Parameter identifiability problems can plague biomodelers when they reach the quantification stage of development, even for relatively simple models. Structural identifiability (SI) is the primary ...question, usually understood as knowing which of P unknown biomodel parameters p1,…, pi,…, pP are-and which are not-quantifiable in principle from particular input-output (I-O) biodata. It is not widely appreciated that the same database also can provide quantitative information about the structurally unidentifiable (not quantifiable) subset, in the form of explicit algebraic relationships among unidentifiable pi. Importantly, this is a first step toward finding what else is needed to quantify particular unidentifiable parameters of interest from new I-O experiments. We further develop, implement and exemplify novel algorithms that address and solve the SI problem for a practical class of ordinary differential equation (ODE) systems biology models, as a user-friendly and universally-accessible web application (app)-COMBOS. Users provide the structural ODE and output measurement models in one of two standard forms to a remote server via their web browser. COMBOS provides a list of uniquely and non-uniquely SI model parameters, and-importantly-the combinations of parameters not individually SI. If non-uniquely SI, it also provides the maximum number of different solutions, with important practical implications. The behind-the-scenes symbolic differential algebra algorithms are based on computing Gröbner bases of model attributes established after some algebraic transformations, using the computer-algebra system Maxima. COMBOS was developed for facile instructional and research use as well as modeling. We use it in the classroom to illustrate SI analysis; and have simplified complex models of tumor suppressor p53 and hormone regulation, based on explicit computation of parameter combinations. It's illustrated and validated here for models of moderate complexity, with and without initial conditions. Built-in examples include unidentifiable 2 to 4-compartment and HIV dynamics models.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
MicroRNAs (miRs) are a class of small noncoding RNAs whose expression changes have been associated with cancer development and progression. Current techniques to isolate miRs for expression analysis ...from blood are inefficient. We developed a reverse-transcription quantitative real-time PCR (RT-qPCR) assay for direct detection of circulating miRs in serum. We hypothesized that serum concentrations of miR-21, a biomarker increased in breast tumors, would correlate with the presence and extent of breast cancer.
The RT-qPCR applied directly in serum (RT-qPCR-DS) assay for circulating miR-21 was tested in sera from 102 patients with different stages of breast cancer and 20 healthy female donors.
The assay was sensitive for detection of miR-21 in 0.625 μL of serum from breast cancer patients. For differentiation of samples from patients with locoregional breast cancer from those from healthy donors, the odds ratio was 1.796 and the area under the curve was 0.721. In a multivariate analysis that included standard clinicopathologic prognostic factors, high circulating miR-21 concentrations correlated significantly (P < 0.001) with visceral metastasis.
A novel RT-qPCR-DS can improve the efficiency of miR assessment. Use of this assay to detect circulating miR-21 has diagnostic and prognostic potential in breast cancer.
Aberrations in the methylation status of noncoding genomic repeat DNA sequences and specific gene promoter region are important epigenetic events in melanoma progression. Promoter methylation status ...in long interspersed nucleotide element-1 (LINE-1) and absent in melanoma-1 (AIM1;6q21) associated with melanoma progression and disease outcome was assessed. LINE-1 and AIM1 methylation status was assessed in paraffin-embedded archival tissue (PEAT; n=133) and in melanoma patients’ serum (n=56). LINE-1 U-Index (hypomethylation) and AIM1 were analyzed in microdissected melanoma PEAT sections. The LINE-1 U-Index of melanoma (n=100) was significantly higher than that of normal skin (n=14) and nevi (n=12; P=0.0004). LINE-1 U-Index level was elevated with increasing American Joint Committee on Cancer (AJCC) stage (P<0.0001). AIM1 promoter hypermethylation was found in higher frequency (P=0.005) in metastatic melanoma (65%) than in primary melanomas (38%). When analyzed, high LINE-1 U-Index and/or AIM1 methylation in melanomas were associated with disease-free survival (DFS) and overall survival (OS) in stage I/II patients (P=0.017 and 0.027, respectively). In multivariate analysis, melanoma AIM1 methylation status was a significant prognostic factor of OS (P=0.032). Furthermore, serum unmethylated LINE-1 was at higher levels in both stage III (n=20) and stage IV (n=36) patients compared with healthy donors (n=14; P=0.022). Circulating methylated AIM1 was detected in patients’ serum and was predictive of OS in stage IV patients (P=0.009). LINE-1 hypomethylation and AIM1 hypermethylation have prognostic utility in both melanoma patients’ tumors and serum.
Dairy cows are usually culled and transported from the farm when they no longer meet the farm's standards for production or are not needed for milk production. Some cows are transported while in poor ...condition and may deteriorate further during transport. In February 2020, Canadian federal animal transport regulations were revised with the aim to minimise risks to livestock during transport; changes that may impact cull dairy cows included defining compromised cattle and limiting their maximum transport time. This study conducted semi-structured interviews with dairy farmers (n = 6) and cattle haulers (n = 4) in British Columbia, Canada, to gain an in-depth understanding of the effect of the regulations on their practices when shipping and transporting dairy cows to slaughter. Interviews were transcribed in Otter.ai and thematically coded in NVivo 12. While farmer and hauler participants recognised the importance of animal welfare during transport and described practices such as shipping mobile animals to reduce the risk that cows would become non-ambulatory during transport, they also described little change in shipping and transport practices due to the new regulations. Among interviewed participants, barriers to compliance with the regulations appear to be low knowledge of, and mixed or negative attitudes towards the regulations. Participants also described how they felt a lack of communication along the transport chain and limited transport and slaughter infrastructure made compliance difficult. Possible suggestions to remedy these barriers include providing educational resources about the regulations and encouraging communication about cow fitness for transport between responsible parties in the transport chain.
Hypermethylation of the promoter region of tumor-related genes (TRGs) has been shown to silence gene expression during melanoma progression, whereas microRNA-29(miR-29) has been found to downregulate ...DNA methyltransferases DNMT3A and DNMT3B which were shown as essential to the methylation of TRGs. We hypothesized that the expression level of miR-29 is associated to TRG methylation status and may have prognostic utility in melanoma. AJCC stage I-IV cutaneous melanoma paraffin-embedded archival tissue (PEAT) specimens (n=149) were assessed. Expression of miR-29 isoforms a, b, and c were analyzed by reverse-transcription quantitative real-time polymerase chain reaction(RT-qPCR). Expression of DNMT3A and DNMT3B was assessed by immunohistochemistry(IHC) on defined clinically annotated tissue microarrays (TMA) of AJCC stage III melanoma lymph node metastases. Promoter region CpG island methylation status of RASSF1A, TFPI-2, RAR-β, SOCS, GATA4 and genomic repeat sequence MINT17 and MINT31 were previously evaluated in melanoma tissues. Only miR-29c isoform expression was correlated to advancing AJCC stages in melanoma. miR-29c expression was significantly downregulated in AJCC stage IV melanoma tumors compared to primary melanomas. Hypermethylation status of TRGs and non-coding MINT loci in different stages of melanoma showed an inverse association with miR-29c expression. Overall, an increase in miR-29c expression inversely correlated to both DNMT3A and DNMT3B protein expression in melanomas. Expression of DNMT3B and miR-29c were significantly (p=0.004 and p=0.002, respectively) associated with overall survival(OS) in AJCC stage III melanoma patients by multivariate analysis. The studies demonstrated that both miR-29c and DNMT3B have significant roles in melanoma progression, and may be useful epigenetic biomarkers for disease outcome.
Expression of specific breast cancer stem cells (BCSCs) is seen in aggressive tumors, but their regulation is unclear. Epigenetic changes influence gene expression and are implicated in breast cancer ...progression. We hypothesized that promoter methylation regulates specific BCSC-related genes CD44 , CD133 , CD24 , MSH1 (alias, Musashi-1 ), and ALDH1 and that this epigenetic profile can identify aggressive subtypes, such as triple-negative breast cancer (TNBC). Methylation analysis was performed using MassARRAY EpiTYPER sequencing; CpG-rich sites were identified in the promoter regions of BCSC genes, except ALDH1 . These sites were screened by treatment with 5-aza-2′-deoxycytidine in four TN and five non-TNBC cell lines. The specific regulatory CpG site demonstrating the most significant inverse correlation between CpG site methylation and mRNA expression was identified for CD44 , CD133 , and Musashi-1 , but not for CD24 . Methylation of CD44 , CD133 , and Musashi-1 was evaluated in 91 American Joint Committee on Cancer stage I to III primary breast cancer tumors, and these sites were significantly hypomethylated in TNBC versus non-TNBC. The IHC staining of primary tumors with the highest and lowest methylation levels revealed the strongest staining in hypomethylated specimens, suggesting that hypomethylation leads to gene activation. We demonstrate that methylation is a significant mechanism regulating CD44 , CD133 , and Musashi-1 , and that gene hypomethylation correlates with TNBC. Assessment of epigenetic changes in BCSC genes may provide a more accurate classification of TNBC and could be developed as potential therapeutic targets.
Do-not-resuscitate (DNR) status has been shown to be an independent risk factor for mortality in the post-operative period. Patients with DNR orders often undergo elective surgeries to alleviate ...symptoms and improve quality of life, but there are limited data on outcomes for informed decision making.
Retrospective cohort study.
A multi-institutional setting including operating room, postoperative recovery area, inpatient wards, and the intensive care unit.
A total of 566 patients with a DNR status and 316,431 patients without a DNR status undergoing elective procedures using the American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) from 2012.
Patients undergoing elective surgical procedures.
We analyzed the risk-adjusted 30-day morbidity and mortality outcomes for the matched DNR and non-DNR cohorts undergoing elective surgeries.
DNR patients had significantly increased odds of 30-day mortality (OR 2.51 1.55–4.05, p < 0.001) compared with non-DNR patients. In the DNR versus non-DNR cohort there was no significant difference in the occurrence of a number of 30-day complications, the rate of resuscitative measures undertaken, including cardiac arrest requiring CPR, reintubation, or return to the OR. The most common complications in both DNR and non-DNR patients undergoing elective procedures were transfusion, urinary tract infection, reoperation, and sepsis. Finally, the DNR patients had a significantly increased total length of hospital stay (7.65 ± 9.55 vs. 6.87 ± 9.21 days, p = 0.002).
DNR patients, as compared with non-DNR patients, have increased post-operative mortality but not morbidity, which may arise from unmeasured severity of illness or transition to comfort care in accordance with a patient's wishes. The informed consent process for elective surgeries in this patient population should include a discussion of acceptable operative risk.
•DNR patients undergoing elective procedures showed increased mortality without increased morbidity.•DNR patients had a longer total length of hospitalization after elective procedures.•Significant racial disparities exist in the DNR patient population.
The outcomes of patients with melanoma who have sentinel lymph node (SLN) metastases can be highly variable, which has precluded establishment of consensus regarding treatment of the group. The ...detection of high-risk patients from this clinical setting may be helpful for determination of both prognosis and management. We report the utility of multimarker reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) detection of circulating tumor cells (CTCs) in patients with melanoma diagnosed with SLN metastases in a phase III, international, multicenter clinical trial.
Blood specimens were collected from patients with melanoma (n = 331) who were clinically disease-free after complete lymphadenectomy (CLND) before entering onto a randomized adjuvant melanoma vaccine plus bacillus Calmette-Guérin (BCG) versus BCG placebo trial from 30 melanoma centers (United States and international). Blood was assessed using a verified multimarker RT-qPCR assay (MART-1, MAGE-A3, and GalNAc-T) of melanoma-associated proteins. Cox regression analyses were used to evaluate the prognostic significance of CTC status for disease recurrence and melanoma-specific survival (MSS).
Individual CTC biomarker detection ranged from 13.4% to 17.5%. There was no association of CTC status (zero to one positive biomarkers v two or more positive biomarkers) with known clinical or pathologic prognostic variables. However, two or more positive biomarkers was significantly associated with worse distant metastasis disease-free survival (hazard ratio HR = 2.13, P = .009) and reduced recurrence-free survival (HR = 1.70, P = .046) and MSS (HR = 1.88, P = .043) in a multivariable analysis.
CTC biomarker status is a prognostic factor for recurrence-free survival, distant metastasis disease-free survival, and MSS after CLND in patients with SLN metastasis. This multimarker RT-qPCR analysis may therefore be useful in discriminating patients who may benefit from aggressive adjuvant therapy or stratifying patients for adjuvant clinical trials.
Liver metastasis is the predominant cause of colorectal cancer (CRC) related mortality. Chemokines, soluble factors that orchestrate hematopoetic cell movement, have been implicated in directing ...cancer metastasis, although their clinical relevance in CRC has not been defined. Our hypothesis was that the chemokine receptor CXCR4 expressed by CRC is a prognostic factor for poor disease outcome.
CRC cell lines (n = 6) and tumor specimens (n = 139) from patients with different American Joint Committee on Cancer (AJCC) stages of CRC were assessed. Microarray screening of select specimens and cell lines identified CXCR4 as a prominent chemokine receptor. CXCR4 expression in tumor and benign specimens was assessed by quantitative real-time reverse transcription polymerase chain reaction and correlated with disease recurrence and overall survival.
High CXCR4 expression in tumor specimens (n = 57) from AJCC stage I/II patients was associated with increased risk for local recurrence and/or distant metastasis (risk ratio, 1.35; 95% CI, 1.09 to 1.68; P = .0065). High CXCR4 expression in primary tumor specimens (n = 35) from AJCC stage IV patients correlated with worse overall median survival (9 months v 23 months; RR, 2.53; 95% CI, 1.19 to 5.40; P = .016). CXCR4 expression was significantly higher in liver metastases (n = 39) compared with primary CRC tumors (n = 100; P < .0001).
CXCR4, a well-characterized chemokine receptor for T-cells, is differentially expressed in CRC. CXCR4 gene expression in primary CRC demonstrated significant associations with recurrence, survival, and liver metastasis. The CXCR4-CXCL12 signaling mechanism may be clinically relevant for patients with CRC and represents a potential novel target for disease-directed therapy.
Purpose: Somatic B-RAF gene mutation has been identified in many malignancies and detected at a high frequency in cutaneous malignant melanoma.
However, the significance of the B-RAF mutation ( B-RAF ...mt) in terms of its prognostic and predictive capabilities for treatment response or disease outcome is not known. We hypothesized
that circulating serum B-RAF mt ( B-RAF smt) at V600E, detected in serum, predicts response in melanoma patients receiving concurrent biochemotherapy.
Experimental Design: A real-time clamp quantitative reverse transcription-PCR assay was designed to assess B-RAF smt by peptide nucleic acid clamping and a locked nucleic acid hybrid probe. Normal ( n = 18) and American Joint Committee on Cancer stage I to IV melanoma patients ( n = 103) were evaluated. These included stage IV patients ( n = 48) with blood drawn before and after biochemotherapy. Patients were classified as biochemotherapy responders or nonresponders.
Responders ( n = 24) had a complete or partial response following biochemotherapy; nonresponders ( n = 24) developed progressive disease.
Results: Of the 103 melanoma patients, 38 (37%) had B-RAF smt DNA, of which 11 of 34 (32%) were stage I or II, and 27 of 69 (39%) were stage III or IV. Of the 48 biochemotherapy patients,
10 of 24 (42%) patients were positive for the B-RAF smt in the respective responder and nonresponder groups before treatment. After biochemotherapy, B-RAF smt was detected in only 1 of 10 patients (10%) in the responder group and 7 of 10 patients (70%) in the nonresponder group.
B-RAF smt is associated with significantly worse ( P = 0.039) overall survival in patients receiving biochemotherapy.
Conclusion: These studies show the presence and utility of circulating B-RAF smt DNA in melanoma patients.
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