs ranging from to for individual cancer sites for the four most common cancers breast lung colorectal and prostate ppvs were at least for all case definitions minimal differences in ppvs were observed between age groups years and sexes online supplementary appendix figures figure presents sensitivity values for each case definition sensitivity was generally higher for the case definitions that included ncras data ranging from to for individual cancer sites except bladder cancer identified using ncras data alone and ¥ for the four most common cancers breast lung colorectal and prostate sensitivity was also generally high for definitions using a combination of cprd gold hes apc and ons mortality data ranging from to ¥ for the four most common cancers sensitivity was lower for case definitions that used cprd gold alone range to for individual cancer sites or hes apc alone range to sensitivity values for cprd gold alone and hes apc alone increased slightly in younger patients and more recent years no differences were observed between men and women online supplementary appendix figures post hoc analysis suggested that the low sensitivity of cprd gold only definitions for kidney cancer sensitivity n false negatives was driven by missing n1136 or incompletely specified urinary organ cancer codes n1108 in cprd gold rather than contradictory information about the first cancer record n625 these incompletely specified codes are less likely to be used for bladder cancers n85 than kidney cancers n1108 bladder cancers that were not recorded in ncras data n3445 were commonly recorded in both hes apc and cprd gold n2228 or in hes apc only with a subsequent unspecified or bladder cancer record in hes apc within months n995 table describes the number of days median iqr and 5th95th percentile lag between the date of incident cancers from the gold standard definition and the date of cancer arising from each case definition ie the first record within the specific combinations of data sources used case definitions using ncras alone and combinations of ¥ data sources captured cancers close to the gold standard date median lag ¤ days for all cancer sites whereas median lags were generally longer for the case definitions using cprd gold alone and hes apc alonefigure describes mortality over time following incident cancer diagnoses ascertained from each case definition minimal differences in mortality were observed between cancers identified from different case definitions where variability was observed cancers identified using cprd gold only had the lowest mortality rates eg kidney cancer and cancers identified using hes apc only or ncras only had higher mortality rates eg prostate cancer and bladder cancer respectivelyfigure describes completeness of grade and stage for cancers identified using ncras only recording of grade was highly variable between cancers with gradual increases in completeness over time completeness of staging information was low in earlier calendar years but improved substantially from around especially for the four most common cancers minimum in and in post hoc logistic regression models adjusted for year and cancer site indicated that completeness of stage and grade were associated with each other and these variables were least complete in patients aged stage data was more complete for higher grade tumours whereas grade data was more complete for lower stage tumours online supplementary appendix figure online supplementary appendix figure describes recording of treatment modalities identified using ncras strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure positive predictive value of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm percentage of incident cancers defined using the first ever record in each combination of sources confirmed by a gold standard algorithm that considers confirmatory and contradictory data from each source cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system nhl non hodgkin's lymphoma cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service ons office of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access figure sensitivity of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm percentage of incident cancers identified using the main gold standard algorithm that considers confirmatory and contradictory data from each source that are identified using the first ever record in each combination of sources cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system nhl non hodgkin's lymphoma cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service onsoffice of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0celitnecrep hthtrqi inademelitnecreprqi inadem hthtelitnecrep htht inademrqielitnecrep hthtcpaseh dlogdrpc ytil atromsnodna cpaseh dlogdrpc cpasehdnasarcn secruos fonoitanbmoc hcae nii iidrocer reve tsrfi ot etad ssongaddradnats dog nammorf syad n ili emti l ebatopen access recnac lanoitan sarcn atad erac tneitapdetti mda scitsitats edospei latipsoh cpaseh knil atadhcraeser ecitcarp lacniilc drpc metsys suovren lartnec sncl tluafed ybnoitinfieddradnats dog eht sa emas eht si etad ssongad sa nwohs tonnoitinfied secruos ll iia setis recnac nommoc tsom ruofdcii nosrev sesaesdi fonoitacfissacliscitsitats lanoitan rof ecfifo snoi atadnoitartsgerrecnac ecvres ssyanadna liinoitartsgeri lanoitanretni eht imorf sedoc gndnopserroc ot gndrocca deredro era setis recnaci snoitinfiedde ilii ppa dna etad ssongaddradnats dog nam neewteb syadil fo rebmun ot ot ot ot ot cl amoeym epitluml ot ci ameakuel ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot inademrqi ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot elitnecrep hthtsarcn inademrqi ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot c ytivac laroc laegahposeoc hcamots cc latcerooclrecnac amonaeml tnangilamc saercnapc gnulc suretuc etatsorpc seiravoc yendkic tsaerbci xvreccc sncnarbic reddablc amohpmyl s'inkgdo hnonc idoryhtc revlistrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access figure mortality following first ever record of cancer in each combination of sources cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service nhl non hodgkin's lymphoma ons office of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure completeness of grade and stage for cancers identified using ncras data only cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites grading information is not applicable to braincns sarcoma or haematological cancers and not required by in the national data standard cosd for prostate cancer core staging is not applicable to haematological and gynaecological cancers other types of staging are recommended by cosd cns central nervous system cosd cancer outcomes and services data set ncras national cancer registration and analysis service nhl non hodgkin's lymphomastrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access only missing records may indicate that the patient did not receive that treatment modality or that the treatment modality was not recordeddiscussionstatement of principal findingswe investigated the use of different sources of electronic health record data to identify incident cancers for all case definitions using individual or combined data sources a minimum of of incident site specific cancers were confirmed using the gold standard algorithm this rose to of the four most common cancers use of cancer registration data alone or in any combination of data sources captured at least of site specific cancers identified by the gold standard algorithm excepting bladder cancer and of cases for the four most common cancers combining cprd gold hes apc and ons mortality data captured at least of site specific cancers excepting kidney oral cavity and ovarian cancers and captured of cases for the four most common cancers sensitivity was much more variable when using primary care or hospital data alone and dropped to when identifying bladder cancers using cancer registration data alone use of primary care or hospital data alone resulted in a small lag in identifying cancers of interest compared with the gold standard dates but other case definitions captured cancers close to the gold standard date finally while we observed minimal changes in ppvs and sensitivities between and completeness of ncras cancer registration stage and grade data increased markedly from onwards for specific cancer types demonstrating that initiatives to improve data can have a profound impact on the quality of the data4 completeness of cancer treatment recording was difficult to assess due to the absence of a missing categorystrengths and weaknesses of the studythe main strength of this study is that we have developed a gold standard algorithm using the entirety of the evidence available from cprd to demonstrate the impact of choice of data sets in identifying incident cancers for real life studies we have also assessed the value of using ncras cancer registration data to measure stage grade and cancer treatment modalitiesa limitation of the study is that our gold standard algorithm is not validated we feel that we were justified in pre weighting ncras data as more reliable that other data sources as ncras is a highly validated data set that matches merges and quality checks data from multiple sources4 we did not consider ncras to be the outright gold standard as it is plausible that ncras does not identify all tumours diagnosed and treated in primary and secondary care for most cancer sites our gold standard algorithm identified a small proportion of cancers that are recorded in hes apc cprd gold or ons mortality data but not in ncras these tumours may have been diagnosed and coded as invasive in primary or secondary care but not by ncras been incorrectly coded in hes apc cprd gold or ons mortality data not have been notified to ncras eg tumours treated in private hospitals or be the result of linkage errors between the data sets the proportion of cancers identified in hes apc but not in ncras is particularly high for bladder cancer this is likely to be the result of difficulties inconsistencies and changes in the pathological definition and coding of cancers over time in ncras which are greatest for bladder cancer4 this explanation is supported by the higher mortality rates that we observed in bladder cancer cases identified in ncras compared with other data sources to identify incident cancers we required months of research quality follow up in cprd gold prior to inclusion in the study previous research has demonstrated that historic data is generally incorporated within the patient record with this time frame15 the identification of first ever cancers will also have been affected by different lengths of follow up data available in linked data sources as ncras data collection started in hes apc in and ons mortality data in and by the inclusion of all diagnostic codes in hes apc assuming that the first ever primary or secondary record identified incident cancer reassuringly ppvs for liver and brain cancer were high for all individual and combinations of data sets suggesting that these were not unduly misclassified as primary incident cancers despite being common sites for metastases requiring internal confirmation within months for cancers recorded in cprd gold alone in our gold standard definition is more likely to discount cancers with poorer prognoses and those recorded in the last months of follow up our data cut only included ncras data for the top cancers earlier cancers at other sites will have been missed in this studyit is also important to note that as the gold standard algorithm uses data recorded after the first record of the cancer site in any source index date it cannot be used to identify outcomes in applied studies and follow up of cohort studies with cancer as an exposure would need to start at least months after diagnosis our first ever cancer record in any source definition would be more appropriate for most studiesstrengths and weaknesses in relation to other studies discussing important differences in resultsthe most up to date study describing concordance between linked cprd gold hes apc and ncras data sets demonstrated that to of the five most common cancers recorded in cprd gold are not confirmed in either hes apc or cancer registration data and to of registered cancers are not recorded in cprd gold8 for cancers recorded in both sources the diagnosis date was a median of to days later in cprd gold than in the cancer registration data using cprd gold alone to identify these strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0ccancers marginally over represented younger healthier patients and identified to fewer deaths in the first years after diagnosis use of hes apc only identified a higher proportion of patients with the correct diagnosis date than cprd gold but over represented older patients and those diagnosed through the emergency route the majority of registered cancers were picked up using both cprd gold and hes apc ranging from for lung cancer to for breast cancer previous research demonstrated similar results with substantial differences between cancer types5 additionally a study using data from to found that using hes data in addition to ncras data identified an additional and of surgically treated colorectal lung and breast cancer cases respectively16our study is consistent with these results and provides more complete and practical evidence of the strengths and limitations of using individual and combinations of linked data sets to identify and characterise the most common incident cancerswe have also demonstrated the added value of using cancer registration data to measure stage and grade of incident cancers from about onwards levels of data completeness of staging information in the cprd extract in were similar to those reported by the united kingdom and ireland association of cancer registries ukaicr9meaning of the study possible explanations and implications for clinicians and policymakersuse of ncras cancer registration data maximised the proportion of cases confirmed as true positive based on all available linked information and captured the highest proportion of true positive cases highly complete staging and grading information is available from this source from approximately case definitions based on a combination of cprd gold hes apc and ons mortality data also had acceptable validity for the majority of cancer sites including the four most common cancersthese findings should be considered when deciding which data sources to include in research studies and which sources to use to define cancer exposures outcomes and covariatesunanswered questions and future researchfurther research is required to investigate the validity of cancer recorded in cprd gold and hes apc that are not recorded in the ncras data and to understand differences in cancer data recording with cprd gold and cprd aurum cprds recently launched primary care database based on records from practices that use emis software17 further investigation would be required to confidently identify subtypes of cancer either using codes available in each data set eg colon and rectal cancer or additional information available in hes apc or ncras data use of ncrass recently open accesslaunched systemic anti cancer therapy sact18 and national radiotherapy data sets will also improve ascertainment of therapies for futu | 0 |
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comorbidities that are particularly dangerous in covid19 patients including induced lung inflammation fibrosis and immune system suppression propolis components have inhibitory effects on the ace2 tmprss2 and pak1 signaling pathways in addition antiviral activity has been proven in vitro and in vivo in preclinical studies propolis promoted α this immunoregulation involves monocytes and macrophages as well as jak2stat3 nfkb and inflammasome pathways reducing the risk of cytokine storm syndrome a major mortality interaction with cellular angiotensinconverting enzyme ace2 and serine protease tmprss2 this mechanism involves pak1 overexpression which is a kinase that mediates coronavirusimmunoregulation of proinflammatory cytokines including reduction in il6 il1 beta and tnfrespiratory diseases hypertension diabetes and cancer standardized propolis products with consistent bioactive properties are now available given the current emergency caused by the covid19 pandemic and limited therapeutic options propolis is presented as a promising and 0crelevant therapeutic option that is safe easy to administrate orally and is readily available as a natural supplement and functional food keywords propolis sarscov2 covid19 antiviral antiinflammatory pak1 blocker introduction the covid19 pandemic is of grave concern due its impact on human health and on the deadly disease most require more robust data in clinical trials before they can be widely and safely used isolation and stayathome measures do not effectively protect essential workers economy it is much more deadly than influenza and other types of diseases that recently have had worldwide impact forcing countries to take unusual measures such as limiting travel closing schools businesses and other locations where many people can come into contact with each other various public healthcare strategies have been adopted in an attempt to reduce the impact of the disease but with limited effectiveness as the virus continues to spread often through asymptomatic patients unfortunately tests to determine if people are infectious or were previously infected are not widely available often are costly and frequently do not provide timely and accurate results various therapeutic alternatives have been proposed and tested however preproofnonessential professions return to the workplace they become more at risk for infection in this scenario any options that could help ameliorate disease progression and its consequences even marginally would be useful the world needs safe alternatives to help reduce the impact of this especially health care personnel who have become infected and are dying at alarming rates economic and other necessities limit how well and how long these isolation measures can be maintained especially in poor countries and in poor communities such as slums and favelas [ ] as populations gradually try to get back to normalcy reducing social distancing and people in natural products which have historically been widely used to help avoid and alleviate diseases are among the options being considered as an adjuvant treatment for sarscov2 infection because they are generally inexpensive widely available and rarely have undesirable side effects some have proven antiviral activity an important advantage of using natural remedies is that people who have other health problems or who have mild flurelated 0c infection infection by sarscov2 the virus that causes covid19 is characterized by binding symptoms but do not have the means or courage to visit an already overcrowded medical facility could take simple and inexpensive measures to help reduce the impact of infection with sarscov2 considering the large number of deaths and other types of damage that the covid19 pandemic is causing there is an urgent need to find treatments that have been approved as safe and potentially able to inhibit the new coronavirus reduce its infectivity andor alleviate the symptoms of infection [ ] along this line propolis and its components emerge as potential candidate materials that could help to reduce the pathophysiological consequences of sarscovfactors increased pak1 levels also suppress the adaptive immune response facilitating viral replication [ ] sarscov2 infection is associated with increased levels of chemokines and activated proinflammatory cytokines that lead to the development of atypical pneumonia with rapid respiratory impairment and pulmonary failure immunologicalinflammatory between viral spike proteins and angiotensinconverting enzyme ace2 activation of the spike protein is mediated through proteases such as tmprss2 which play important roles in the viral infection after entry followed by endocytosis coronavirus infection causes pak1 upregulation a kinase that mediates lung inflammation lung fibrosis and other critical mortality preproofphenomena such as cytokine release syndrome have been shown to be important in the spectrum of sarscov2 infection these mechanisms are associated with an dysfunction more than the viral load per se along this line a retrospective observational study found higher serum levels of proinflammatory cytokines such as il6 il1 and tnfα in patients with severe inflammatory diseases by affecting various metabolic cycles recently several studies have shown that propolis extract and some of its components act against several important targets in the pathophysiological context of the disease caused by sarscov2 such as reducing tmprss2 expression and reducing ace2 anchorage which would otherwise facilitate entry of covid19 compared to individuals with mild disease there is considerable evidence that propolis can reduce and alleviate the symptoms of the virus into the cell this is in addition to immunomodulation of monocytes macrophages reducing production of and eliminating il1 beta and il6 reduction of the transcription factors 0cviral replication and antiinflammatory action [ ] propolis and its properties are particularly relevant to sarscov2 infection such as immune system fortification reduced nfkb and jak2 stat3 and blocking pak1 which determine inflammatory activities and fibrosis caused by covid19 various comorbidities have been associated with severe covid19 symptoms and a greater chance of patients requiring intensive care these include hypertension and diabetes also mortality rates of covid19 patients are much higher in those with cardiovascular disease chronic respiratory disease and diabetes [ ] there is considerable evidence that these conditions could be alleviated by treatment with propolis this includes research in animal models of diabetes [ ] hypertension [ ] and cardiovascular disease [ ] propolis has properties that preproofthese plant products to make propolis which they use to protect the colony the production and use of propolis by honey bees evolved to the point that these social bees have considerably fewer immune genes than solitary insect species bees in colonies that produce more propolis are healthier and live longer and propolis consumption by the bees augments their immune [ ] as this variability can affect their medicinal properties standardized propolis products have been developed to help meet the need for a product that does not vary in the main bioactive components and is safe with minimal interaction with pharmaceutical drugs and proven efficacy in clinical trials in recent decades it has been shown to have antimicrobial including the composition of propolis varies according to the plant species available in each region propolis is a product derived from resins and plant exudates plants defend themselves from pathogens mainly by producing phytochemicals many of which have been extracted and used in medicine plant defense substances collected by bees include phenols and terpenoids phytochemical compounds that show promise for the inhibition of coronavirus in humans include quercetin myricetin and caffeic acid all components of propolis honey bees and many other species of social bees recognize these antimicrobial properties and selectively collect and process response to bacterial challenge antiviral antiinflammatory immunomodulatory antioxidant and anticancer properties propolis has historically been widely used to alleviate various diseases [ ] it also has been considered among other natural alternatives as an adjuvant treatment for sarscov2 infection 0ccompared to the propolis that the bees make from these plant materials because it is generally inexpensive widely available and rarely causes undesirable side effects some types of propolis that are highly valued for their medicinal properties such as brazilian green propolis are mainly produced by bees from materials they collect from specific plants in this case baccharis dracunculifolia after the botanical origin of the propolis has been identified extracts of the plant can be made to develop useful products such as a medicinal mouthwash however the medicinal properties of these plant extracts are often inferior can be safely consumed these propolis products and the raw material for their manufacture are extensively exported by brazilian companies especially to asian countries including japan south among natural medicine alternatives propolis has been widely studied and is already extensively consumed in many countries [ ] for example propolis products such as throat sprays and extracts are produced by hundreds of companies in brazil and are sold as a health aid in nearly every pharmacy throughout the country demonstrating on a practical basis that they preproofin fact propolis has a wide spectrum of pharmacological properties and is a dietary supplement that is commonly consumed by both healthy and sick people as a preventative precaution and for treatment it is also used in veterinary medicine due its antibacterial antifungal antiviral korea and china [ ] the importance of propolis in chinese japanese russian and korean medicine is reflected in the number of patents for propolis containing products registered by including about by china and each for japan russia and korea since about new propolisrelated patents were applied for in the us patent office it is a key ingredient in traditional chinese medicine japanese scientists have isolated and patented various brazilian propolis components for cancer treatment demonstrating their usefulness why propolis may be a good fit for dealing with covid19 antiparasitic hepatoprotective and immunomodulatory activities in the wake of the coronavirus outbreak south korea has seen a boon in the use of functional foods according to their ministry of food and drug safety health functional foods are nutrients that have been proven to be beneficial to health in march of this year in response to the coronavirus pandemic the ministry eased regulations for propolis which is considered a 0ccould help fight against the covid19 pandemic active site of this enzyme is a relevant target for drug discovery functional food and allowed new oral formulations however despite considerable evidence that propolis can reduce and alleviate disease symptoms its acceptance as a healthpromoting supplement in human medicine has been limited in many countries such as the usa because of a relevant criticism that propolis products are not standardized and vary in their components and biological activity in part this is because propolis varies with the species of plants available in each region from which the bees collect resins to produce it [ ] however standardized propolis products have recently become available to help fill the need for a product that does not vary in the main bioactive components and effectiveness [ ] one such option a standardized brazilian propolis extract blend has been tested for safety and effectiveness in clinical trials for treating kidney disease and diabetes denture stomatitis and burn patients therefore propolis as a nutraceutical or functional food should be considered as a resource that preproofcaffeic acid phenethyl ester cape galangin chrysin and caffeic acid substances found in several different types of propolis around the world appeared as potential drugs against this viral target table specifically cape was predicted to interact with sarscov2 mpro in a potential targets in order to inactive the virus and reduce the damage that it causes the main protease of coronavirus sarscov2 mpro 3chymotrypsinlike cysteine enzyme is essential similar study therefore although it will be necessary to run in vitro assays to evaluate the potential antisarscov2 effects of propolis andor its constituents these in silico results are along this line hashem evaluated various natural compounds with an in silico approach molecular docking to try to find useful options for treating sarscov2 infection curiously for coronavirus processing of polyproteins and for its life cycle and therefore inhibition of the some propolis compounds can potentially interact with sarscov2 mpro the research community has examined the genetic code of coronavirus and the mechanisms underlying the damages caused by sarscov2 to help search for drugs andor well boding propolis can interact with ace and tmprss2 potentially blocking or reducing sarscov2 invasion of the host cell 0c sarscov2 strongly binds to angiotensinconverting enzyme ace2 using this enzyme as a receptor for invasion and replication in the host cell [ ] causing damage and increasing interpersonal transmission [ ] consequently ace inhibitors have been considered as useful drug alternatives however potential deleterious effects on users of angiotensinconverting enzyme ace inhibitors and angiotensin receptor blockers arbs have tool against potential cardiovascular events inhibition of ace2 enzyme is an important target for treatment against sarscov2 myricetin caffeic acid phenethyl ester hesperetin and pinocembrin rutin interacts with zinc fingers of the active sites of ace2 a metalloprotease that presents the same zinc finger in ace1 emerged as a concern for treatment of covid19 patients an observational study involving patients did not confirm this suspicion and therefore these classes of drugs remain an important infection [ ] güler et al prepared an alcoholic extract of propolis and identified some hydroxycinnamic acids caffeic acid pcoumaric acid tcinnamic acid and cape the flavanons rutin and myricetin and the flavones hesperidin chrysin and pinocembrin using molecular docking evaluations they found that rutin had the highest binding energy to ace2 followed by preproofvarious characteristics including inhibition of ace they found strong inhibition for most of the propolis types they studied with higher than ace inhibition the best results were found in addition to the in silico evidence osés et al evaluated several types of propolis for with the propolis components catechin and pcoumaric acid ace2 and tmprss2 transmembrane serine protease on the surface of host cells are used by sarscov2 via interaction with spike glycoproteins in order to proceed with invasion and replication vardhan sahoo studied several molecules commonly found in medicinal herbs using molecular docking procedures with relevant targets such as rnadependent rna polymerase rdrp ace2 and spike glycoproteins and compared the resulting scores with those of hydroxychloroquine limonin was the most active compound however quercetin and kaempferol also propolis compounds gave high docking scores kaempferol was studied in prostate cancer models and the expression of tmprss2 was reduced showing a potential mechanism of action for an antitumoral effect kaempferol could be an important propolis component for use against covid19 since it is involved in the inhibition of tmprss2 0c potentially interacting with ace2 rdrp and spike glycoprotein sgp besides its antiviral activity table propolis blocks pak1 potentially avoiding lung fibrosis and restoring a normal immune response among the possible targets for controlling covid19 damage the major pathogenic contributes to their suppression pak1 inhibitors can both help combat the virus and restore a normal immune response kinase pak1 is key it is an essential component in malaria and viral infections but it is also involved in a wide variety of other diseases and disease conditions including cancer inflammation and immunosuppression when abnormally activated consequences of pak1 activation include lung fibrosis which is an aggravating factor in covid19 pak1 is activated by rac xu et al demonstrated that caffeic acid and its ester cape components of propolis can inactivate rac consequently inhibiting pak1 the inactivation of pak1 directly or upstream can potentially attenuate coronavirus pathogenesis bcells and tcells are lymphocytes that produce specific antibodies against viruses and other intruders and pak1 preproofimprove the immune response its components increase neutralizing antibody titers activate phagocytosis and increase ifnγ levels and the number of lymphocytes an increase in ifnγ levels was also detected by shimizu et al who evaluated the mechanisms involved in the cape caffeic acid phenethyl ester is a potent inhibitor of activation of nfkb in myelomonocytic cells anse et al demonstrated that propolis cape quercetin hesperidin and some other propolis flavonoids can inhibit the cytokine production of th1 and th2 type t cells while increasing tgfbeta an important antiinflammatory cytokine moreover cape can attenuate oxidative stress and inflammation through downregulation of jak2stat3 signaling propolis from europe and temperate asia usually made by bees from resins collected from poplar trees has predominantly flavonoid compounds while green propolis from baccharis dracunculifolia a propolis exclusively found in brazil has various kinds of flavonoids and prenylated phenylpropanoids such as artepellin c baccharin and drupanin these and all other types of propolis can inactivate pak1 artepillin c selectively inhibits pak1 table some studies have shown that propolis can act as an immunostimulant with the ability to effects of some types of propolis in a herpes simplex animal model 0c as well as having an immunomodulatory effect in which cape inhibits il6 phosphorylation and stat3 which are important for proinflammatory th17 development besides the antiinflammatory effect of cape and kaempferol paulino et al evaluated the antiinflammatory effect of artepellin c in rat paw edema and in cell cultures demonstrating that the activity was at least in part mediated by prostaglandin e2 and no inhibition through nfkb modulation artepillin c is an important biomarker of brazilian green propolis botanical source baccharis dracunculifolia immune modulation is desirable since coronavirus infection dysregulates the immune response in the initial phases of infection which facilitates viral replication however in later stages of covid19 the body develops an exaggerated inflammatory response which can greatly damage the lungs and other ans propolis different from typical immunosuppressants can help avoid immunosuppression during the initial phases of disease and in later stages reduce an exaggerated host inflammatory response inhibiting excess il6 il2 and jak signaling cape a propolis component is also known as an immunemodulating agent and should be considered as an alternative to help reduce an exaggerated inflammatory response in a mouse model propolis had immunomodulatory action in vivo on tolllike receptor expression and on preproofreplication and infectivity potentially decreasing lung inflammation due to antiinflammatory properties while promoting immune system fortification these are useful properties that could there is ample evidence for interference of propolis andor its components with viral propolis has been tested against various viral disease anisms initial successes have prompted research to determine the most useful components which may be modified to produce more active and specific pharmaceuticals viruses that were controlled by propolis in animal models with suggestion for control in humans include influenza [ ] herpes simplex virus type and hiv [ ] shimizu et al evaluated three different types of propolis in ethanol extracts using a murine model of herpes simplex virus type despite the chemical differences proinflammatory cytokine production help minimize the symptoms and deleterious effects of covid19 figure figure propolis as an antiviral substance 0cdue to the different plant origins of the resins the bees used to produce the propolis baccharis dracunculifolia baccharis eriodata and myrceugenia euosma all three propolis extracts not only had direct antihsv1 effects but also stimulated immunological activity against intradermal hsv1 infection in mice antiviral activity of propolis has been reported for dna and rna viruses poliovirus herpes simplex virus and adenovirus in an in vitro model cultured cells the best results were obtained against poliovirus and herpes virus with inhibition of the latter at a propolis concentration of ugml the propolis components chrysine and kaempferol caused a concentrationdependent reduction of intracellular replication of herpesvirus strains when host cell monolayers were infected and subsequently cultured in a drugcontaining medium quercetin another propolis component had the same effect but only at the highest concentrations tested ugml against various human herpes simplex virus strains with a intracellular replication reduction of approximately while it reduced the infectivity of bovine herpes virus human adenovirus human coronavirus and bovine coronavirus about the reduction was in the preproofimpairment and pulmonary failure inflammatory response to infection sarscov2 infection is associated with increased levels of chemokines and activated proinflammatory cytokines that lead to the development of atypical pneumonia with rapid respiratory immunologicalinflammatory phenomena such as cytokine release syndrome have been shown to be important mortality factors in sarscov2 infection higher serum levels of proinflammatory cytokines such as il6 il1 and tnfα are found in patients with severe covid compared to those of individuals with mild disease molecular mechanisms involved in this immune process are the targets of various synthetic medicines being tested in patients including ciclesonide hydroxy chloroquine ivermectin and ketorolac which are pak1 blockers pak1 raccdc42activated kinase is overexpressed in the lung in response to sarscov2 infection and is a critical mediator of the cytokine storm that frequently results in mortality the most critical cases of covid19 which require ventilatorassisted intensive care and often result in prolonged ventilator dependency and death are a result of an exaggerated case of rotavirus antiinflammatory and immunomodulatory properties of propolis 0cin hospitalized patients fortuitously propolis components are effective pak1 blockers table there is considerable evidence that propolis can reduce and alleviate the symptoms of inflammatory diseases and has immunomodulatory properties [ ] however these properties can vary according to the plant origin of the propolis as well as the extraction processsolvent used and the inflammatory protocol cell culture animal models induction by lipopolysaccharides when the propolis extracts are tested tests with animal models have shown that propolis can reduce the levels of il6 and tnfalpha which are key proinflammatory mediators and increase the levels of the regulatory cytokine il10 kaempferol a propolis component reduces il6 tnfalpha and vegf vascular endothelial growth factor via the preproofifnγ in serum fernandes et al found that propolis exerted a positive adjuvant effect on vaccines that were developed against canine coronavirus they assayed ifnγ which is an effective way to measure the cellular response induced by a vaccine in a mouse model propolis added as an adjuvant to inactivated swine herpesvirus type vaccine stimulated increased cellular propolis is considered a safe immunostimulant and a potent vaccine adjuvant propolis has been widely tested as a vaccine adjuvant because it induces an earlier immune response and provides a longer protection period it is also included as an adjuvant ingredient in traditional chinese medicine propolis flavonoids have potential as adjuvants enhancing igg il4 and propolis has potential as a vaccine adjuvant erknfkbcmycp21 pathway table tests on macrophage cell cultures also demonstrated that propolis inhibits the production of il1 beta an important component of the inflammasome inflammatory pathway in diseases such as rheumatoid arthritis lupus and other autoimmune diseases although the mechanisms of action are not well elucidated these propolis components have potential as complementary supplements in the preventive treatment of chronic inflammatory diseases and humoral responses increasing ifnγ [ ] propolis enhanced the immune response to inactivated porcine parvovirus vaccine in guinea pigs when added to a trichomonas vaginalis protein vaccine propolis increased the igg antibody response times in mice 0ccancer is considered a relevant comorbidity factor for covid19 cancer patients have a patients cancer patients compared to the protein alone propolis was also effective as an adjuvant in the immunization of cattle with bovine herpesvirus it improved the humoral and cellular responses in mice inoculated with inactivated virus vaccines propolis as an adjuvant gave a similar immune response increasing ifnγ levels to alum and freunds adjuvant in mice vaccinated with an hiv1 polytope vaccine candidate with less risk of undesirable side effects comorbidities and evidence of how propolis can help reduce their impact in covid19 to risk a visit to a clinic or hospital to determine if they have cancer alternative therapies could help retard cancer or reduce the impact of cancer and cancer treatment in covid19 times higher risk of progressing to severe covid19 disease than patients without comorbidities also the hospital environment during the coronavirus pandemic can interfere with or delay the treatment that cancer patients should receive patients with symptoms may choose not preproofacid cape quercetin and chrysin propolis and its components normally have little impact on normal cells displaying differential cytotoxicity in liver cancer melanoma and breast cell carcinoma cell lines [ ] propolis enhances the activity of tumor various types including bladder blood brain breast colon head and neck kidney liver pancreas prostate and skin cancers propolis could help prevent cancer progression in various parts of the world it is considered an alternative therapy for cancer treatment propolis extracts have been found to inhibit tumor cell growth both in vitro and in vivo including inhibition of angiogenesis demonstrating potential for the development of new anticancer drugs various components of propolis have been shown to inhibit cancer cell growth including cinnamic propolis has potential as a complementary therapy for cancer it has shown efficacy against necrosis factor related apoptosis inducing ligand trail in cancer cells hypertension and cardiovascular disease 0c hypertension and cardiovascular disease are considered relevant comorbidities for covid19 propolis has demonstrated antihypertensive effects in rat models [ ] in cameroon it is used in popular medicine to treat various ailments including high blood pressure propolis has been widely used as a dietary supplement for its health benefits including cardiovascular protective effects [ ] in a human trial consumption of propolis improved critical blood parameters including hdl gsh and tbars levels demonstrating that obesity a natural antiobesity agent it could contribute to a reduced risk for cardiovascular disease obesity is a major comorbidity and predictor of increased mortality in covd19 patients obesity and sarscov2 both induce an inflammatory process exacerbating sarscov2 infection in the obese propolis reduced inflammation and prevented hyperlipidemia and metabolic syndromes in highly caloric diet induced obesity in mice body weight gain visceral adipose tissue liver and serum triglycerides cholesterol and nonesterified fatty acids were all reduced in the propolis fed mice [ ] caffeic acid phenethyl ester a propolis component is preproofplatelet aggregation was reduced by propolis in tests on human blood in vitro and in other in vitro tests caffeic acid phenethyl ester cape a wellstudied bioactive propolis component inhibits collagen induced platelet activation thromboembolism thrombosis and microthrombosis microthrombosis disseminated intravascular coagulation and consequent multian failure are common in severely affected covid19 patients with associated high mortality rates anticoagulants are sometimes prescribed to such patients because they can reduce mortality tang et al an elevated level of plasminogen activator inhibitor1 pai1 is a biomarker and risk factor for thrombosis and atherosclerosis [ ] various types of evidence demonstrate that propolis can reduce platelet aggregation and other thrombosisrelated parameters propolis decreased thrombotic tendencies in mice by suppressing lipopolysaccharideinduced increases in pai1 levels [ ] propolis downregulated plateletderived growth factor and platelet endothelial cell adhesion molecules in lowdensity lipoprotein knockout mice 0c old age the elderly are more often affected by chronic inflammation characterized by systemically increased levels of proinflammatory cytokines which can contribute to development of a cytokine storm a major cause of covid19 mortality propolis has antioxidant properties which could help retard o | 0 |
clinical manifestations of sarscov2 infection include more frequently fever and cough failure can occur in persons with additional comorbidities liver dysfunction is one of the most striking affections among patients suggesting that sarscov2 may represent a new king of liver aggressor however the molecular process underlying this phenomenon is 0cstill unclear in this work we overview the most recent findings between the molecular biology of the virus pathogenic mechanisms and its relationship to liver disease observed in patients abbreviations aado2 ace2 aih alt ast covid19 ggt gi gtex alveolararterial oxygen gradient angiotensinconverting enzyme autoimmune hepatitis alanine transaminase aspartate aminotransferase coronavirus infectious disease gamma glutamyl transpeptidase gastrointestinal genotypetissue expression metabolicassociated fatty liver disease nonstructural proteins open reading frame preproofcomplex disease in many severely ill patients in other infected subjects an infection is keywords sarscov2 liver liver impairment covid19 ace2 mafld nsp orf introduction sarscov2 is the etiological agent of the disease known as covid19 which causes a disease characterized by pneumonia cough fever occasional diarrhea headache cardiac injury and in some patients liver alterations covid19 has been found to be an extremely reported to be so severe that it can lead to a disproportionate and mortal reaction of the immune system known as a cytokine storm all of these factors make covid19 highly unpredictable it is what specialists call a multisystem disease 0caround the world cases of liver dysfunction denoted by elevated hepatic enzymes in serum such as ast aspartate aminotransferase and alt alanine transaminase have been documented among patients infected with sarscov2 there is still no certainty whether the covid19related liver damagedysfunction is due mainly to the viral replication per se drugs toxicity or other coexisting comorbidities whether sexrelated difference could help to explain why infected men are more healthy or harmful relationship between the liver and its viral aggressor in this paper we describe a brief overview of the implications for researchers in the field of it is important to understand how liver function can be altered by direct infection with this predisposed to develop covid19associated liver dysfunction than infected women to analyze if there is any genetic predisposition related to impaired liver function during the respiratory virus which mechanisms of viral pathogenesis are involved to evaluate disease and of course the crosstalk between viral and cellular proteins that mediate this preproofliver disease of the most recent findings between the molecular biology of the virus this emerging viral illness is typically characterized by fever dry cough myalgia headache sore throat diarrhea and may be aggravated with shortness of breath and respiratory failure the angiotensinconverting enzyme ace2 the functional receptor of the spike glycoprotein of sarscov2 is widely distributed in the anism historically hamming and colleagues reported ace2 expression in the surface of lung alveolar epithelial cells enterocytes of the small intestine arterial and venous endothelial pathogenic mechanisms and its relationship to liver disease observed in patients clinical characteristics and liver injury in patients with covid19 0ccells and arterial smooth muscle cells posterior transcriptomic and proteomic analyses confirmed their findings and added high ace2 expression in adipose tissue bone marrow duodenum endometrium heart kidney small intestine smooth muscle testis and thyroid ace2 is also expressed in liver but in lesser extent one of the most worrisome severe cases of covid19 regarding the gastrointestinal gi tract and liver over covid19associated liver injury is defined as any liver damage that occurs during disease progression andor covid19 treatment in patients with or without a history of previous complications is the unusual formation of blood clots in many patients with covid19 even those who were receiving anticoagulants researchers at mount sinai in new york published studies suggesting that clots in the lungs play an important role in the most of covid19 patients develop gi symptoms such as anorexia diarrhea nausea and vomiting and a significant proportion present with altered liver function tests preproofdecreased albumin levels are associated with severe infection and poor prognosis still ast elevation is the most common abnormality in patients presenting with covid19 observed more frequently in men and is mainly documented in more severe cases liver disease in general the incidence of increased liver biochemical markers in hospitalized patients with covid19 mainly ast and alt and slightly elevated bilirubin varies between to of cases the increase in liver enzymes is there are no reports of acute or subacute liver failure in patients with covid19 the largest cohort study that included cases of covid19 from china showed that had preexisting chronic liver disease lei and colleagues reported that impaired liver function was related to mortality in covid19 patients elevated ast was more frequent and significant than the increase of alt in severe 0chospitalized patients moreover elevated ast was shown to be associated with highest mortality risk in the study reported by yijin wang they found that of covid patients had elevated ast activity the median levels of alt were ul vs ul respectively ast were ul vs ul respectively in abnormal and normal aminotransferase groups liver enzymes abnormality were associated with disease severity protein levels in addition they found by ultrastructural examination of coronavirus ps in hepatocytes in covid19 cases sarscov2 infected hepatocytes displayed as well as a series of laboratory tests including higher alveolararterial oxygen gradient aado2 higher gamma glutamyl transpeptidase ggt lower albumin and lower total conspicuous mitochondrial swelling endoplasmic reticulum dilatation and glycogen granule decreased histological findings showed apoptosis and binuclear hepatocytes preproofshowed a disease course similar to that reported in non immunosuppressed population coronavirus the interaction of its proteins with cellular proteins and consequently the immunosuppressive therapy for autoimmune hepatitis aih developing covid19 taken together both ultrastructural and histological evidence indicated a typical lesion of viral infection all these findings by different reports demonstrates that sarscov2 infection in liver is a crucial cause of hepatic impairment in covid19 patients however alteration of cellular metabolism that give rise to systematic alterations and metabolic report from alessio gerussi demonstrated that patients under today the cellular and molecular mechanisms that are altered by infection with this changes are still unknown 0cmolecular biology of sarscov2 coronaviruses are enveloped viruses that contain a positively polarized unsegmented rna genome belonging to the coronaviridae family and the order of nidovirales they are distributed in humans and other mammals the size of the sarscov2 virions is approximately to nm in diameter [] sarscov2 has a genome that consists polymerase rdrp which is nsp12 and is responsible of the replication and transcription of the virus which are encoded by the various genetic loci on the genome at the center of the virion lies a nucleocapsid composed of the genomic rna and the nucleocapsid protein the virus glycoprotein s consists of two subunits s1 which is at the amino terminus and that encodes for structural proteins and a larger region that encodes two open reading frames orf 1a and 1b which together encode for the nonstructural virus proteins from nucleocapsid protein which is within the phospholipid bilayer and nonstructural proteins nsp1 to nsp16 the virions have a structural sspike protein outer spiky glycoprotein mmembrane protein a type iii transmembrane glycoprotein nof nucleotides encoding amino acids and it is composed of a region preproofvirus as well as protein m which is a type iii transmembrane glycoprotein and participates in the cellular membrane rearrangements for the replication and transcription complexes among the encoded proteins is an rnadependent rna provides the receptor binding site and s2 which is at the carboxyl terminus responsible for membrane fusion the envelope protein e has a role in the assembly and release of the nonstructural proteins have several functions during de viral cycle for example nsp 0cthe virus enters the cell by endocytosis through the interaction between envelope glycoprotein s with the cell receptor ace2 and with the participation of the type ii transmembrane serine protease tmprss2 once it enters the cell the n protein with viral genome are released within the cytoplasm then cellular proteases degrade the capsid and the virus genome is left free next the polyprotein containing the viral proteins that are how does the virus select which cells to infect viruses can infect only certain species of hosts and only permissive cells within that host permissive cells make all the necessary proteins and viral factors to allow virus to replicate once a virus gets inside a cell it hijacks the cellular processes to produce virally encoded proteins that will replicate the viruss genetic material viral replication may cause exocytosis will translate into viral proteins this entire process will occur in the cell cytoplasm the processed to form the replication complex is translated and then the complementary strand of negative sense pregenomic rna is synthesized to be used as a template to replicate the structural proteins that will be synthesized in the endoplasmic reticulum membrane to assembly the viral p and finish the cycle through the release of the viruses by positive sense viral genome furthermore the replication and transcription complex will lead to a series of smaller positive sense subgenomic rnas these are the ones that preproofboth sarscov and the new sarscov2 are very similar in structure and pathogenicity but the major structural protein s protein is slightly different between them compared to other beta coronaviruses the presence of a furinlike cleavage site in sarscov2 enables the s protein priming and facilitates an increase on the efficiency of the spread of sarscov2 as is reported wide world 0cbiochemical changes producing cell damage called cytopathic effects like other coronaviruses sarscov2 requires cellular receptors to initiate its internalization to the cells that carry these factors li sarscov2 uses the angiotensinconverting enzyme ace2 as a host cell receptor sarscov2 spike s protein binds ace2 with significantly high affinity in addition the main host protease that suggested to promote the pathogenesis of this coronavirus program httpsportalgdccancergov they compared ace2 expression levels across human tissues between males and females and between younger and older persons in these individual tissues furthermore other reports have analyzed the correlations between ace2 in order to provide insights into the mechanism of sarscov2 infection li analyzed the expression of ace2 in various normal human tissues using the datasets from the genotypetissue expression gtex project and the cancer genome atlas tcga transmembrane serine protease other host proteases such as furin have also been mediates sprotein activation on primary target cells and initial viral entry is the type ii preproofreported by li ace2 expression levels showed no significant difference between have no significant association with sex age or race is the liver a direct target for sarscov2 males and females between younger and older persons or between asian and nonasian races this finding suggests that the infection risk of sarscov2 and sarscov may expression levels and immune signature enrichment levels in individual tissues as as we expected because the systemic manifestations of covid19 it has been reported that sarscov2 has an anotropism beyond the respiratory tract including the kidneys 0cliver heart and brain and possibly that anotropism influences the course of covid19 disease and aggravates preexisting conditions the ace2 protein is found at high levels in the gi tract as the colon biliary system and liver on the other hand it is well documented a sarscov2 rna shedding in the gi tract supporting its tropism for architecture express ace tmprss1 receptors the presence of these two host factors in the liver suggests that a direct viral cytopathic effect occurs also in sars infection the presence of viral rna in liver tissue was documented but not as extensively as the new coronavirus data published by gordon suggest that mitochondrial proteins interact directly with the virus which helps to understand the potential mechanism by which elevated ast the gi tract and liver cells and these may be sites of active viral replication and either direct or indirect tissue injury indeed a large part of the cells distributed in the liver preproofeffect the exacerbated inflammatory response in covid19 may play a central role in profiles are detected in these patients furthermore in addition to this intracellular more recently identified the clinical and laboratory characteristics of covid19 patients with abnormal liver transaminases and they reported that sarscov2 is able to which high levels of il6 have been reported which are involved in both infect liver cells and cause liver impairment by direct cytopathic effect inflammatory and repair responses in liver disease mechanisms of liver pathogenicity 0cif sarscov2 replication has direct adverse effects on liver function it is still unknown findings in liver biopsy of patients killed by covid19 showed moderate micro vesicular steatosis and mild portal and necroinflammatory activity this seems to indicate that a direct injury occurred while the infection that could have been directly caused by sarscov2 another possibility is that a druginduced liver injury occurred to date there are the following possible mechanisms figure infection the massive release of cytokines by the immune system in response to the viral infection can result in a cytokine storm and symptoms of sepsis that are the cause of death in of fatal covid19 cases in these cases uncontrolled inflammation induces multian damage leading including liver failure biomarkers of inflammation such as creactive protein pcr serum ferritin ldh ddimer il6 il2 are have been found to be significantly elevated in immune damage from exacerbated inflammation in response to sarscov2 preproofpathogenesis of sars cov related liver disease more studies should be liver is a potential target for direct infection with this virus to understand the performed for evidence of viral mechanisms of replication in different cell anelles as cytoplasm endoplasmic reticulum golgi apparatus and lipidrafts cov2 enters cells through the ace2 molecule which is abundantly expressed in the liver and in particular in bile epithelial cells based on this expression the direct cytopathic effect due to active viral replication in various liver cells sarscritically ill patients with covid19 into hepatocytes and liver histology characterization it is also important to know in cells their capacity to efficiently produce both infectious and defective non 0cinfective whole virions there are not yet enough data to know the viral dynamics in the different tissues and the associated pathogenesis anoxia respiratory failure is one of the main characteristics of covid19 anoxic hypoxic hepatitis is common in patients with severe symptoms reactivation of preexisting liver disease patients with preexisting chronic liver medications such as tocilizumab and baricitinib used to combat the adverse immune cholestatic liver disease various studies such as lopinavir ritonavir remdesivir chloroquine tocilizumab uminefovir traditional chinese medicine so it is important to consider that they could be potentially hepatotoxic in some patients druginduced liver damage dili initial clinical guidelines recommended antiviral agents for covid19 so a variety of drugs have been administered in disease may be more susceptible to liver damage from sarscov2 biological preproof genetic factors genetics may well be one of the determining factors in some reaction may also cause hbv reactivation and induce eventual impairment of liver function in those patients with hbv on the other hand it is still unknown whether sarscov2 infection exacerbates cholestasis in people with underlying patients who become seriously ill with covid19 but until now we cannot be sure it is possible for example that the genetic variation that makes an individual more susceptible to high blood pressure or diabetes also makes him more vulnerable to the virus it will be important to find out what role genetic factors predisposing to liver steatosis have and their sensitivity to severe symptoms of covid19 ji and 0ccolleagues showed that subjects with metabolicassociated fatty liver disease mafld have a higher risk of covid19 severity disease and abnormal liver blood tests than patients without mafld in contrast louise biquard demonstrated that mafld is not associated with changes in liver expression blood test abnormalities reported by ji and colleagues is thus likely not explained by concluding remarks the scenario is not yet complete which does not allow us to establish or understand the natural history of the disease and the participation or commitment of the liver in this disease certainly the application of new technological platforms such as singlecell increased hepatic sarscov2 uptake still several contradictory reports will help of genes implicated in sarscov2 infection the observed persistence of liver to find the real role of genetic factors in the evolution of this disease preprooftranscriptomic assays will allow us to quickly know the commitment of each cell type in affected ans and the meaning of viral dynamics in the various affected systems including the liver however as we have already learned from the old hepatotropic viruses history still is ongoing and we have much to learn and understand about the virologic characteristics of this emerging rna virus that allow us to develop specific antivirals such as the case of hcv and the vaccine to decrease the impact of this acute infection declarations of interest none ethical approval not required 0c references chen n zhou m dong x qu j gong f han y epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan china httpsdoi101002path1570 wang d eraslan b wieland t hallström b hopf t zolg dp a deep proteome and transcriptome abundance atlas of healthy human tissues mol syst hamming i timens w bulthuis mlc lely at navis gj van goor h tissue distribution of ace2 protein the 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h clinical and chai x hu l zhang y han w lu z ke a specific ace2 expression in invest httpsdoi101172jci137244 death cases with covid19 medrxiv httpsdoi1011012020022620028191 immunological features of severe and moderate coronavirus disease j clin preproofcholangiocytes may cause liver damage after 2019ncov infection biorxiv patients medrxiv httpsdoi1011012020040120047381 httpsdoi10110120200203931766 herold t jurinovic v arnreich c hellmuth jc von bergweltbaildon m klein m level of il6 predicts respiratory failure in hospitalized symptomatic covid grein j ohmagari n shin d diaz g asperges e castagna a compassionate use of remdesivir for patients with severe covid19 n engl j med httpsdoi101056nejmoa2007016 u s food and drug administration fact sheet for health care providers emergency 0cuse authorization eua of hydroxychloroquine sulfate supplied from the strategic national stockpile for treatment of covid19 in certain hospitalized patients varona pérez j rodriguez chinesta jm riesgo de reactivación de la hepatitis b asociado al tratamiento con corticoides frente a sarscov2 covid19 rev clÃnica española httpsdoi101016jrce202004012 ji d qin e xu j zhang d cheng g wang y nonalcoholic fatty liver httpsdoi101016jjhep202003044 sarscov2 in metabolicassociated fatty liver disease j hepatol diseases in patients with covid19 a retrospective study j hepatol preproof biquard l valla d rautou pe no evidence for an increased liver uptake of httpsdoi101016jjhep202004035 0cfigure legends preprooffig1 proposed mechanisms of liver pathogenicity of sarscov2 in infected cells sars cov2 infection2 cytokinestorm3 drugeffects4 hypoxia5 previousliverdamagebiochemicallabmarkerswhite bloodcellsgenomereleasereplicationtranslationvirionassemblyviral proteinsmaturevirus release\uf0e9aado2mitochondrialproteinshypoxicisquemicliverinjuryliver damagelopinavirritonavirremdesivirchloroquinetocilizumaboxidativeimbalancesteatosisace2s proteincytopathiceffect\uf0e9gmcsf\uf0e9il6\uf0e9il1β\uf0e9il2\uf0e9il8\uf0e9ccl2\uf0e9ccl3\uf0e9ccl5\uf0e9cxcl \uf0e9alt\uf0e9ast\uf0eaalbumin\uf0e9pcr\uf0e9ldh\uf0e9ddimer\uf0e9ferritin\uf0e9bilirubin 0c' | 0 |
" variability in the health effects of dietary fiber might arise from interindividual differences in the gutmicrobiotas ability to ferment these substrates into beneficial metabolites our understanding of what drives thisindividuality is vastly incomplete and will require an ecological perspective as microbiomes function as complexinterconnected communities here we performed a parallel twoarm exploratory randomized controlled trial in adults with overweight and classi obesity to characterize the effects of longchain complex arabinoxylan n at high supplementation doses female gday male gday on gut microbiota composition and shortchainfatty acid production as compared to microcrystalline cellulose n nonfermentable control and integratedthe findings using an ecological frameworkresults arabinoxylan resulted in a global shift in fecal bacterial community composition reduced αdiversity andthe promotion of specific taxa including operational taxonomic units related to bifidobacterium longum blautiaobeum and prevotella copri arabinoxylan further increased fecal propionate concentrations p friedmanstest an effect that showed two distinct groupings of temporal responses in participants the two groups showeddifferences in compositional shifts of the microbiota p permanova and multiple linear regression mlranalyses revealed that the propionate response was predictable through shifts and to a lesser degree baselinecomposition of the microbiota principal components pcs derived from community data were better predictors incontinued on next page correspondence jenswalteruccie nguyen k nguyen and edward c deehan contributed equally to this work1department of agricultural food nutritional science university of albertaedmonton ab t6g 2e1 canada13department of biological sciences university of alberta edmonton abt6g 2e1 canadafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cnguyen microbiome page of continued from previous pagemlr models as compared to single taxa indicating that arabinoxylan fermentation is the result of multispeciesinteractions within microbiomes this study showed that longchain arabinoxylan modulates both microbiota composition and theoutput of healthrelevant scfas providing information for a more targeted application of this fiber variation inpropionate production was linked to both compositional shifts and baseline composition with pcs derived fromshifts of the global microbial community showing the strongest associations these findings constitute a proofofconcept for the merit of an ecological framework that considers features of the wider gut microbial community forthe prediction of metabolic outcomes of dietary fiber fermentation this provides a basis to personalize the use ofdietary fiber in nutritional application and to stratify human populations by relevant gut microbiota features toaccount for the inconsistent health effects in human intervention studiestrial registration clinicaltrialsgov nct02322112 registered on july keywords arabinoxylan dietary fiber gut microbiota interindividual variability overweight adults shortchainfatty acids epidemiologic studies consistently associate dietary fiberconsumption with a reduced incidence of obesityassociated pathologies [ ] in largescale observationalstudies whole grains and cerealderived fibers eg arabinoxylan and glucan showed stronger associationswith reduced risk of developing cardiovascular diseasetype ii diabetes gastrointestinal cancers and of allcausemortality when compared to other fiber sources [ ] asubstantial body of animal research further consolidatedthe mechanisms by which fiber reduces metabolic pathologies despite these convincing associations findings obtained from human dietary intervention trialsaimed to improve metabolic risk markers by supplementing isolated dietary fibers remain inconsistent possibly due to an individualized clinical response [ ]owing to their chemical structure dietary fibers resistdigestion in the smallintestine and reach the colonwhere they become substrates for the gut microbiotathe microbial fermentation of fiber to shortchain fattyacids scfas has been implicated in the prevention ofobesityassociated pathologies propionate and butyrate are two scfas that are especially relevant as theyhave been linked to beneficial immunological and metabolic effects intervention studies with arabinoxylanisolated from wheat endosperm for instance have demonstrated increased fecal concentrations of both butyrateand propionate dietary fibers can further modulategut microbiota composition in a structuredependentway through the enrichment of bacterial taxa that utilizethe substrate and tolerate or benefit from the environmental changes caused by fiber fermentation [ ] forexample dietary interventions with shortchain fractionsof arabinoxylan resulted in an enriched abundance ofbacterial species that can either utilize arabinoxylan oliaxos directly eg bifidobacteriumgosaccharidesadolescentis and bifidobacterium longum or benefitfrom metabolic byproducts released during axos degradation eg anaerobutyricum hallii and faecalibacterium prausnitzii although fiberinduced alterationsto the gut microbiota are significant the effects are alsohighly individualized and this variability might haveclinical ramifications that could explain the individualized clinical responses to understand the individualized response of the gutmicrobiota to dietary fiber an ecological perspective isrequired as fiber fermentation is determined by complexinterspecies interactions between members of the gutmicrobiota the process is often based on a crossfeeding cascade where primary degraders that access thefiber provide breakdown products oligosaccharides disaccharides and monosaccharides to other microbesand metabolites that result from the fermentation ofthese products also serve as substrates interindividual variation in gut microbiota composition mayresult from the absence of keystone species that initiatethe degradation of recalcitrant fibers differences inunrelated species with similar ecological functions thatcompete for the same substrate or variation instrains of the same species that differ in their capacity tometabolize the substrate these compositional variations likely determine both the competitive and cooperative relationships between community membersthat form trophic networks some of which anize intoecological guilds that collaborate to degrade complexfibers although interindividual variation in the response of the gut microbiota to fiber can influence metabolite outputs relevant to health ie propionate orbutyrate this topic and the underlying ecologicalprinciples have received little attentionthe objective of this study was to apply an ecologicalframework to characterize the compositional and metabolic responses of the human gut microbiota to a longchain arabinoxylan isolated from corn bran compared to 0cnguyen microbiome page of a fiber that is not fermented by the gut microbiotamicrocrystalline cellulose mcc we further assessedwhether nutritional and microbiotarelated factors couldexplainamongindividualsresponsesobservedthevariableresultssubject characteristics and protocol adherenceto compare the effects of arabinoxylan and mcc weconducted a 6week parallel twoarm exploratory randomized controlled trial in individuals with overweightand classi obesity where females received gday andmales gday of either fiber fig of the subjectsenrolled and randomized to an intervention arm sevenwithdrew from the study in the arabinoxylan groupthree experienced challenges consuming the supplementand one reported constipation in the mcc group twowithdrew due to personal reasons and one due to constipation and weretherefore excluded from analysesadditional file fig s1 subjects that completed thestudy protocol n included females and males aged ± years with a body mass indexbmi of ± kgm2 no differences in age sex orbmi were detected between the intervention groups atbaseline additional file table s1 overall protocoladherence assessed by the amount weight of returnedsupplement was ± and ± in the arabinoxylan and mcc arms respectivelyeffect on the composition of the fecal microbiotafecal microbiota diversitynonmetric multidimensional scaling analysis of euclidean distances between subjects based on centered logratio clrtransformed operationaltaxonomic unitotu data showed that the two treatment groups harbored bacterialcould not becommunitiesthatdifferentiated at baseline p permutational multivariate analysis of variance [permanova] fig 2aoneweek supplementation with arabinoxylan alteredthe global fecal bacterial community which became significantly different from the fecal microbiota of subjectsreceiving mcc p this effect was maintaineduntil the end of the fiber intervention p thesechanges occurred by arabinoxylan inducing temporalshifts in fecal microbiota composition determined as theaverage diversity between the individuals treatmentand baseline samples which were significantly largerwhen compared to the mcc group p mannwhitney test fig 2b in addition while mcc increasedinterindividual differences diversity between subjectsp generalized estimated equation [gee] modelarabinoxylan reduced it p fig 2canalysis of αdiversity showed that arabinoxylan reduced fecal bacterial diversity shannons index p gee model fig 2d but not richness total otusafter weeks of supplementation overall these findingsshowed that while the nonfermentable mcc had no detectable effects on measures of bacterial diversity arabinoxylan altered the global bacterial community within week inducing temporal shifts in composition and a reduction of both interindividual variation and αdiversityeffect on the relative abundance of bacterial taxa and coabundance response groupsneither arabinoxylan nor mcc altered microbiota composition at the phylum level at lower taxonomic levelschanges in the relative abundance of two bacterial families were detected at weeks of arabinoxylan relative tobaseline and mcc namely an increase in bifidobacteriaceae q wilcoxon test fig 2e additional file table s2 and a decrease in erysipelotrichaceae q fig study design shaded study week blocks indicate a scheduled clinic visit the x indicates the task was completed during the study weekcdhq ii canadian diet history questionnaire ii stool characteristics selfreported stool consistency and bowel movement frequency 0cnguyen microbiome page of fig arabinoxylan alters the global composition of fecal bacterial communities and induces distinct shifts in taxa a nonmetricmultidimensional scaling nmds plot based on euclidean distance metrics of arabinoxylan and microcrystalline cellulose groups at each timepoint intersubject diversity showing changes in the distance between subjects over time euclidean distances b between fecal microbiotas ofsubjects at each study time point intersubject and c between each subjects fecal microbiota at baseline and during w1 and w6 of treatmentintrasubject d αdiversity displayed as shannon index and total otus of the fecal microbiotas of subjects at each time point e absolutechange δw6bl in relative abundance of bacterial taxa affected by the dietary intervention data analyzed using permanova for a gee modelswith bonferroni correction for b and d and mannwhitney tests for c for e data were analyzed using either wilcoxon tests to assess withingroup changes relative to baseline or mannwhitney tests to assess betweengroup changes ie ax vs mcc with fdr correction diversityand compositional data were reported as mean ± sd and centered logratio transformed prior to the statistical analyses bl baseline otuoperational taxonomic unit w1 week w6 week at the genus level arabinoxylan increased thegenera bifidobacterium and prevotella when comparedto both baseline and mcc and enriched blautia whencompared to mcc otu level analysis revealed that otus changed during arabinoxylan treatment relative tobaseline henceforth referred to as significant otusin particular otus related to bifidobacterium longumotu4 prevotella copri otu6 bacteroides plebeiusotu53 bacteroides sp otu56 bacteroides ovatussuccinatutensotu26otu38 blautia obeum otu85 subdoligranulum spotu11 clostridium leptum otu46 mollicutesotu32 and muribaculaceae otu79q phascolarctobacteriumbecame enriched while otus related to ruminococcusbromii otu5 eubacterium oxidoreducens otu41bacteroides uniformis otu7 and faecalibacillus sppotu21 declined in relative abundance supplementation with mcc only increased the family lachnospiraceaegenus parasutterella q numerically the dominant compositional effects of arabinoxylan were to a large degree specific to b longumotu4 and p copri otu6 as these taxa increased inrelative abundance by an average of 46fold and 4fold while other otus increased by and thein an attempt to identify groups of cooperating species that could function as ecological guilds in the 0cnguyen microbiome page of degradation of arabinoxylan we adapted a clustering approach conceptually similar to that described by tong instead of absolute proportions of taxa weused arabinoxylaninduced shifts to identify clusters ofspecies whose responses were intercorrelated this analysis revealed a total of seven coabundance responsegroups cargs fig 3a five of which showed statistically significant responses to arabinoxylan while noneresponded to mcc additional file table s2 thecarg that showed the largest increase in relative abundance was carg1 p wilcoxon test whichconsisted of six out of the eleven otus that increasedthrough arabinoxylan fig 3b among those six otusbshift andshowed significant connections to all but one member ofcarg1 rs q spearmans correlations usingpermutation tests suggesting arabinoxylan may be degraded through cooperative interactions between theselongum otu4 exhibited the largesttaxa in carg6 p copri otu6 exhibited the largestresponse but only showed one strong connection withanother member of the carg bacteroides massiliensisotu98 rs q which suggests that pcopri might act to a larger degree independently to degrade arabinoxylan fig 3b the majority of taxa thatdecreased during arabinoxylan consumption particularlyb uniformisotu7 clustered within carg7 andshowed negative correlations with taxa of carg1carg2 and carg6 suggesting competitive or antagonistic interactionstemporal responses of otus and cargsto determine if short and longterm treatment witharabinoxylan and mcc differed in their effects on thefecal microbiota we compared shifts from baseline toweek w1 with those from baseline to week w6however there were no detectable differences betweenfig identification of coabundance response groups cargs during arabinoxylan supplementation a heatmap shows the change δw6bl inrelative abundance of otus affected by arabinoxylan p wilcoxon test the hierarchical dendrogram shows clustering of centered logratio clr transformed otus rows based on spearmans correlation distances by the completelinkage clustering algorithm and then groupedon the dendrogram into seven cargs by permanova p subjects columns clustered based on euclidean distances colors from blue tored indicate the direction and magnitude of change b coresponse network analysis each node represents an otu where the size isproportional to the change δw6bl in relative abundance the shape indicates direction of change positive circle negative square and thecolor references the respective carg to which it was clustered lines between nodes represent significant positive red line or negative blueline spearmans correlations rs values ¥ or and q values bl baseline otu operational taxonomic unit w6 week 0cnguyen microbiome page of the two time frames q wilcoxon test data notshown in addition comparison of baseline w1 andw6 values by friedmans test indicated that the effectsof arabinoxylan occur rapidly within week with nofurther detectable changes at weeks fig 4a considering these findings analyses on compositional changeswere performed with w6 data unless otherwise statedinterindividual variation in responses to arabinoxylanbacterial shifts in response to arabinoxylan and theirmagnitude were highly individualized fig 4b for instance absolute increases in relative abundance rangingfrom to to 429fold change were detected inseven subjects for the otu classified as blongumotu4 while other subjects showed either a muchsmaller increase a decrease or the species was undetectable otus related to b obeum otu85 subdoligranulum sp otu11 b ovatus otu26 and c leptumotu46 were enriched by arabinoxylan in around twothirds of the subjects less frequently enriched wereotus classified as potu6 b plebeiusotu53 and bacteroides sp otu56 p copri otu6responded in only four subjects but effects were largewith the species expanding beyond to 7foldchange ofthe total bacterial community in threesubjectscoprito determine drivers of these individualized responseswe used multiple linear regression mlr analyses totest if responses in otus that showed numerically thelargest shifts p copri b longum b obeum and subdolifig temporal and individualized responses of the otus and cargs affected by arabinoxylan and microcrystalline cellulose a plots show thetemporal response of the ten most abundant otus detected in of subjects and the seven cargs centered logratio transformed datawere analyzed by friedmans test with dunns correction to assess withingroup changes between time points ie δw1bl and δw6w1 bbubble plot shows individualized differences δw6bl in relative proportions of the ten most abundant otus percentage of total microbiotacomposition and cargs sum of otus detected after weeks of arabinoxylan and microcrystalline cellulose supplementation the size of thebubble is proportional to the change in abundance relative to baseline while the color of the bubble represents the direction of the change redincrease black decrease the x indicates that the otu was either undetected or the change was relative abundance bl baseline cargcoabundance response group otu operational taxonomic unit w1 week w6 week 0cnguyen microbiome page of granulum sp and in cargs with significant responsescargs and could be predicted by baselinediet or microbiota composition baseline microbiota allotus and significant otus and diet variables werefirst reduced in their dimensionality by principal component analysis pca and then treated as predictors thisanalysis revealed that individualized responses of bacterial taxa and cargs to arabinoxylan and mcc could notbe predicted by baseline diet or microbiota compositionq additional file fig s2effect on stool characteristics and bowel movementswhile fecal moisture content was not changed by eitherfiber q wilcoxon test additional file table s3subjects consuming arabinoxylan reported softer stoolconsistencies when compared to subjects consumingmcc treatment effect p gee model additional file fig s3a both arabinoxylan and mcc ledto an increase in bowel movements relative to baselinep gee model additional file fig s3b withno difference detected between treatment groups treatment effect p effect on fecal ph and scfasfecal ph and scfa concentrations did not change after weeks of either fiber treatment q wilcoxon testadditional file table s3 considering that absoluteconcentrations of fecal scfas are affected by their absorption in the gut we additionally assessedchanges in the percentages of acetate propionate andbutyrate relative to total scfa concentrations at w6which has been previously shown to vary little across colonic regions this analysis revealed an increase inthe percentage of propionate produced through arabinoxylan when compared to mcc q mannwhitney test and a reduction in the percentage ofbutyrate relative to baseline q wilcoxon test although differences in butyrate were not detected whencompared to mcc q further investigation ofthe ratio between propionate and butyrate showed an increase in propionate relative to butyrate when comparedto baseline q and mcc q suggestingthat arabinoxylan supplementation directed the outputof scfas in favor of propionatecharacterization of the temporal response in the threeprimary scfas also showed an increase in fecal propionate concentrations by arabinoxylan at w1 p friedmans test fig 5a although propionate concentrations remained elevated at w6 this increase was notstatistically significant when compared to baseline p this loss of significance was caused by an increasein the interindividual variation at w6 fig 5b visualevaluation of the individualized temporal response ofpropionate to arabinoxylan revealed clear separation offig temporal and individualized output of fecal scfas in response to arabinoxylan and microcrystalline cellulose supplementation a line plotsshow the temporal response of acetate propionate and butyrate reported as mean ± sd b individualized temporal propionate response of w6responders red and w6nonresponder black grouped based on δw6w1 data analyzed for a and b using friedman test with dunnscorrection to assess withingroup changes between time points and for b using mannwhitney tests to assess differences betweengroup ateach time point bl baseline carg coabundance response group otu operational taxonomic unit scfa shortchain fatty acid w1 week w6week 0cnguyen microbiome page of subjects into two distinct patterns fig 5b based on thedirection of change from w1 to w6 ie positive ornegative subjects were grouped into w6respondersδ w6w1 and w6nonresponders δ w6w1 in general w6responders showed a higher outputof propionate at w6 p friedmans test butnot at w1 while the opposite isseen in w6nonresponders p the two groups differed bypropionate concentrations at w6 p mannwhitney testw6propionate responders and nonresponders differ intheir microbiota response to arabinoxylanmicrobiota compositionalbaseline and shifts anddiet data were ordinated using pca and then differencesandbetween w6propionaterespondersnonresponders were tested using permanova thisthe bacterial communities ofanalysis revealed thatw6responders wereindistinguishablefrom w6nonresponders at baseline but differed in their response to arabinoxylan δw6baseline fig thiswas detected if analysis was based on all otus p the significant dietresponsive otus p or the seven cargs p in contrastneither baseline microbiota composition fig nordietary factors additional file fig s4a separatedaccording to w6 response p in addition comparing w6respondersinterms of their baseline total grain whole grain andtotal fiber consumption or their stool consistency andbowel movement frequency during treatment did notreveal any differences either p mannwhitneyand w6nonrespondersfig the individualized temporal propionate response to arabinoxylan associates with compositional responses in the fecal microbiota principalcomponent analysis plots based on euclidean distance comparing the relative abundance of fecal microbiota both at baseline and arabinoxylaninduced shifts δw6baseline between w6responders red and w6nonresponders black microbiota variables ie otu or carg thatcontributed the most to intersubject variation were shown as vectors on the plot when statistical significances were determined by permanovap carg coabundance response group otu operational taxonomic unit w1 week w6 week 0cnguyen microbiome page of test additional files and fig s3c and fig s4btogether these findings indicate that the temporal response in fecal propionate concentrations is primarilyassociated with the shifts in the microbiota and notbaseline microbiota composition or dietindividualized scfa responses can be predicted by gutmicrobiota featuresas with compositional responses gut microbiota functional responses to fiber interventions have been shownto be individualized [ ] but what drives this variation is poorly understood we applied mlr to determine whether fecal scfa responses could be explainedby stool consistency and bowel movement frequencydiet or microbiotarelated factors and then comparedthe quality ofthe models using corrected akaikeinformation criterion aicc values where lower valuesmean higher quality these analyses revealed that thew6 scfa response to arabinoxylan could be predictedby the fecal microbiota fig additional file fig s5but not by baseline diet stool consistency or bowelmovement frequency reported during treatment additional file fig s6a and fig s6b the best modelswere achieved for propionate especially when principalcomponents pcs generated from w6 shifts of all otuswere used as predictors fig 7a additional file tables4 models were of lower quality when w6 shifts of significant otus cargs pcs of cargs or single otuswere used suggesting that global community measuresexhibited stronger linear relationships with the propionate response than single or groups of taxa although themodels that used baseline and w1 shifts of otus asfig individualized arabinoxylaninduced propionate responses could be explained by baseline gut microbiota composition and microbiotashifts a heatmap shows the linear associations between the individualized propionate response δw6bl dependent variable columns andmicrobiota profiles bl δw1bl δw6bl predictors rows cells represent individual multiple linear regression models with fdr correction thatassess whether the predictors explain the individualized propionate response multivariate microbiota data were simplified into principalcomponent pc variables pc1 pc2 and pc3 prior to analysis each model contained the best one or two predictors of pcs individual cargs orsignificant otus selected by stepwise regression all models were adjusted by fiber dosesex colors from white to red indicate relative aicchighest aicc value x lower aicc values red indicate higher quality models b scattercorrected akaike information criterion values calculated byplots show the linear relationship between propionate responses δw6bl and either the baseline contribution of all otus to pc1 or the shiftsof carg1 color and size of each point indicate propionate response magnitude and the shaded area specifies the confident interval thetop six otus that contributed the most to either pc1 of all otus or carg1 are further provided ax arabinoxylan bl baseline carg coabundance response group mcc microcrystalline cellulose otu operational taxonomic unit w1 week w6 week aicc value 0cnguyen microbiome page of predictors were of lower quality than those based on w6shifts they are still valid showing q values less than after benjaminihochbergs false discovery rate fdrcorrection linear relationships between propionate responses and significant predictors using baseline pc1 ofall otus and shifts carg1 were further visualizedusing scatter plots fig 7b reaffirming the quality ofthe analysis as a majority of subjects fall within the confidence regionssignificant models could also be designed for acetateand butyrate responses to arabinoxylan additional file fig s5 interestingly in contrast to propionate the bestmodels to predict butyrate responses were achieved usingshifts of a single otu e oxidoreducens otu41 aknown butyrate producer however overallthemodels for acetate and butyrate were of much lower quality than those for propionate in summary while individualized responses in scfas showed no association withdiet they could be predicted by microbiota shifts andbaseline composition in contrast to the analysis of the effects of arabinoxylan not one single mlr model wasfound to be significant for mcc indicating that the statistical approach based on mlr models did not detect anyassociations independent of fiber fermentationdetermining the role of bacterial taxa in propionateresponsemlr analyses were applied to determine connectionsbetween arabinoxylan responding otus within cargs and and fecal propionate concentrations fig 8athis analysis revealed that shifts in p copri otu6 didnot predict propionate responses while blongumotu4 and correlated taxa in carg1 showed strongerlinear relationships the highest quality models were obtained with b obeum otu85 b plebeius otu53and p succinatutens otu38 all of which encodemetabolic pathways for propionate production such analysis provides a potential explanation for themetabolic interactions between proposed primary degraders secondary fermenters and metabolite utilizersthat result in the promotion of propionate in responseto arabinoxylan fig 8bdiscussionin the present study we characterized the impact of a6week highdose corn bran arabinoxylan supplementation on the composition and function of the fecalbacterial community in healthy adults with overweightand classi obesity arabinoxylan treatment changedfig relationship between propionate responses to arabinoxylan and proposed primary degraders secondary fermenters and metaboliteutilizers a individual multiple linear regression models determine otu responses δw6bl that predict the fecal propionate response δw6blyaxis shows the coefficient for each predictor as in the average propionate response when otu relative abundance increases xaxis showsthe p value for each predictor all models were adjusted by fiber dosesex where bubble size represents the adjustedr2 b proposed model ofbacterial crossfeeding in the gut during degradation of complex soluble arabinoxylans otu operational taxonomic unit 0cnguyen microbiome page of showing two distinctcommunity structure and induced specific shifts inthe composition of the gut microbiota that manifestedthemselves after week of treatment without furtherchanges at w6 arabinoxylan induced increases inpropionate output both compositional and functionalresponses were highly individualized with propionateresponsestemporal patternscompositional responses to arabinoxylan could not bepredicted and functional responses were independentof stool consistency bowel movement frequency andbaseline diet however baseline microbiota composition and especially the compositional shifts correlatedwith propionateresponses the nonfermentablemcc showed virtually no effect on gut microbiotacomposition or functionan understanding of compositional and functional responses of the gut microbiota to changes in diet requiresan ecological framework arabinoxylan supplementation provides resources that can be used by microbesthat possess the traits to either access the chemicalstructures directly or utilize public goods released duringarabinoxylan degradation in our study the dominant effects of arabinoxylan were directed toward twobacterial species b longum and p copri while nine additional otus showed smaller increases including threebacteroides species eg b ovatus b plebeius and bacteroides sp this high degree of specificity toward blongum over other bifidobacterium species is in agreement with other studies testing longchain arabinoxylans[] and genomic analyses that showed that genesencoding arabinoxylandegrading glycosidase eg xylosidase and αarabinofuranosidase are conservedonly among b longum strains [ ] in contrast to thespeciesspecific enrichment of b longum arabinoxylanenriched several species within the phylum bacteroidetesthat possess the genetic and functional traits necessaryfor accessing arabinoxylan [] although arabinoxylan utilization is not universally conse | 0 |
" preproofsevere acute respiratory syndrome coronavirus sarscov2 is a highly contagious zoonotic pathogen that has exacted heavy public health social and economic tolls in february the world health anization acronymed the disease caused by sarscov2 as covid19 for coronavirus disease the number of confirmed covid19 infections which has been detected in at least countries has reached worldwide as of april with deaths according to the us centers for disease control and prevention cdc1 many cases of covid19 resolve quickly however the disease which like other respiratory pathogens that cause common cold symptoms is believed to be transmitted through respiratory droplets infection with covid19 can also lead to significant morbidity and death this is particularly the case for cancer patients moreover because the signs and symptoms of covid are easily misattributed to the sequelae of cancer itself such as pulmonary embolism or its treatment such as nausea and diarrhea diagnosis may be delayed or missed potential covid rule out criteria based on the wells criteria for pulmonary embolism another protean disease entity are provided as a decisionmaking aid this review summarizes the current understanding of the transmission clinical presentation diagnosis and differential diagnosis pathogenesis rationale to treat the cancer or not treatment and prevention of covid19 with an emphasis on implications in cancer keywords covid19 sarscov2 cancer introduction sarscov2 the rna virus responsible for the illness which has been named covid19 for coronavirus disease2019 the year it was diagnosed has sent shockwaves and dominated the news cycle due to its pandemic spread from the point of origin in wuhan china to the rest of the world declared a public health emergency of international concern pheic by the world health anization who2 sarscov2 is the third highly pathogenic novel zoonotic bat coronavirus sonamed because of the crownlike spikes on its surface to have emerged the first was sars coronavirus now named sarscov1 in with a fatality rate of and 0c preproofthe second was middle east respiratory syndrome mers in with a fatality rate of where the camel was the intermediate host3 sarscov1 merscov and sarscov2 belong to the betacoronavirus genus which are enveloped positivestranded rna viruses whose approximately nucleotide genome serves as an mrna template for the translation of viral proteins4 the virion contains four proteins spike envelope membrane and nucleocapsid and the host receptor with which the spike surface glycoprotein of sarscov2 engages is angiotensin converting enzyme ace25 the biology of sarscov2 is described in more detail in figure a zinc metallopeptidase enzyme ace2 which is abundantly present in lung and gastrointestinal epithelial cells6 not only mediates viral entry through receptormediated endocytosis7 but also the efficiency of viral replication8 its expression is upregulated with older age smoking the antihypertensives angiotensin converting enzyme ace inhibitors and angiotensin receptor blockers arbs thiazolidinediones tzds a class of oral antidiabetic drugs and ibuprofen risk factors which may increase susceptibility to the covid19 virus infection and which are common in generally elderly multimorbid cancer patients9 preproofthe clinical spectrum of sarscov2 ranges from mild upper respiratory tract infection with fever sore throat headache cough and potentially nausea and diarrhea the majority of cases recover without serious complications to severe pneumonia with sequelae that include acute respiratory distress syndrome ards cytokine storm and death10 because sars is an acronym for severe acute respiratory syndrome digestive manifestations including inappetence nausea abdominal pain and diarrhea of covid19 resulting from binding of the virus to the ace2 receptor in the gi tract may precede respiratory symptoms11 gastrointestinal manifestations are potentially underappreciated and overlooked as sentinel symptoms that herald the onset or persistence of disease especially in cancer patients and may contribute to delayed or missed opportunities for testing diagnosis and containment unlike sarscov1 which seems to have disappeareddied out12 and mers which reappears only sporadically sarscov2 is less lethal with a fatality rate between although this number is highly uncertain and debated13 but much more infective consequently public panic and economic disruption have ensued resulting in wartimelike mobilization efforts to mitigate its spread old age smoking and comorbidities such as diabetes morbid obesity immunosuppression frailty and cardiovascular disease appear to predispose to worse outcomes possibly secondary to impaired t and b cell responses notably covid19 infection is associated with lymphopenia and delayed development of the adaptive immune response which appears to correlate with prolonged virus clearance and more severe disease progression15 the first pillar of defense against infection is hand washing avoidance of face touching and minimization of close contact ie social distancing with selfquarantine and selfisolation16 in case of exposure or evidence of covid19 symptoms respectively the second prophylactic pillar is vaccination with specific viral antigens or mrnas which are not yet publicly available although the company moderna has reportedly started testing an mrna vaccine in healthy volunteers and multiple other vaccination strategiesplatforms appear to be in progressunder development17 0c transmission and prevention according to the world health anization who21 sarscov2 is spread persontoperson mainly via aerosol inhalation from sneezing coughing or exhalation 22and via fomitetoface contact since depending on the surface material the virus may remain viable and infectious for hours to days figure fecaloral transmission has also been hypothesized because diarrhea was a common feature with sars and mers and diarrhea and other digestive issues have also been reported in patients with covid1925 26notably transmission of sarscov2 is not limited to symptomatic individuals ie those with fever cough sore throat myalgias or dyspnea but also to asymptomatic or subclinically infected carriers of the virus which is problematic from the perspective of disease control 27and highlights the importance of containment measures including isolation and quarantine the basic reproduction number r0 of sarscov2 is which means that on average for every patient an additional individuals are infected because coronaviruses may persist on inanimate surfaces like metal glass or plastic for up to days careful disinfection with or greater ethanol for small surfaces or sodium hypochlorite for larger surfaces is recommended preproofdiagnosis and clinical features preproofthe available evidence is limited but clinical courses and outcomes of covid19 are likely to be worse in patients with cancer especially given the clear association between severity of disease and older age and higher levels of comorbidity the overall case fatality rate cfr for the general covid19infected population is around but in cancer it rises to overall and to in italy19 this cfr in cancer patients compares to for no comorbid conditions for cardiovascular disease for diabetes for hypertension and for chronic respiratory disease the aim of this review is to summarize and condense the current understanding of the transmission clinical presentation diagnosis and differential diagnosis pathogenesis treatment and prevention of covid19 with a special focus on cancer in the united states the test of choice for sarscov2 is a nasopharyngeal swab specimen or sputum if a productive cough is present on which a reversetranscriptase pcr rtpcr assay or an enzymelinked immunoassay eia directed particularly at the envelope e rnadependent rna polymerase rdrp spike protein s and nucleocapsid n genes is performed the fda has also approved an antibody test29 a positive test for sarscov2 in a symptomatic patient generally confirms the diagnosis of covid19 with the caveat that false positive and false negative tests have been documented if initial testing is negative but a high index of suspicion and pretest probability for covid19 remains on the basis of patient signs and symptoms then retesting is indicated in patients with high indexes of clinical suspicion and equivocal or negative test results the who recommends that lower respiratory tract specimens which contain the highest viral loads should be obtained since nasopharyngeal swabs may miss some infections30 according to cdc guidelines disease is excluded on the basis of two 0c preproofconsecutive negative tests respiratory tests separated by ¥ hours however in the presence of suggestive symptoms rectal swabs may also be indicated since the ace2 enzyme to which the virus binds is abundantly present in rectal epithelia cells31 the differential diagnosis for sarscov2 in cancer is extremely broad and includes conditions such as foreign body aspiration toxicities from chemotherapy and radiation tumor progression postobstructive pneumonia malignant obstruction atelectasis pulmonary embolism pneumonitis pulmonary edemafluid overload immunotherapyrelated pneumonitis copd exacerbation q fever adenovirus bocavirus coronavirus 229e hcov 229e coronavirus hku1 hcov hku1 coronavirus nl63 hcov nl63 coronavirus oc43 hcov oc43 human metapneumovirus hmpv influenza a influenza a subtype h1n1pdm09 influenza a subtypes h1 and h3 influenza b parainfluenza virus piv respiratory syncytial virus ab rsv ab rhinovirusenterovirus hrvev bordetella pertussis legionella pneumophila and mycoplasma pneumoniae32 the diagnosis of sarscov2 is complicated by the possibility of simultaneous coinfection with other respiratory viruses33 which is especially true for immunosuppressed cancer patients whose susceptibility to microanisms is increased the heightened infectious risk for cancer patients underscores the importance of screening them at presentation with extended viral respiratory panel testing given that coinfection may impact management decisions since conceptually at least the morbidity of covid19 and the risk of severe illness should increase in the presence of a second or third virus preproofunlike infection with influenza for example covid19 signs and symptoms may vary considerably depending on the dose of viral inoculum route of inoculation concomitant medications and underlying health status34 to include fever dry cough fatigue sputum production shortness of breath sore throat headache myalgia or arthralgia chills nausea or vomiting nasal congestion diarrhea hemoptysis and conjunctival congestion with an incubation period of to days after exposure36 presymptomatic or minimally symptomatic infection may majorly drive transmission especially since detected viral loads are similar in both symptomatic and asymptomatic patients3738 populations of concern include the elderly smokers vapers and dual users those of any age with preexisting chronic medical conditions those receiving particular medications or therapies which upregulate the ace2 receptor or suppress the immune system and those from lower socioeconomic classes a conglomeration of factors which are often present in cancer patients as depicted in figure while the surveillance focus for covid19 is on the respiratory tract enteric symptoms are a potentially underappreciated overlooked and misattributed manifestation of disease as stated earlier and this is especially the case for cancer patients where gastrointestinal toxicity occurs routinely from chemotherapy ie cisplatincarboplatinoxaliplatin irinotecan 5fluorouracil ifosfamide from targeted agents ie erlotinib imatinib bortezomib temsirolimus sunitinib regorafenibsorafenib and bevacizumab39 and from locally advanced or metastatic disease therefore abdominal complaints in cancer patients which are potentially but not automatically attributable to underlying disease justify further investigation especially if persistent worsening or new particularly because sarscov2 transmission may occur via the fecaloral route40 0c preproofabnormal laboratory findings in covid19 include lymphopenia percent prolonged prothrombin time percent elevated lactate dehydrogenase percent elevated ast and alt percent elevated highly sensitive hs crp and elevated procalcitonin however because these parameters routinely fall well outside of the normal reference range in cancer patients it is difficult to confirm or refute the presence of disease on this basis alone chest radiographs and chest ct abnormalities are similarly nonspecific since the most common features multifocal groundglass opacities and consolidation mimic other pneumonias41 significant antibody production is observed after infection but it is unknown whether this helps or harms since antibodydependent enhancement ade may potentiate viral entry and the induction of a severe inflammatory response42 universal screening of cancer patients for covid19 is desirable but logistically impossible for the foreseeable future since diagnostic tests are in short supply and simply not always readily available43 hence covid19 rule out criteria are proposed in table as a potential decisionmaking aide mémoire which separates patients into low and highrisk groups by analogy to the wells criteria for pulmonary embolism4445 preventive measures focus on selfisolation social distancing with a 6foot 2m separation46 frequent hand washing with soap and water andor use of hand sanitizers patient isolation during clinical care use of masks to help prevent aerosol transmission and flushing with the lid closed to control socalled toilet plume in an asco guidance immunocompromised cancer patients are advised to minimize exposure to sick contacts and large crowds48 for healthcare personnel the use of personal protective equipment such as n95 masks ffp3 masks gowns eye protection gloves and gowns is mandated49 preproof vaccination and immunity vaccination efforts and the related topic of whether those who have recovered from covid19 develop protective immunity have drawn great attention the latter has implications on whether people who test positive for sarscov2 antibodies can be safely assumed to be immune and at negligible risk of contracting or transmitting the disease there have been case reports of patients who have recovered from covid19 and had recurrence of rtpcr positivity approximately one month after initial diagnosis with only one patient exhibiting significant clinical symptoms and another having a mild intermittent cough50 but while not zero the risk of transmissibility or recurrence of symptomatic disease in recovered patients has yet to be quantified and the paucity of currently available reports of recurrence in the setting of a pandemic suggests that it is low a separate practical question will be whether antibodybased tests prove to have sufficient sensitivity and specificity to identify people who had asymptomatic infections developed immunity and can return to normal activities without jeopardizing disease containment efforts immunity may be due to antibodies cell mediated immunity or a combination of the two previous experience with using plasma from convalescent patients to treat severe cases of the first sars and mers as well as limited experience with covid19 suggests that antibody mediated immunity alone is clinically beneficial even during acute infection51 safety concerns about antibodies have been raised based on preclinical studies of sarscov vaccination in 0c preproofferrets showing hepatotoxicity52 and of vaccination against feline infectious peritonitis virus another coronavirus leading to more severe disease when kittens were subsequently challenged with the virus53 although animal models may not be representative of human hostpathogen interactions the nature of sarscov and sarscov2 antibodies are likely different as crossneutralization was not observed invitro54 and experience with convalescent plasma has not borne evidence of antibody mediated enhancement of infection in acutely infected patients the potential risk deserves attention if vaccination is proposed for the entire population t cell responses are also readily observable in patients who recover from coronavirus infections55 and memory t cell responses alone were protective in mice56 with the potential advantage of longer persistence of memory t cell responses compared to humoral immunity when clinical data on vaccine candidates becomes available cancer patients may face different considerations surrounding vaccination than the general population particularly patients with hematologic malignancies being treated with agents targeting b cells who would derive greater benefit from vaccines eliciting cell mediated than antibody responses preproofpathogenesis and pathology relating to ace2 and ras signaling the ace2 enzyme a key regulator of the reninangiotensin system ras57 to which the virus binds through its surface spike proteins is particularly abundant in the digestive tract lungs kidney heart and blood vessels where pathology from sarscov2 occurs58 a peptidase that catalyzes the conversion of angiotensin ii angii referred to as the quintessential perpetrator of inflammation to angiotensin ang ace2 mediates antiproliferative and vasodilatory functions that oppose the vasoconstrictive and inflammatory functions of angiotensin converting enzyme ace60 the binding of sarscov2 to ace2 leads to downregulation of ace2 expression potentially through increased internalization and shedding from the cell surface with decreased ang1 generation and increased ang ii levels as a consequence61 this unfavorably shifts the balance of the renin angiotensin system ras from the vasoprotective ace2ang17 axis to the aceang iiangiotensin at1 receptor axis and drives a proinflammatory profibrotic and proliferative response62 as shown in figure fang et al63 contend that because thiazolidinediones ibuprofen and angiotensin converting enzyme ace inhibitors and angiotensin ii typei receptor blockers arbs substantially increase the expression of ace2 they facilitate sarscov2 infection and therefore the risk of severe and fatal covid19 in contrast alghatrif et al64 present a diametrically opposed hypothesis that downregulated ace2 signaling is responsible for sarscov2induced acute lung injury ali acute respiratory distress syndrome ards and cytokine storm and that aceis and arbs are beneficial precisely because they increase ace2 expression and activity furthermore according to alghatrif et al lower ace2 levels and hence higher baseline oxidative stress and inflammation6566 are present in older comorbid individuals such as cancer patients which renders them more susceptible to severe covid19 than younger noncomorbid individuals with increased ace2 levels and lower baseline inflammation as shown in figure furthermore low ace2 may promote tumor progression and conversely ace2 overexpression is associated with antiangiogenesis and tumor regression67 in summary then 0c preproofdespite the concerns and controversy68 surrounding the use and continuation of aceisarbs during the sarscov2 epidemic it is likely that the pros outweigh the cons especially in cancer patients due to their potential antitumor and anticovid19 effects69 in line with ras involvement emerging data suggest that sarscov2 infection may induce serious cardiovascular injury or exacerbate existing cardiovascular disease cardiovascular sequela includes heart failure arrythmias disseminated intravascular dissemination dic and troponin elevation which may closely correlate with disease severity and the likelihood of inhospital death70 liu et al71 propose a mechanism whereby the virus which lowers hemoglobin hb levels72 binds to the porphyrin of heme and displaces iron thereby compromising the oxygencarrying capacity of red blood cells and exacerbating the hypoxemia since chloroquine and the experimental anticancer agent rrx001 also bind to porphyrins they may competitively interfere with binding by the virus rationale for continuation or discontinuation of cancer therapy preproofthe benefitrisk calculus that informs the decision whether and how to treat with anticancer therapy falls into a gray zone about which no consensus exists leading to a therapeutic dilemma on the one hand zhang et al73 in annals of oncology reported a strong association in patients of them with lung cancer between antineoplastic therapy in the past days and severe effects of covid19 hr4079 ci p0037 on this basis the authors recommend treatment interruption dose reduction or substitution of cytotoxic chemotherapy with nonimmunosuppressive options eg checkpoint inhibitors if available especially in the case of lung cancer patients that are already prone to develop respiratory infections and complications74 similarly heavily immunosuppressed patients such as those who have undergone hematopoietic stem cell transplantation are also particularly susceptible to viral respiratory infections these findings are supported by a nationwide analysis of data75 in china from covid19 patients of which were diagnosed with cancer this patient cohort experienced a higher incidence of severe events vs p and the administration of chemotherapy or surgery was found to have increased the risk of death andor intensive care unit admission even after adjusting for age sex and comorbidities odds ratio or ci p while these studies are limited by small sample sizes the data suggests that cancer predisposes to more severe disease therefore since inperson contact increases the risk of transmission several institutions have mandated realtime video or telephone interactions alternatively referred to as telehealth77 postponed surgeries biopsies endoscopies scans and routine investigations when possible and in line with esmo guidelines78 encouraged conversion from the intravenous to the oral route eg 5fluorouracil to capecitabine etoposide and vinorelbine on the other hand the immediate existential threat of progressive disease for which death is an impending imminent certainty rather than a remote possibility in the absence of treatment likely outweighs the theoretical risk of sarscov2 infection even in lower risk disease for example in situ or localized prostate breast and head and neck cancer delayed treatment is 0c preproofpotentially conducive to tumor development and progression and thus may unfavorably impact prognosis79 hanna et al have proposed a triage strategy80 which prioritizes treatment for those patients with imminent risk of early mortality from acute leukemias aggressive lymphomas metastatic germ cell tumors oncologic emergencies such as spinal cord compression chemoradiotherapeuticresponsive cancers such as head and neck cervical and anal cancers and neoadjuvant or adjuvant therapyresponsive tumor types such as stage iii colon cancer and deprioritizes visits for surveillance and survivorship however in the absence of a one size fits all consensus recommendation which is unlikely since cancer is so genetically diverse and heterogeneous the decisionmaking process and the subsequent treatment plan are individualized and to be determined tbd on casebycase basis taking into account multiple factors including the risk of cancer recurrence if therapy is delayed modified or interrupted the type of therapy eg surgery radiation chemotherapy checkpoint inhibitors and stem cell transplantation extent of comorbidities concomitant medications patient preferences physicianpatient relationship race age the number of cycles of therapy completed and treatment tolerance in terms of specific cancerrelated conditions asco makes the following heavily qualified recommendations81 \uf0b7 growth factor prophylaxis for neutropenia and neutropenic fever even at lower levels of risk as well as empiric antibiotics for acute care \uf0b7 erythropoietinstimulating agents for anemia prophylaxis and transfusion when necessary depending on the patient context and underlying comorbidities preproof treatment based on the high transmissibility of the virus82 the main nonpharmacologic countermeasures to mitigate or delay the impact of covid19 include rigorous hand hygiene use of facemasks respiratory etiquette ie coughing or sneeze into the upper sleeve or elbow not the hands flushing with the lid down to prevent bioaerosolization as well as quarantine stay at home policies and workplace and school closures which have upended the social cultural political and economic status quo no specific treatment or vaccine is currently available although promising activity has been reported for hydroxychloroquine chloroquine arbidol remdesivir convalescent sera and favipiravir the mainstay of medical therapy includes symptomatic care such as supplemental oxygen antibiotics and hemodynamic and mechanical ventilatory support if indicated for septic shockmultiple an failure and respiratory failure respectively83 over active clinical treatment trials are underway84 these include vaccines as well as a number of different agents some with promising preliminary data as mentioned above and also those with potential anticancer activity which will hopefully serve a double purpose first to treat covid19 and second as an adjunct to bridge the time gap until the patient is recovered and the primary antineoplastic is startedrestarted as shown in table 0c preproof conclusions the alarming spread of the covid19 pandemic has disproportionately affected cancer patients an atrisk population both from the standpoint of increased disease severity and disruption to care which includes widespread suspension of clinical trials in the united states that are already fraught with barriers to enrollment and participation85 86because the symptoms of covid19 are nonspecific underlying symptoms from the cancer eg dyspnea cough fever fatigue diarrhea etc which overlap with those from the viral infection may obscure and delay the diagnosis hence if the covid19specific rapid reverse transcriptase polymerase chain reaction rtpcr test is not readily available andor in short supply which is currently the case diagnosis will depend on the maintenance of a high index of clinical suspicion especially in advanced cancer patients who check all the boxes for risk factors such as older age frailty disability immunosuppression generalized systemic inflammation and multiple comorbidities eg hypertension diabetes and cardiorenovascular diseases that predispose to severe disease and death preproofthese comorbidities are commonly treated with renin angiotensin system blockers such as angiotensinconverting enzyme inhibitors aceis or angiotensinreceptor blockers arbs which increase levels of ace2 the continued use of aceisarbs is the centerpiece of an intense debate because on the one hand sarscov2 coopts ace2 for target cell entry but on the other ace2 overexpression may counterbalance vasoconstriction and profibrotic processes and thereby reduce the incidence or mortality associated with covid19associated ali or acute respiratory distress syndrome another controversy involves whether or not to continue cancer treatment given the high transmissibility potential of the virus however since no expert consensus recommendations have been issued to date and prognosis stage and responses to therapy are highly heterogeneous the riskbenefit tradeoff and subsequent treatment plan are highly individualized and context dependent currently the focus of treatment is infection control appropriate symptomatic care and oxygen therapy no approved medication or vaccine has been developed but promising activity has been reported for hydroxychloroquine chloroquine arbidol remdesivir convalescent sera and favipiravir and several repurposed agents with antitumor properties are under investigation including thalidomide and rrx001 which may hopefully bridge the gap from the time covid is first diagnosed until the primary anticancer therapy is restarted finally multiple comparisons have been made between the allout mobilization efforts to combat covid19 with the massive scaleup of human and material resources that occurred during world war ii8788 in the words of winston churchill prime minister of great britain from whose intrepid fighting spirit iron will and intransigent defiance of tyranny galvanized the resolve of an entire nation to fight on in the face of seemingly impossible odds oncologists on the frontlines that have answered the call should never worry about action only inaction 0c preproof declaration of interests the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper references sitammagari k skandhan a dahlin awhat hospitalists need to know about covid19 medscape mar 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2019ncov transmission through the ocular surface must not be ignored the lancet preproof 0c preproof holshue ml et al first case of novel coronavirus in the united states n engl j med yeo c kaushal s yeo d enteric involvement o | 0 |
" clinical trials have been conducted to clarify the beneficial effects of vd3 1α25dihydroxy vitamind3 also known as calcitriol treatment in prostate cancer however the molecular mechanisms underlying theseeffects are not fully understood recent studies on igfbp3 have indicated its intracellular functions in cell growthand apoptosis the aim of this study was to confirm the benefits of lowdose vd3 treatment and clarify themolecular mechanisms underlying these beneficial effects in prostate cancer cellsmethods the molecular effects of simultaneous treatment of lncap cells and their genetically modified cell lineswith low concentration of docetaxel and vd3 were biologically and biochemically analyzed to further determinethe effects of vd3 treatment on igfbp3 induction system cells were temporarily treated with vd3 in combinationwith a transcriptional inhibitor or protein synthesis inhibitor bcl2 protein and its mrna behavior were also observed inigfbp3 expressionmodified lncap cells to determine the involvement of igfbp3 in the suppression of bcl2 by vd3treatmentresults changes in igfbp3 expression levels in lncap cells indicated that it mediated the inhibition of cell growthinduced by vd3 treatment igfbp3 was also found to be a mediator of the enhanced cytotoxicity of prostate cancercells to vd3 in combination with the anticancer drug we further identified the distinct property of the igfbp3induction system wherein temporal vd3 stimulationinduced prolonged igfbp3 expression and vd3 treatmentinduced increase in igfbp3 expression were optimized based on the protein concentration rather than the mrnaconcentration meanwhile bcl2 expression was downregulated by vd3 treatment in an igfbp3independent manner these findings indicate the molecular mechanisms of igfbp3 induction stimulated by vd3 and igfbp3independent bcl2 suppression by vd3 treatment in prostate cancer cells the results could prompt a reevaluation ofvd3 usage in therapy for patients with prostate cancerkeywords vitamin d nonlinear igfbp3 induction bcl2 suppression prostate cancer treatment correspondence satorusasagawatokushukaijp1molecular biology laboratory research institute nozaki tokushukai hospitaltanigawa daito osaka japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cigarashi bmc cancer page of vitamin d has a central role in calcium and skeletalhomeostasis [ ] its pleiotropic role both in physiological and pathological phenomena such as cell growthimmune function and tumorigenesis has also been examined [] which revealed that exposure of cancercells to vitamin d significantly reduces the cell growthrate in multiple cancer types [] indeed recent epidemiologicalinvestigations have reported that highervitamin d concentration could prevent multiple types oftumorigenesis consistent with such finding for example an increase in colon cancer incidence with lowervitamin d dietary habits has been reported [ ]however suppressive effect on prostate cancer is stillunder discussion [ ]in turntraditional methodscurrently prostate cancer is one of the most commonthecancers in men worldwide clinical use ofprostatespecific antigen psatest dramatically improved the screening sensitivity of prostate cancer compared to that ofthenumber of patients with earlystage prostate cancer hasrapidly increased since the mid1990s [ ] unlikeother cancer types most cases of prostate cancer haveslow progression or have nonprogressive indolentsymptom and are localized in the prostate thus they areunlikely to cause poor physical condition or death therefore patients with prostate cancer need a less burdensome treatment in order to avoid potential harmfrom excessive treatmentalthough the impact of vitamin d as a single agent onprostate cancer has been investigated its significance remains under discussion [ ] meanwhile the synergistic or additive effects of vitamin d and its derivativeswith anticancer drugs on prostate cancer have beenclinically studied and encouraging results have been reported [ ] however the results of larger trials thatevaluated the synergistic effect of vitamin d in combination with docetaxel one of the firstline anticancerdrugs in prostate cancer chemotherapy showed limitedor nonsignificant benefit of vitamin d efficacy in castration or androgen deprivation therapyresistant prostatecancer [ ] furthermore overconsumption of 1α25dihydroxy vitamin d3 vd3 also known as calcitriolthe biologically active form of vitamin d3 from food orprolonged treatment with vd3 derivatives could triggerhypercalcemia resulting in physiological side effects therefore to date vd3 has not been proactivelyused in the treatment of patients with prostate cancerthe biological function of vitamin d is mainly mediatedby vitamin d receptor vdr which acts as a transcriptional factor vitamin d receptor elements vdreon the promoter region of target genes are recognizedand transcriptionally activated by vitamin dcoupledvdr consistent with the diverse physiological functionof vd3 vdre was identified not only in the gene related to calcium and skeletal homeostasis but also in thegene related to fundamental cellular functions includingcell growth igfbp3 is one of the families of sixhigh affinity igfbps and was originally found in plasmaas a stabilizer and transporter of igfs in the bloodstream interestingly vdre was found on the prothe igfbp3 gene and recent studies havemoter ofrevealed that igfbp3 functions inside the cell as wellregulating cell growth and apoptosis [ ]methodsthis study aimed to investigate igfbp3 induction byvitamin d treatment and determine its role in prostatecancer treatment with vitamin d in combination withanticancer drugs in order to provide molecular biologicalevidence of benefit of vitamin d and to suggest effectivevitamin d usage in prostate cancer treatmentchemicals and reagentsdihydrotestosterone dht and calcitriol vd3 purchased from tokyo chemical industry tokyo japanwere resolved in ethanol as a stock solution pei maxmolecular weight was purchased from polysciences pa usa the other chemicals and reagentswere purchased from wako pure chemicalosakajapan and sigmaaldrich st louis mo usaincluding testosteronecharcoal stripping of fetal bovine serum fbsfbs was purchased from gibco waltham ma usato deplete hormonesin fbsdextrancoated charcoal powder was added to theserum and the mixture was incubated with rotation at degree overnight thereafter the mixture was centrifugedto pellet charcoal and the supernatant was filtered througha 022μm polyvinylidene difluoride membrane thecharcoalstripped serum was used for all experimentstotalthe concentrations oftestosterone and totalvitamin d in the serum were determined using a totaltestosterone test kit abbott japan chiba japan and atotal vitamin d test kit roche basel switzerland according to manufacturers instructions the concentrations of total testosterone in the pre and posttreatmentserum were nm and less than nm limit of detection respectively the concentrations of total vitamin d in the pre and posttreatment serum were and nm respectively thus the basal concentrationsin the culture medium supplemented with fbs wereless than nm total testosterone and approximately nm total vitamin dcell culturethe lncap cell line was obtained from american typeculture collection and cultured in dulbeccos modified 0cigarashi bmc cancer page of eagle medium dmem sigmaaldrich supplementedwith 10charcoalstripping fbs the 293ft celllinewas purchased from invitrogen waltham ma usaand cultured in dmem supplemented with fbs mm of lglutamine sodium pyruvate and nonessentialamino acids the cells were cultured at a temperature of °c in co2humidified condition the mycoplasma contamination was routinely checked and confirmed as negativecell growth assaydmem supplemented with charcoalstripping fbs was used for the cell growth assay the cells were seededat à cells per well in a 6well plate the next daythe medium was replaced with ml of fresh mediumand nm dht andor nm vd3 were added the cellculture was continued throughout the indicated periodthe cultured cells were trypsinized and the number ofcells was assessed using an automated cell countercountess iitm fl invitrogen each assay was repeatedat least three times and similar results were obtainedwestern blottingthe antibodies used for western blotting were mouseantiigfbp3 santa cruz biotechnology mouseantiβactin santa cruz biotechnology mouseantibcl2 santacruz biotechnology and horseradish peroxidaseconjugated secondary antibodies jackson immunoresearch laboratories westgrove pa usa the collected cells were resuspendedin ripa buffer supplemented with protease and phosphatase inhibitors roche basel switzerland and lysedusing bioruptortm ii sonicator cosmo bio tokyojapan cell lysates were resolved by nupagegels invitrogen and transferred onto polyvinylidenefluoride membrane millipore burlington ma usathe signals were developed using enhanced chemiluminescence reagent perkinelmer waltham ma usaand luminograph i atto tokyo japan was used forimage capture quantification of band signal wasanalyzed using cs analyzer software atto at leasttwo biological replicates of each experiment were performed with similar resultsrealtime reverse transcriptionpolymerase chain reactionthe total rna from the cultured cells was extractedusing trizol reagent invitrogen according to the manufacturers instruction the rna was reverse transcribedby the primescript rt reagent kit takara bio shigajapan using oligodt quantitative reverse transcriptionpolymerase chain reaction rtpcr reaction wasperformed using tb green premix ex taq ii takaraand genespecific primers supplementary table bycfx96 touch realtimepcr system bioradlaboratories hercules ca usa gene expression datawere normalized against glyceraldehyde 3phosphate dehydrogenase or hprt1 as internal control at leastthree biological replicates of each experiment were performed and similar results were obtainedflow cytometric analysis of cell cycle and apoptosisfor cell cycle analysis and apoptotic cell detection flowcytometric analysis was performed using the guavaeasycyte plus flow cytometry system millipore andguava cell cycle reagent and annexin v fitc apoptosiskit millipore according to manufacturers instructionas previously described at least three biologicalreplicates of each experiment were performed and similar results were obtainedlentiviral construction and transductionbackbone vectors plko1 puro plasmid andplenti cmv puro dest w118 plasmid were provided by drs bob weinberg eric campeau andpaul kaufman respectively via addgene ref pentr1a plasmid and lentiviral packaging mix plp1plp2 and plpvsvg were purchased from invitrogenfulllength igfbp3 was cloned from lncap cell complementary dna cdna using kod fx neo toyoboosaka japan with a specific primer set supplementarytable and inserted into the pentr1a vector thenthe sequence was confirmed the igfbp3 cdna was introduced into the plenti cmv puro dest vector by recombinaseenzymeinvitrogen to generate the lentiviral expression vectorspecific short hairpin rnas shrnas were designedusing invitrogen or biosettia websites the selectedtarget sequence oligos supplementary table wereannealed and inserted into the plko1 puro vector according to addgenes instruction the lentiviral expressionvector or shrna vector was cotransfected with the lentiviral packaging mix into the 293ft cells using pei maxinstead of lipofectamine according to invitrogensinstruction twentyfour hours posttransfectionthemedium was replaced with the culture medium forlncap cells one day later the lentiviruscontaining supernatants were collected and filtrated through a 045mmpolyvinylidene fluoride filter milliporereaction using lr clonaseiilentivirus infectionone day before infection à lncap cells wereplated into a 10cm dish then the culture medium wasreplaced with the lentiviruscontaining supernatant andculture was continued twentyfour hours post infectionreplaced with fresh culturemedium two days later the medium was replaced withfresh culture medium that contained μgml of puromycin and culture was continued until the noninfectedthe medium was 0cigarashi bmc cancer page of control cells were completely killed the puromycinselected cells were then subjected to each assaystatistical analysisresults are presented as mean ± standard deviation unless otherwise specified pairs of groups were comparedusing a twotailed unpaired students ttest onewayanalysis of variance was used for multiplegroup comparisons rather than specifying three a pvalue was considered statistically significant all statistical analyses were performed using excel software microsoftredmond wa usa and statcel3 addin for exceloms publishing tokyo japanresultsvd3 reduces cell growth rateconsistent with a previous report that treatmentwith vd3 inhibits growth of prostate cancer cells ourresults showed that vd3 treatment reduced the cellgrowth rate in a dosedependent manner and nm fig 1a left as shown below igfbp3 induction activity of vd3 was reached to plateau at nm concentration fig 4a on the other hand testosterone hasbeen reported to stimulate the growth of prostate cancer and the results of this study confirmed that dhttreatment from very low concentration nm stimulated cell growth rate and its activity was reached toplateau at less than nm concentration fig 1a centerthe purpose of our study was to investigate the role ofvd3igfbp3 induction system in cell growth inhibitionand to propose the potency oflowdose vd3 usagewhich could evade sideeffect of vd3 treatment such ashypercalcemia in therapy for patients with prostate cancer thus nm of dht and nm of vd3 concentrations were chosen as minimum but stably workingconcentration for following experiment previouslyithas been demonstrated that simultaneous treatmentwith vd3 and dht enhanced the reduction of cellgrowth rate compared to treatment with vd3 alone anda similar result was reproduced with lowdose dht nm and vd3 nm in this study fig 1a right tofurther characterize growth inhibitory effect with lowdose of dht nm and vd3 nm cell cycle andapoptotic analyses were performed with flow cytometrythe cell cycle analysis revealed that there was no significant change in the cell cycle phase distribution amongfig cellular response of lncap cells treated with vd3 and dht a effect of combined treatment of vd3 and dht on cell growth in lncapcells left vd3 treatment reduced cell growth in a dosedependent manner center dht treatment increased cell growth at lowerconcentrations right simultaneous treatment of vd3 and dht enhanced the reduction of cell growth compared to treatment with vd3 aloneb change in cell cycle phase by vd3 or dht treatment neither vd3 nor dht treatment significantly changed the cell cycle phase c induction ofapoptosis by vd3 or dht treatment neither lowdose vd3 nor dht treatment influenced apoptosis at shortterm d induction of igfbp3expression by vd3 or dht treatment vd3 treatmentinduced igfbp3 expression and cotreatment with dht enhanced the expression level ofigfbp3the ratio indicates the density of igfbp3 band normalized by corresponding βactin band the all experiments were performed in serumcontaining medium condition the uncropped fulllength blot images are presented in supplementary fig 5a 0cigarashi bmc cancer page of control no treatment dht nm vd3 nm anddhtvd3 treatment conditions fig 1b suggestingthat lowdose of vd3 or dhtvd3 treatment did notarrest the cell cycle at a specific phase previous reportshave shown that longterm vd3 treatment has apoptosisinducible activity and dht has apoptosis inhibitingactivity in a dosedependent manner in lncapcells however it was unclear or controversial whetherlowerconcentration of vd3 and dht at shorttermcould influence apoptosis respectively in lncap cellsthus the apoptosis assay was performed with nm ofdht and nm of vd3 for shortterm and found thatneither lowerdose dht vd3 nor dhtvd3 treatmentfor shortterm influenced apoptosis fig 1c these results suggested that the decrease in the cell number induced by lowdose vd3 or dhtvd3 treatment wasmainly due to a decrease in the cell growth rate to further address what was occurring at the molecular levelduring lowdose dht andor vd3 treatment the genesknown to regulate the cell cycle and inducible by dhtandor vd3 treatment were chosen and messengerrna mrna induction was quantitatively measuredfig 1d upper supplementary fig 1a the quantitativertpcr results showed that igfbp3 mrna inductionwas positively correlated to cell growth suppression inresponse to lowdose vd3 or dhtvd3 treatment andthe expression strength was dramatically sensitive tovd3 or dhtvd3 treatment consistent with thatigfbp3 protein was markedly induced by vd3 treatment and it was enhanced by simultaneous treatment ofvd3 with dht a similar response was observed in theexpression of ar the receptor of dht which wasknown to a one oftarget of vdr supplementaryfig 1bmultiple recent studies have revealed that igfbp3functions in cellular response including cell growth andapoptosisin an insulinlike growth factor igfindependent manner considering these findings we believethat igfbp3 can be a key molecule for vd3 treatmentin prostate cancer cellsigfbp3 was a dominant factor in cell growth suppressionto confirm ifigfbp3 dominantly suppresses cellgrowth in lncap cells we applied the gainoffunctionand lossoffunction approach using a lentivirus systemfirst we generated igfbp3overexpressing lncapcells and found that the expression of igfbp3 mrnawas about higher compared to that by lowdosedhtvd3 treatment fig 2a as an infection controlegfpoverexpressing lncap cells were also generatedand it was confirmed that lentivirus infection per se didnot induce igfbp3 expression using these cell linesthe effect of igfbp3 on cell growth was observed fig2b results showed that the cell number of egfpoverexpressed cells treated with nm of dht and nm of vd3 for days was decreased to comparedwith that of untreated cells and the igfbp3overexpressing cells showed comparable cell growth decreasewithout dhtvd3 treatment next we generatedshrna for igfbp3 shigfbp3expressing lncap cellsthe knockdown of igfbp3 mrna and protein inducedby lowdose dhtvd3 treatment was confirmed inshigfbp3expressing lncap cells fig 2c using thiscell line the effect of lowdose dht and vd3 treatmenton cell growth was observed as we expected the suppressive efficacy of lowdose vd3 on cell growth wasweakening and simultaneous treatment with nm ofdht and nm of vd3 increased cell growth fig 2dtaken together these data indicated igfbp3dominantfactor of cell growth suppression induced by lowdosevd3 treatment in lncap cellsacceleration of anticancer drug effect by vd3as previously reported and we demonstrated abovevd3 alone is not cytotoxic at physiological and pharmacological concentrations meanwhile simultaneous treatment with vd3 has been reported to improve theefficacy of anticancer drugs including docetaxel howeverits molecular mechanisms were remained not fully uncovered here we supposed that igfbp3 might be amediator of vd3induced sensitization to anticancerdrugs in prostate cancer cells to confirm this hypothesis lncap cells were treated with lowdose of dhtvd3 in combination with several concentrations of docetaxel to determine the appropriate concentration ofdocetaxel for evaluating the vd3 effect we first screenedthe concentration of docetaxel based on cytotoxic activity and found that a docetaxel concentration nmkilled bulk of the cells treated fig 3a here ic50 ofdocetaxel was nm in our assay and it was consistent with previous reports nm thus a docetaxel concentration nm was chosen to observe theeffect of combinatoriallowdose dhtvd3 treatmentfor the following assay to evaluate the synergistic effectof dhtvd3 on cytotoxicity by docetaxel lncap cellswere treated with or nm of docetaxel withor without dhtvd3 and results showed that lowdosedhtvd3 with docetaxel reduced the living cell numberat the concentration range of nm but the effectwas masked when nm docetaxel was applied fig 3bsimilarlylowdose dhtvd3 with cisplatin reducedthe living cell number at the concentration range of nm supplementary fig to see if these enhancedcytotoxicity effects were dependent on igfbp3 igfbpoverexpressed or shigfbp3expressed lncap cellswere analyzed in the same manner indeed in igfbp3overexpressed cells the living cell number was reducedby docetaxel without dhtvd3 addition fig 3c 0cigarashi bmc cancer page of fig igfbp3 mediates the effect of vd3 on cell growth in lncap cells a overexpression of igfbp3 in lncap cells lncap cells were infectedwith lentivirus containing the igfbp3 gene and its overexpression was confirmed by quantitative reverse transcriptionpolymerase chainreaction b suppression of cell growth by igfbp3 igfbp3overexpressed cells were cultured as they were and control cells were cultured withdhtvd3 for days and then the cell number was measured c knockdown of igfbp3 in lncap cells lncap cells were infected with lentiviruscontaining shrna for igfbp3 and igfbp3 knockdown was confirmed by quantitative reverse transcriptionpolymerase chain reaction andwestern blotting the ratio indicates the density of igfbp3 band normalized by corresponding βactin band d the igfbp3 knockdown cells weretreated with dht andor vd3 for days and then the cell number was measured the all experiments were performed in serumcontainingmedium condition the uncropped fulllength blot images are presented in supplementary fig 5bcorrespondingly in the shigfbp3expressed cells reduction of living cell number by dhtvd3 addition wascanceled rather the cell living number was increasedliving cell number despitefig 3d the increase ofdhtvd3 addition in docetaxeltreated shigfbp3expressed cells was assumed by cancelation of vd3 effect and emerging of dht effect on cell growth basedthese findings lowdose dhtvd3induced enhancedcytotoxicity by docetaxel on lncap cells was dependenton igfbp3 expressioncharacterization of the igfbp3 induction mechanismas demonstrated above igfbp3 had a pivotal role inlowdose dhtvd3induced enhanced cytotoxicity byantitumor drugs to further dissect the igfbp3 induction mechanism and to provide the molecular evidenceof vd3 treatment for clinical research mechanisms ofigfbp3 induction by dht and vd3 were analyzed aspreviously reported in prostate cancer cells vd3 treatment induces cyp24a1 as well an enzyme that catalyzes vd3 to its inactive form as a negative feedbackfactor the induced cyp24a1 limits the efficacy of vd3meanwhile activated ar induced by dht treatmentsuppresses cyp24a1 transcription thus cancelling thenegativefeedback loop to inactivate vd3 consistentwith that cyp24a1 induction by vd3 treatment and itssuppression by simultaneous treatment with dht wereconfirmed even when we applied lowdose dht and 0cigarashi bmc cancer page of fig vd3 enhanced cytotoxicity of docetaxel a dosedependent cytotoxicity of docetaxel lncap cells were treated with docetaxel for daysand then the living cell number was measured ic50 was nm b simultaneous treatment of vd3 with dosedependent docetaxelsimultaneous treatment of vd3 with docetaxel reduced the living cell number at nm range c igfbp3 overexpression was conducive todhtvd3 treatment on docetaxel treatment igfbp3overexpressed or control cells were treated with dhtvd3 and nm of docetaxel andcultured for days and then the cell number was measured d igfbp3 knockdown canceled the effect of vd3 on docetaxel treatment in igfbp knockdown cells dhtvd3 treatment increased the cell number compared to that of the notreatment control and the cell number wasalmost equal to that of the dht treated sample the concentration of docetaxel used was nm the all experiments were performed in serumcontaining medium conditionvd3 which were enough to induce and suppresscyp24a1 expression supplementary fig meaningthat h lowdose dhtvd3 treatment could cancel thecyp24a1driven negativefeedback loop to further dissect the mechanism of igfbp3 expression in lncapcells the cells were treated with vd3 alone or dhtfixed in nm and vd3 in a dosedependent manner and nm when treated with vd3alone the induced igfbp3 reached a plateau at to nm in contrast when treated with vd3 together withdht the amount of induced igfbp3 was increased according to the increment of vd3 concentration fig 4athese results indicated that lowdose dht could improve igfbp3 induction activity of vd3 throughcyp24a1 suppressionclinically highdose vd3 or its derivatives for treatment can cause hypercalcemia thus its continual usageshould be carefully monitored to avoid sideeffect ofvd3 here we wondered if continual vd3 treatmentfig 4btop herewould be required for maintaining igfbp3 induction inprostate cancer cells to address this lncap cells weretreated with vd3 alone or lowdose dhtvd3 for or days followed by washout which was done by replacing the culture medium and continuing the culturefor days in totalintracellularigfbp3 protein was observed by western blottinginterestingly 1day treatment of vd3 or dhtvd3 induced stable igfbp3 expression fig 4b bottom although treatment of vd3 alone showed mild igfbp3induction compared to that by dhtvd3 note thatigfbp3 showed similar strength of expression between and days of vd3 or dhtvd3 treatment this resultclearly indicated that temporal vd3 treatment couldinduce prolonged stable igfbp3 expressionthis nonlinear response suggested the presence of aunique molecular property underlying the igfbp3 expression mechanism generally nonlinear cellular response such as sustained protein expression by temporal 0cigarashi bmc cancer page of fig characterization of igfbp3 expression induced by vd3 and dht treatments a effect of dosedependent vd3 treatment with or withoutdht on igfbp3 induction lncap cells were treated with vd3 alone at the indicated concentration or with vd3 and nm dht top westernblotting image of igfbp3 and bottom quantified graph of igfbp3 induction open circles vd3 alone filled circles dht vd3 b effect oftemporal treatment of dhtvd3 on igfbp3 induction top schematic time course of temporal treatment of vd3 and bottom western blottingimage of igfbp3 the ratio indicates the density of igfbp3 band normalized by corresponding βactin band c effect of mrna transcription andprotein synthesis on the stability of igfbp3 expression induced by dhtvd3 lncap cells were treated with vd3dht for day followed byactinomycin d actd μm or cycloheximide chx μm for another day after vd3dht was washed out or left as is top quantification graphof igfbp3 mrna induction middle western blotting image of igfbp3 and the combination of treatments bottom the ratio indicates thedensity of igfbp3 band normalized by corresponding βactin band the all experiments were performed in serumcontaining medium conditionthe uncropped fulllength blot images are presented in supplementary fig 5ctriggered by positivefeedback loopstimulation wasmechanismin which protein synthesis and transcriptional regulation was included as hysterical responsedriver thus to further dissect if protein synthesis ortranscriptional regulation or both are involved in nonlinear vd3igfbp3 induction actinomycin d and cycloheximide which are transcriptionalinhibitor andprotein synthesis inhibitor respectively were added withvd3 or dhtvd3 and behavior of igfbp3 proteinand its mrna was observed experimentally actinomycin d or cycloheximide was added day after treatmentwith vd3 alone or dhtvd3 and the culture was continued for another day when interfered with μm ofactinomycin d there was no change in igfbp3 mrnaor protein expression fig 4c by contrast when interfered with μm of cycloheximide the igfbp3 proteinwas immediately reduced howeverthe mrna wasunexpectedly increased several times higher than thosewithout cycloheximide interference fig 4c the unexpected mrna increase became stronger when dhtvd3 washout was performed ahead of the cycloheximideinterference these cellular responses on igfbp3 induction interfered by actinomycin d and cycloheximidesuggested that the cells had a protein abundancebasedpositivefeedback loop to maintain the total amount ofigfbp3 via transcriptional controligfbp3independent bcl2 suppression by vd3as shown above although lowdose dht andor vd3treatment did not induce apoptosis fig 1d vd3 treatment rendered lncap cells sensitive to the antitumordrugs fig 3b and supplementary fig suggesting thatany apoptosisrelated factor might be influenced to addressinvestigated the behavior ofidea wethis 0cigarashi bmc cancer page of apoptosisrelated molecules in response to lowdosevd3dht treatment consistent with previous report although the concentration of vd3 was higher thanthat we used here bcl2 protein an antiapoptotic molecule was downregulated by lowdose vd3 treatmentfig 5a compared to that by vd3 alone it seemed thatbcl2 downregulation by dhtvd3 was not seemed tobe enhanced unlike to igfbp3 expression suggestingthat bcl2 downregulation by vd3 was igfbp3 induction independent manner to see whether bcl2 downregulation was igfbp3dependent or not the behaviorof the expression of bcl2 protein and mrna was observed in shigfbp3expressedcells interestingly despitethe igfbp3 disappearance bcl2 downregulation wasobserved according to vd3 or dhtvd3 treatmentfig 5b bottom and it was not significantly different atinigfbp3expressionenhancedprotein and mrna expression level compared to that inshctrlexpressing cells moreover in order to confirmthatconditionshcyp24a1 expression cells were established in whichvd3 effect on igfbp3 induction was expected tostrengthen the knockdown of cyp24a1 under the vd3treatment condition was confirmed by qrtpcr supplementary fig indeed the amount of igfbp3 protein wasincreased in shcyp24a1expressing cellscompared to that in shctrl cells fig 5c bottom despiteigfbp3 expression downregulation bcl2 protein by lowdose vd3 or dhtvd3treatment was not observed compared to that in shctrlcells fig 5c bottom also the expression of bcl2mrna was not significantly changed fig 5ctopthese results suggested that the downregulation of bclenhancement offig igfbp3independent reduction of bcl2 protein expression induced by vd3 a western blotting image of bcl2 reduction by vd3treatment the ratio indicates the density of bcl2 band normalized by corresponding βactin band b effect of igfbp3 suppression on bcl2reduction by vd3 treatment the cells were infected with lentivirus encoding of shrna for igfbp3 and then treated with vd3 and dht topquantification graph of bcl2 mrna expression and bottom western blotting image of bcl2 and igfbp3 induced by vd3 and dht treatmentthe ratio indicates the density of bcl2 band normalized by corresponding βactin band c effect of igfbp3 overexpression on bcl2 reduction byvd3 treatment the cells were infected with lentivirus encoding of the cyp24a1 gene then the cells were treated with vd3 and dht topquantification graph of bcl2 mrna expression and bottom western blotting image of bcl2 and igfbp3 induced by vd3 and dht treatmentthe ratio indicates the density of bcl2 band normalized by corresponding βactin band the all experiments were performed in serumcontainingmedium condition the uncropped fulllength blot images are presented in supplementary fig 5d e 0cigarashi bmc cancer page of protein by lowdose vd3 treatment was independentof igfbp3 induction besides the mrna of bcl | 0 |
Millions of people are suffering from cancers but accurate early diagnosis and effectivetreatment are stilllong noncoding RNAslncRNAs have been proven to play an important role in diseases especially cancersThese lncRNAs execute their functions by regulating gene expression Thereforeidentifying lncRNAs which are related to cancers could help researchers gain a deeperunderstanding of cancer mechanisms and help them ï¬nd treatment options A largenumber of relationships between lncRNAs and cancers have been veriï¬ed by biologicalexperiments which give us a chance to use computational methods to identifycancerrelated lncRNAs In this paper we applied the convolutional neural network CNNto identify cancerrelated lncRNAs by lncRNAs target genes and their tissue expressionspeciï¬city Since lncRNA regulates target gene expression and it has been reportedto have tissue expression speciï¬city their target genes and expression in differenttissues were used as features of lncRNAs Then the deep belief network DBN wasused to unsupervised encode features of lncRNAs Finally CNN was used to predictcancerrelated lncRNAs based on known relationships between lncRNAs and cancersFor each type of cancer we built a CNN model to predict its related lncRNAs Weidentiï¬ed more related lncRNAs for kinds of cancers Tencross validation has beenused to prove the performance of our method The results showed that our method isbetter than several previous methods with area under the curve AUC and areaunder the precisionrecall curve AUPR To verify the accuracy of our results casestudies have been doneKeywords long noncoding RNA lncRNA cancer convolutional neural network CNN deep belief network DBNmachine learningINTRODUCTIONFour to nine percent of the sequences transcription are long noncoding RNAs lncRNAs inmammalian genomes Canzio Ji lncRNA was regarded as the noise ofgenome transcription and did not have biological functions at ï¬rst However an increasing numberof studies have reported that lncRNA is widely Robinson involved in chromosomeEdited byLei DengCentral South University ChinaReviewed byHao LinUniversity of Electronic Science andTechnology of China ChinaInner Mongolia University ChinaJuan WangCorrespondenceNan Dudunan05aliyuncomGanfeng Xiexiegfaliyuncom These authors share ï¬rst authorshipSpecialty sectionThis was submitted toMolecular Medicinea section of the journalFrontiers in Cell and DevelopmentalBiologyReceived June Accepted June Published August CitationLiu Z Zhang Y Han X Li C Yang XGao J Xie G and Du N Identifying CancerRelated lncRNAsBased on a Convolutional NeuralNetwork Front Cell Dev Biol 103389fcell202000637Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alA Method to Identify CancerRelated lncRNAsgenomicimprintingchromatin modiï¬cationsilencingtranscriptional activationinterference andnuclear transport Cheng 2018a Recently it has beenproven to be associated with many kinds of cancerstranscriptionalThe secondary structure spliced form and subcellularlocalization of most lncRNAs are conserved Karner which is very important for lncRNA to execute functionsHowever compared to the functions of microRNAs miRNAsand proteins the function oflncRNA is more diï¬cult todetermine According to the position of lncRNA in the genomerelative to proteincoding genes it can be divided into ï¬ve typessense antisense bidirectional intronic and intergenicMany researchers have found lncRNAs play an important rolein cancers Avgeris Cheng 2018b Zhao and neurodegenerative diseases Peng and Zhao as other biological molecules Zhang T Bai Cheng 2019a Liang Although manyresearchers have veriï¬ed many associations between lncRNAsand cancers by biological experiments compared with ourknowledge about diseaserelated genes we still do not knowenough about diseaserelated lncRNAs Considering the timeand money cost of ï¬nding diseaserelated lncRNAs more andmore researchers tend to use computational methods to identifydiseaserelated lncRNAs These methods could be divided intothree categories machine learning methods network methodsand other methodsMachine learning methods build models based on thesimilarities of diseases orlncRNAs and their biologicalcharacteristics Cheng Cheng 2019b Zeng Zou Lan developed thelncRNAdisease association prediction LDAP which is amethod based on bagging support vector machine SVM toidentify lncRNAdisease associations They used similarities oflncRNAs and diseases as the features Yu developedcollaborative ï¬ltering naive Bayesian classiï¬er CFNBC based onnaive Bayesian They integrated miRNAlncRNA associationsmiRNAdisease associations and lncRNAdisease associationsto infer more lncRNAdisease associations Considering thediscriminative contributions of the similarity association andinteraction relationships among lncRNAs disease and miRNAsXuan 2019a developed a dual convolutional neuralnetwork CNN with attention mechanisms to predict diseaserelated lncRNAsNetwork methods are the most common way to identifyassociations between diseases and lncRNAs nowadays Gu Yu Zhang J Kuang Wang L Liu Thiskind of method would build one or multiple networks toinfer new information Wang L built a lncRNAmiRNAdisease interactive network and used their novel methodLDLMD to predict associations between lncRNAs and diseasesSumathipala used a multilevel network topologywhich includes lncRNAprotein proteinprotein interactionproteindisease relationship to use network diï¬usion algorithmto predict diseaserelated lncRNAs The graph convolutionalnetwork GCN and CNN were used on a lncRNAmiRNAdisease network by Xuan 2019b Deng builtlncRNA similarity network disease similarity network miRNAsimilarity network and their associations Then they calculatedthe metapath and feature vector for each lncRNAdisease pair inthe heterogeneous information networkOther methods may borrow the feature extraction methodor similarity conjecture of network methods but the core ofthis method is matrix decomposition or matrix completionLu developed the geometric matrix completionlncRNAdisease association GMCLDA which is a methodbased on geometric matrix completion They calculated diseasesimilarity based on Disease Ontology DO and calculatedthe Gaussian interaction proï¬le kernel similarity for lncRNAsThen they inferred diseaserelated lncRNAs based on theassociation patterns among functionally similar lncRNAs andsimilar diseases Wang Y proposed a weightedmatrix factorization to capture the interintraassociationsbetween diï¬erent types of nodes Then they approximated thelncRNAdisease association matrix using the optimized matricesand weights to predict diseaserelated lncRNAs Localityconstrained linear coding label propagation Latent DirichletAllocation LLCLPLDA was developed by Xie Firstly localconstraint features of lncRNAs and diseases wereextracted by localityconstrained linear coding LLC Thenthey predicted diseaserelated lncRNAs by label propagationLP strategyHowever previous methods did not consider the regulatingtarget gene expression of lncRNA which is an important functionof lncRNA and plays an important role in associations betweenlncRNAs and diseases In addition deep learning methods arean important tool and have shown their power in bioinformaticsChen Lv Wei Wu Zhao 2019abc Therefore in this paper we used thisinformation as features of lncRNA In addition the expressionof lncRNA in diï¬erent tissues were also used as the featuresof lncRNA Then the deep belief network DBN was used toencode and the CNN was used to classifyMETHODSFeature ExtractionTissue Expression Speciï¬city of Long NoncodingRNACompared with proteincoding geneslncRNA shows strongtissue speciï¬city The speciï¬city of lncRNAs in diï¬erent kindsof tissues and cell types has been proven by many biologicalexperiments The diï¬erent expression also plays an importantrole in essential cellular processes Sasaki testedthe expression of lncRNAs in diï¬erent tissues and found lncRNAs exhibited tissuespeciï¬c expression and oflncRNAs were only expressed in one discrete tissue Thereforethe expression of lncRNAs in diï¬erent tissues were used asthe featuresWe obtained the expression of lncRNAs in diï¬erenttissues which included adipose adrenal breast colon heartkidney liver lung lymph node ovary placenta prostate testisand thyroidTherefore the dimension of each lncRNAs expression featureis Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alA Method to Identify CancerRelated lncRNAsTherefore the dimension of each lncRNAs target gene featureis Deep Belief NetworkThe DBN can eï¬ectively learn complex dependencies betweenvariables Zhao 2019d The DBN contains many layers ofhidden variables which can eï¬ectively learn the internal featurerepresentation of the data and can also be used as an eï¬ectivenonlinear dimensionality reduction methodWhen the observable variables are known the joint posteriorprobabilities of the hidden variables are no longer independentof each other so it is diï¬cult to accurately estimate the posteriorprobabilities of all hidden variables The posterior probability ofearly DBN is generally approximated by Monte Carlo methodbut its eï¬ciency is relatively low which makes its parameterlearning diï¬cult In order to eï¬ectively train the DBN weconvert the sigmoid belief network of each layer to a restrictedBoltzmann machine RBM The advantage of this is that theposterior probabilities of the hidden variables are independentof each other which makes it easy to sample In this way theDBN can be regarded as being stacked from top to bottom bymultiple RBMs and the hidden layer of the Lth RBM is used asthe observable layer of the L 1th RBM Further the DBN canbe trained quickly by layerbylayer training that is starting fromthe bottom layer and training only one layer at a time until thelast layer The speciï¬c layerbylayer training process is to trainthe RBM of each layer in turn from bottom to top Assuming wehave trained the RBM in the ï¬rst L1 layer we can calculate theconditional probability of the bottomup hidden variablesphihi Ï bi Wihiwhere bi is the bias of ith layer of RBM Wi is the connectionweight hi is the ith layer of RBMThe process of training DBN is as followsFIGURE The number of target genes for each long noncoding RNAlncRNAFIGURE The distribution of the number of target genes lncRNA longnoncoding RNAreverseTarget Gene of Long Noncoding RNAQuantitativechainreaction qRTPCR and Western blot were used to testthe diï¬erentexpression genes after knocking down oroverexpressing lncRNAstranscriptasepolymeraseWe obtained target genes of lncRNA from LncRNA2TargetInput train dataset Ëvn learning rate λJiang As we can see in Figure there are kinds of lncRNAsOne lncRNA has more than target genes Then we drawthe distribution of the number of target genes correspondingto lncRNAAsshown in Figure most ofthe target genes arecorresponding to less than ï¬ve lncRNAs Therefore if we usedthem to be the features of lncRNAs the features would be sparseTherefore we only select the most common target genes to bethe features The genes which are corresponding to more thanï¬ve lncRNAs were selected as the features of lncRNAs There are kinds of genes Then we need to encode these genesF [G1 G2 · · · G45]where G1 denotes the ï¬rst gene of these genes and F denotesthe feature of lncRNA For each lncRNA if G1 is the target geneof it then G1 otherwise G1 Output weight matrix Wl bias al and blFor l 1LInitialization Wi al bi Sample from train dataset Ëh0For i lSample hi based on phi ËhiEndSet hi1as the train sample to train lth layer ofRBMEndSince the dimension of expression feature and target genefeature are diï¬erent we should reduce the dimension of targetgene feature and make it the same as the expression featuresTherefore in this paper two layers of RBM were used to builda DBN modelThe number of nodes oftheand respectively Sigmoid function was used astwo layers was theFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alactivation functionÏ x exTherefore the dimension of ï¬nal features is F cid20 G1 G2 · · · G13E1 E2 · · · E13 cid21A Method to Identify CancerRelated lncRNAsConvolutional Neural NetworkThe power of CNN in dealing with bioinformatic problems hasbeen proven by many researchers We selected CNN as theclassiï¬er based on two reasons The dimension of features is which can be regarded as an image The outstandingperformance of CNN in image classiï¬cationThere are ï¬ve layers in our CNN model The structure of CNNis shown as Table where G1 G2 · · · G13 denotes target gene feature after DBNand E1 E2 · · · E13 denotes the expression of lncRNAs in diï¬erent tissuesTABLE The structure of convolutional neural network CNNLayersParameterConvolutional layerPooling layerConvolutional layerPooling layerFully connected layerOutputFilter kernel size Activation function tanhpool size Activation function tanhFilter kernel size Activation function tanhpool size Activation function tanhUnits Activation function tanhUnits Activation function sigmoidWork FrameFigure shows the work frame of our method DBNCNNThere are three steps of our methods Firstly we should extractfeatures of lncRNAs There are two parts of features expressionfeature and target gene feature Then DBN was used to encodethe target gene feature After encoding the two kinds of featureswere combined together Finally CNN was used to classifyRESULTSData DescriptionThe known associations between lncRNA and diseases wereobtained from LncRNADisease database Bao Wetotally obtained kinds of cancerrelated lncRNAs The numberof their corresponding lncRNAs is shown as Figure As shown in Figure Peoples understanding of cancerrelated lncRNAs varies widely We have known more than lncRNAs for some cancers but few lncRNAs are known for somecancers To better build our model we only selected cancerswhich have more than related lncRNAs Therefore kindsof cancers were selectedFIGURE Work frame of deep belief network DBNconvolutional neural network CNN lncRNA long noncoding RNAFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alA Method to Identify CancerRelated lncRNAsFIGURE The number of long noncoding RNAs lncRNAs for each cancerTABLE The performance of deep belief network DBNconvolutional neuralnetwork CNN in cancersCancerArea undercurve AUCArea under precisioncurve AUPRCervical cancerBreast cancerColorectal cancerStomach cancerUrinary bladder cancerLung cancerOvarian cancerThyroid cancerProstate cancerLiver cancerPancreatic cancerOvarian epithelial cancerGallbladder cancerEndometrial cancerColon cancerEsophageal cancerThetargetgenes oflncRNAs were obtained fromLncRNA2Target database We have discussed about this insection Target Gene of Long Noncoding RNAFIGURE The receiver operating characteristic ROC curves of the threemethods DBN deep belief network CNN convolutional neural network PCAprincipal component analysisFIGURE The area under the precisionrecall curve AUPR of the threemethods DBN deep belief network CNN convolutional neural network PCAprincipal component analysisThe expression oftissues wasobtained from NONCODEV5 Zhao We only usedhuman datalncRNAs in diï¬erentThe Performance of Deep BeliefNetworkConvolutional Neural NetworkWe did 10cross validation on each cancer Area under the curveAUC Cheng Dao Zhang and areaunder the precisionrecall curve AUPR were used to evaluatethe performance of DBNCNN The results are shown in Table As we can see in Table the performance of DBNCNN isquite diï¬erent in diï¬erent cancers This may be caused by thediï¬erent sample sizes The average AUC is and AUPR is Comparison ExperimentsTo verify the superior of DBNCNN we compared it with similarmethods Since the main function of DBN is to reduce dimensionprincipal component analysis PCA has the same functionTherefore instead of using DBN to encode we used PCA thistime and CNN was used to classify the features after PCA We callthis method PCACNN In addition we also used the deep neuralnetwork DNN to replace CNN so this comparison method wascalled DBNDNNWe used these three methods to test on cancers andsummarized the results to get a ï¬nal AUC and AUPR for eachmethod The receiver operating characteristic ROC curves areshown in Figure As shown in Figure the blue curve denotes the results ofDBNCNN The red and black curves denote PCACNN andDBNDNN respectively As we can see DBNCNN performedbest among these three methods The AUC of DBNCNN is which is better than and for PCACNN andDBNDNN respectivelyFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alA Method to Identify CancerRelated lncRNAsAs shown in Figure the AUPR of DBNCNN is the highestwith the least standard errorCase StudyLiu found down syndrome cell adhesion molecule antisense RNA DSCAMAS1 is associated with breast cancerby constructing two suppression subtracted cDNA librariesMartensUzunova reported the associationbetween H19 and bladder cancer They also pointed out that H19could be the biomarker of bladder cancerShi measured the expression level of lncRNAsLoc554202 in breast cancer tissues and found that Loc554202was signiï¬cantly increased compared with normal control andassociated with advanced pathologic stage and tumor sizeCONCLUSIONSIncreasing evidence has shown the relationship between lncRNAsand cancers lncRNAs could be the biomarkers to help diagnosecancer and also help researchers understand the mechanismof cancers Compared with peoples knowledge of diseaserelated protein coding genes we knew few about diseaserelated lncRNAs However the biological experiments for ï¬ndingdiseaserelated lncRNAs are timeconsuming and expensiveTherefore in this paper we proposed a novel method foridentifying cancerrelated lncRNAs We called this methodDBNCNN which is a fusion of DBN and CNN Two kindsof features were used based on the biological background SincelncRNAs have tissuespeciï¬c expression and the expression ofcancer tissues is diï¬erent from normal tissues the expressionoftissues could provide importantin diï¬erentlncRNAsREFERENCESAvgeris M Tsilimantou A Levis P K Tokas T Sideris D C StravodimosK Loss of GAS5 tumour suppressor lncRNA an independentmolecular cancer biomarker for shortterm relapse and progression in bladdercancer patients Br J Cancer 101038s4141601803206Bai Y Dai X Ye T Zhang P Yan X Gong X PlncRNADBa repository of plant lncRNAs and lncRNARBP protein interactions CurrBioinform Bao Z Yang Z Huang Z Zhou Y Cui Q and Dong D LncRNADisease an updated database of long noncoding RNAassociateddiseases Nucleic Acids Res D1034D1037 101093nargky905Canzio D Nwakeze C L Horta A Rajkumar S M Coï¬ey E L Duï¬y EE Antisense lncRNA transcription mediates DNA demethylationto drive stochastic protocadherin α promoter choice Cell 653e15 101016jcell201903008Chen X Shi W and Deng L Prediction of disease comorbidity usinghetesim scores based on multiple heterogeneous networks Curr Gene Ther Cheng L Computational and 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NONCODE an informative and valuable data source of long noncoding RNAs NucleicAcids Res D203D208 101093nargkv1252Zou Q Xing P Wei L and Liu B Gene2vec gene subsequenceembedding for prediction of mammalian N6methyladenosine sites frommRNA RNA 101261rna069112118Conï¬ict of Interest The authors declare that the research was conducted in theabsence of any commercial or ï¬nancial relationships that could be construed as apotential conï¬ict of interestCopyright Liu Zhang Han Li Yang Gao Xie and Du This is an openaccess distributed under the terms of the Creative Commons Attribution License CCBY The use distribution or reproduction in other forums is permitted providedthe original authors and the copyright owners are credited and that the originalpublication in this journal is cited in accordance with accepted academic practiceNo use distribution or reproduction is permitted which does not comply with thesetermsFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0c' | 2 |
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