M1 Group Research Articles

Effect of Differently Polarized Macrophages on Proliferation and Differentiation of Ependymal Cells from Adult Spinal Cord.

Ependymal cells (EpCs) are a kind of multi-potent stem cells in the central canal of adult spinal cord, which proliferate following spinal cord injury (SCI). Although they can differentiate into functional neurons in vitro, EpC progeny differentiate mainly into astrocytes after SCI, and the mechanism remains unclear. The present study aimed to explore whether neuroinflammation induced by classically activated macrophages (M1) or alternatively activated macrophages (M2) had an effect on EpC proliferation and/or differentiation. EpCs were isolated from intact spinal cord of adult mice and co-cultured with M1 or M2, respectively, in vitro. EpC proliferation was detected using a Cell Counting Kit-8 (CCK8) assay and Ki67 staining. Expression of Sox2 (SRY-box 2) in EpCs derived from different groups was detected by immunofluorescence and western blotting. Also explored was whether the mitogen activated protein kinase (MAPK) signaling pathway was involved in EpC proliferation. Immunofluorescence staining of βIII-tubulin and MAP2 were performed to assess the differentiation direction of EpCs in different culture conditions. Immunofluorescence and western blotting assays showed much more Sox2-positive EpCs in the group EpCs-M1 than the group EpCs-M2 in vitro (p < 0.01). The percentage of EpCs with positive Sox2 staining was decreased after tumor necrosis factor α (TNFα) antibody was added into the medium of EpCs-M1. Correspondingly, fewer Sox2-positive staining cells were observed in the central canal of TNFα-deficient mice with SCI. M1 co-culture promoted EpC proliferation significantly, which could be downregulated by Sox2 gene silencing (p < 0.01). Interestingly, M1 regulated the expression of Sox2 through the MAPK signaling pathway, especially the activation of ERK and p38 kinase. Co-culture in M2 conditioned medium obviously increased the proportion of βIII-tubulin-positive cells (p < 0.01). Small amounts of MAP2-positive neurons could be detected on day 7 in the M2 group and the control group. M1 conditioned medium could promote EpC proliferation in response to SCI through the TNFα-MAPK-Sox2 signaling pathway; M2 conditioned medium favors EpCs differentiating toward neurons.

The short-term outcomes of primary tumor resection with synchronous metastases from pancreas neuroendocrine tumors: an analysis of the ACS-NSQIP registry

Background: The incidence of pancreatic neuroendocrine tumors (PNET) is rising worldwide, with many patients developing metastatic disease. Resection of the primary tumor despite of unresectable metastases is suggested to improve long-term oncologic outcomes. Assessments of the morbidity of this approach are scarce and limited to small, single-institutional studies. We sought to determine the patient characteristics and short-term outcomes of pancreatectomy for metastatic (M1) PNET, and compare them to non-metastatic (M0) PNET. The comparison was undertaken to provide a reference with which providers are familiar – i.e. outcomes of pancreatectomy for M0 disease, to better appreciate the burden of complications with M1. Methods: We conducted a retrospective cohort study using the ACS-NSQIP targeted pancreatectomy registry. We included all adults undergoing pancreatectomy for PNET between 2014-2016, with complete staging information. M1 and M0 PNET groups were created. Primary outcomes were 30-day major morbidity, mortality, and prolonged length of stay (>75th percentile). The characteristics and outcomes of M1 and M0 groups were compared using Pearson Chi square, Fisher exact test, and Student t test, as appropriate. A propensity score was created with the following potential confounders defined a priori: age, sex, body mass index, ASA classification, T and N stage, surgical procedure, open approach, and functional histology. The association between M1 and outcomes was adjusted for the propensity score using logistic binomial regression. Results: Of 1,381 included patients, 148 (12%) had M1 disease. Patients with M1 were more likely to be younger, have lower hematocrit, higher ASA class, lower body mass index, and have pre-operative weight loss, than M0 patients (all p < 0.05). T1/2 (75.7% vs. 28.8%, p < 0.01), node positive (72.3% vs. 25.9%, p < 0.01), and functional (29.7% vs. 22.1%, p = 0.04) tumors were more common in the M1 group. There were no differences in the type of pancreatectomy (distal pancreatectomy 60.1% Vs. 57.3%, p = 0.46), but there were more open resections (78.4% vs. 55.5%, p < 0.01) and longer median operating time (322.5 vs. 261 minutes, p < 0.01) for M1. No significant difference was detected in overall 30-day major morbidity (38.5% vs. 39.7%, p = 0.77) and mortality (2.7% vs. 1.0%, p = 0.06) between groups. Prolonged length of stay was more common with M1 (35.1% vs. 25.8%, p = 0.01). Adjusted regression analysis revealed no independent association between M1 and 30-day morbidity (relative risk 0.90, 95% confidence interval: 0.71–1.14), or prolonged length of stay (relative risk 2.89, 95% confidence interval: 0.78–10.7). Regression was not conducted on mortality due to too few events. Conclusion: In a multi-institutional analysis of prospectively collected data, resection of the primary tumor for M1 PNET was not associated with different post-operative morbidity and mortality than for M0 disease. The knowledge of short-term outcomes provided herein is important to weigh perioperative risks against the potential benefits of PNET primary tumor resection in the setting of M1 disease. This can support both decision-making and patient counselling.

Effects of Moringa oleifera leave meal in the diet on layer performance, haematological and serum biochemical values

Abstract Utilisation of plant and leaf extracts in animal production is a strategy to improve health status and animals performance. Medical plants for use in animal diets need to be safe for animal health, so the effects on blood parameters need to be investigated. In this study the effect of different levels of Moringa oleifera leave meal (MOLM) in the diet on blood parameters and production performance of laying-type chickens from 1-day-old to 55 weeks of age was investigated. A total of 450 day-old chicks were distributed into three dietary treatment groups with 0% (M0), 1% (M1) and 3% (M3) addition of MOLM, with 5 replicates of 30 birds each. During the experimental period, feed intake (FI), body weight, egg laying rate and feed conversion ratio (FCR) were recorded. Blood samples for analysis of total protein, albumin, total iron and haematology were taken in 15 birds (3/replicate) at the 5 th, 15 th, 25 th, 35 th, 45 th and 55 th weeks of age between 9.00 am and 10.00 am. An increase in MOLM during the growing phase did not affect FI of the birds significantly, but live weights and FCR were improved (p < 0.05). In the laying period, FI was reduced in M1 and M3. Birds fed 1% of MOLM had the lowest FCR. The albumin level increased significantly (p < 0.05) in birds fed 3% of MOLM. Layers in the M1 group had the highest average egg-laying rate (p < 0.05). Red blood cells, white blood cells (WBC), packed cell volume and lymphocytes decreased with the increase in the Moringa oleifera level (p < 0.05). In conclusion, WBC and lymphocyte decreased probably because of a reduction of intestinal microflora. This resulted in an improvement of albumin level which subsequently affected bird’s growth and egg production.

Development of novel predictive miRNA/target gene pathways for colorectal cancer distance metastasis to the liver using a bioinformatic approach.

BackgroundLiver metastases are the major cause of colorectal cancer (CRC)-related deaths. However, there is no reliable clinical predictor for CRC progression to liver metastasis. In this study, we investigated possible predictors (miRNAs and biomarkers) for clinical application.MethodologyThe Gene Expression Omnibus (GEO) datasets GSE49355, GSE41258 and GSE81558 for genes and GSE54088 and GSE56350 for miRNAs were used to identify common differentially expressed genes (DEGs) and miRNAs between primary CRC tissues and liver metastases. The identified miRNAs and their targets from the DEGs were verified in datasets comprising gene, miRNA and miRNA exosome profiles of CRC patients with no distant metastases (M0) and distant metastases (M1); the interaction networks and pathways were also mapped.ResultsThere were 49 upregulated and 13 downregulated DEGs and 16 downregulated and 14 upregulated miRNAs; between the DEGs and miRNA targets, there were five upregulated and four downregulated genes. MiR-20a was strongly correlated with the status of liver metastasis. MiR-20a, miR499a, and miR-576-5p were highly correlated with the metastatic outcomes. MiR-20a was significantly highly expressed in the M1 group. In an analysis of the miRNA target genes, we found that CDH2, KNG1, and MMP2 were correlated with CRC metastasis. We demonstrated a new possible pathway for CRC metastasis: miR-576-5p/F9, miR20a/MMP2, CTSK, MMP3, and miR449a/P2RY14. The regulation of IGF transport and uptake by IGFBPs, extracellular matrix organization, signal transduction and the immune system were the enriched pathways.ConclusionThis model can predict CRC to liver metastases and the pathways involved, which can be clinically applicable.

Effects of replacing fish meal with mussel (Cristaria plicata) meat on growth, digestive ability, antioxidant capacity and hepatic IGF-I gene expression in juvenile Ussuri catfish (Pseudobagrus ussuriensis)

An 8-week feeding trial was implemented to evaluate the effects of replacing fish meal (FM) with mussel (Cristaria plicata) meat (MM) on growth, digestive ability, antioxidant capacity and hepatic insulin-like growth factor I (IGF-I) gene expression of juvenile Ussuri catfish (Pseudobagrus ussuriensis). Three isonitrogenous and isolipidic diets were formulated to include 0, 177.5 and 355.1 g/kg of MM, accordingly, replacing 0% (M0, control), 50% (M1) and 100% (M2) of FM protein, respectively. The results showed that the final body weight, weight gain, specific growth rate and feed intake were gradually decreased with dietary MM protein levels increased, but there were no significant difference between M0 and M1 groups (p > 0.05). The protein efficiency ratio was increased significantly with dietary MM inclusion (p < 0.05). The apparent digestibility coefficient of dry matter, crude lipid and gross energy gradually increased with increasing dietary MM protein levels, but the apparent digestibility coefficient of crude protein was not significantly affected by MM protein supplementation (p > 0.05). Fish fed diet, M0 and M1 remained unaffected significantly on activities of alpha-amylase and pepsin (p > 0.05), but fish fed diet M2 had the highest activities of alpha-amylase and pepsin. Fish fed diet M1 or M2 had significantly lower hepatic total antioxidant capacity, superoxide dismutase and the higher malondialdehyde level compared to fish fed diet M0. In addition, no significant difference was observed in hepatic IGF-I gene expression level for fish fed diet M0 and diet M1, and fish fed diet M2 showed significantly lower hepatic IGF-I gene expression level. Therefore, we can conclude that MM protein can successfully substitute 50% of FM protein without significantly negative effect on growth, nutrient utilization, and hepatic IGF-I gene expression for juvenile Ussuri catfish, but the antioxidant capacity was negatively affected in the present study, what is more, the total replacement of FM by MM in diet may result in the inhibition of the growth and antioxidant capacity of fish.

Is the non-metastatic, locally advanced colon adenocarinoma a distinct biological tumor variant? A study based on pathological evaluation, immunohistochemical panel and survival.

to evaluate the clinical and pathological differences between locally advanced colonic adenocarcinomas (LACA) with adhesions between adjacent organs or structures, and colonic adenocarcinomas with other clinical presentations. we conducted a retrospective study from a convenience sample of patients with colonic adenocarcinoma, pathological stage pT3, distributed according to clinical and pathological characteristics in three groups: locally advanced tumors (LACA), pT3 tumors without adhesions or distant metastases (SF) and tumors with metastatic disease (M1). We evaluated clinical and pathological characteristics and the expression of seven immunohistochemical markers related to proliferation/apoptosis, cell invasion/migration and metastasis. we studied 101 patients: 30 LACA, 44 SF and 27 M1. Locally advanced tumors presented larger dimensions and were associated with increased lymphocyte infiltration rates, lower levels of bax expression, and CD 44v6 when compared with SF and M1 groups. We observed significant differences between LACA and M1 in relation to colonic location, histology, lymph node status and bax and CD44v6 expression. We found differences were observed between the three groups for tumor size and lymphocytic infiltrate. Survival was similar in the LACA and SF groups (p=0.66) and was lower in the M1 group (p<0.001). the data suggest that locally advanced colonic adenocarcinomas with adhesions between adjacent organs or structures represent a distinct entity.

Comparative evaluation of the effect of surface treatment of fiber-reinforced posts and prefabricated metal posts on adhesion of a resin-based luting cement: An in vitro study.

Background:The purpose of this study was to evaluate the effect of surface treatment of fiber-reinforce post and metal post of adhesion of a resin luting cement.Materials and Methods:Sixty methyl methacrylate specimens were fabricated with a customized metal rod. The samples were segregated into six groups (F1, F2, F3, M1, M2, and M3) of 10 specimens (n = 10) each. The first three groups (F1, F2, and F3) were for fiber posts and (M1, M2, and M3) were for metal posts. The postspace preparation was done with dedicated drills supplied by the postmanufacturers to a length of 14 mm. F1 and M1 were the control groups. The posts in F2 and M2 groups were treated with airborne-particle abrasion with 70 μm Al2O3 particles for 5 s at a constant distance of 20 mm, cleaned with alcohol and cemented in the postspace. The posts in the F3 and M3 groups were treated with airborne-particle abrasion followed by primer application. After cementing the posts into the postspace, the acrylic blocks were sectioned with a motor-driven jigsaw to obtain four specimens each of 3-mm thickness. These sections were then subjected to push-out tests on a universal testing machine.Results:There was a statistically significant increase in the bond strength of both fiber and metal posts to resin cement after airborne-particle abrasion with Al2O3 particles and airborne abrasion followed by primer application.Conclusion:There is an increase in the bond strength of the resin cement with the prefabricated posts after the various surface treatments.

Phenotypic changes of interstitial cells of Cajal after intestinal obstruction in rat model.

The objective was to study the effect of mechanical intestinal obstruction in rats on the phenotype of interstitial cells of Cajal (ICC). Healthy Wistar rats were randomly divided into sham-operation group (C), one day obstruction group (M1), two days obstruction group (M2), and three days obstruction group (M3), with 10 rats in each group. The expression of SCF mRNA and c-Kit protein in intestinal tissue was investigated by RT-PCR and immunohistochemistry. Compared with the sham-operation group, the relative expression of SCF mRNA and the expression of c-Kit protein in intestinal tissue were significantly decreased in both obstruction groups. Levels decreased gradually with the prolongation of obstruction time, and significantly decreased on the 3rd day after obstruction (P<0.05). Immunohistochemical staining of the small intestine showed that the number of ICC in the sham-operation group was the highest, and they were gradually decreased with the extension of obstruction time in the M1 to M3 groups. There was a significant difference between groups (P<0.05). Intestinal obstruction caused a decrease in the concentrations of SCF mRNA and c-Kit protein in ICC. With the prolongation of intestinal obstruction, the number of ICCs gradually decreased.

Vaccine containing G protein fragment and recombinant baculovirus expressing M2 protein induces protective immunity to respiratory syncytial virus.

PurposeRespiratory syncytial virus (RSV) can cause serious respiratory illnesses such as pneumonia, asthma, and bronchiolitis in infants and elderly or immunocompromised individuals. An RSV vaccine has yet to be developed; only prophylactic anti-RSV antibody is commercially available. So, we investigated whether our vaccine candidate is able to induce type 1 CD4+ T helper (Th1), CD8+ T-cell responses, and protective immunity without vaccine-enhanced disease (VED) against RSV.Materials and MethodsWe used RSV G protein fragment (Gcf A) with recombinant baculovirus capable of expressing the RSV M2 protein (Bac M2) as a vaccine candidate, and injected this vaccine (Gcf A/Bac M2) intramuscularly, and challenged with RSV intranasally into mice. Enzyme-linked immunosorbent assay, flow cytometry, plaque assay, and weight measurement were performed to confirm humoral immunity, cellular immunity, and protective immunity.ResultsThe Gcf A/Bac M2 formulation induced a stronger IgG response to Gcf A than Gcf A inoculation alone, and the ratio of IgG1/IgG2a indicated that the responses shifted predominantly to Th1. In addition, both RSV G-specific Th1 responses and RSV M2-specific CD8+ T-cell responses were induced, and G protein-associated eosinophilic infiltration was suppressed compared to the control group. Moreover, the Gcf A/Bac M2 group showed effective protection after an RSV challenge.ConclusionBac M2 could serve as a vaccine with intrinsic adjuvant activity, and the Gcf A/Bac M2 shows promise as a vaccine candidate for inducing protective immunity without inciting VED.

Aspirin inhibits cell stemness of esophageal cancer by downregulation of chemokine CCL2

Objective: To investigate the effect of tumor-associated macrophages on the stemness of esophageal cancer cells and the potential mechanism of antiproliferative effects of aspirin (ASA). Methods: The effects of aspirin on the stemness characteristics of KYSE-450 cells and KYSE-450 cells co-cultured with M2 macrophages (KYSE-450+ M2) were performed using spheroid formation assay. After treatment with aspirin, the expression of different chemokines, the core pluripotency gene Nanog and the stem cell marker CD90 in different cell groups were determined by real-time quantitative PCR, flow cytometry and Western blot. Results: The number of spheres formed in the ASA and KYSE-450+ M2 cell groups were 7.00±1.23 and 34.33±2.33, respectively, showing statistically significant difference compared with that of control group (14.50±2.33, all P<0.05). The number of spheres in KYSE-450+ M2+ ASA cell group were 20.67±2.33, which was significantly lower than that of KYSE-450+ M2 group (P<0.05). The expression levels of Nanog gene in control and ASA groups were 1.00 and 0.50±0.10, respectively, and the difference was statistically significant (P<0.05). Moreover, the expression of Nanog gene in cells of KYSE-450+ M2 group and M2+ KYSE-450+ ASA group was 1.74±0.13 and 1.43±0.05, showing statistically significant difference (P<0.05). When chemokine CCL2 was knocked down, the levels of Nanog gene in M2+ shCCL2-KYSE450+ ASA group and M2+ shCCL2-KYSE450 group were decreased to 1.22±0.11 and 1.17±0.08, respectively, and there was no statistically significant difference between them (P=0.69). Flow cytometry analyses showed that the expression levels of CD90 in control and ASA cells were (2.93±0.52)% and (1.30±0.17)%, respectively, and the difference was statistically significant (P<0.05). Moreover, the expression levels of CD90 in M2+ shCCL2-KYSE450 cells and M2+ shCCL2-KYSE450+ ASA cells were (4.07±0.12)% and (4.73±0.38)%, respectively, showing no statistically significant difference (P=0.17). Conclusions: Tumor-associated macrophages enhances the stemness of esophageal cancer cells, whereas aspirin attenuates the stemness by suppressing the expression of CCL2. Aspirin plays an anti-tumor effect in esophageal cancer cells.

Challenging New AJCC 8 Staging For Soft Tissue Sarcoma Using The NCDB

Soft tissue sarcoma (STS) is a rare and heterogeneous entity for which there are many challenges to staging. One of the most significant updates in the AJCC 8th edition is the grouping of N1 and M1 collectively into stage IV disease. The aim of this study is to present an analysis of the impact of N1 vs. M1 disease on the overall survival (OS) of STS patients treated in the modern era and in the context of the contemporary AJCC staging system. Adult STS patients were identified in the 2004 – 2014 NCDB, classified by the WHO 2013 system into 10 major histologic subtypes and grouped according to new AJCC 8 staging. Primary retroperitoneal STS, patients with “other/unusual” histologic subtypes and those who had delays in therapy (>180 days from diagnosis) were excluded. Chi-square tests were used to compare demographic factors, tumor characteristics, and therapy received, while Kaplan-Meier estimates, Cox proportion hazard ratios and log rank tests were used for survival analyses. Of the 82,987 adult STS patients identified, 55,417 met inclusion criteria. On univariable analysis, females, patients >50yo and those with low grade tumors were less likely to be N1 (p< 0.01). No significant difference was found in the likelihood of having N1 disease between tumor sizes </=5cm and those >5cm. Multivariable analysis identified N1 and M1 disease as independent factors associated with overall survival (HR= 1.34, p< 0.001 and HR= 2.52, p< 0.0001, respectively). N1 patients were more likely to have M1 disease (OR= 2.5) compared to N0 patients (p< 0.001). M1 disease was more likely in African Americans than in Whites, in intermediate/high grade than in low grade tumors and in tumors >5cm than in those </=5cm (p< 0.001). There were significant differences in 10-year OS between AJCC 8 stage groups 1-4 (63.3%, 48.7%, 32.3% and 7.9%, respectively); p< 0.001. N1 patients were found to have a significantly shorter 5-year OS (34.4%) and 10-year OS (27.3%) compared with N0 patients (65.3% and 49.9%, respectively); p< 0.001. There were no significant differences in the 5- and 10-year OS of N0M1 patients (10% and 5.4%, respectively) compared with N1M1 patients (11.31% and 0%, respectively); p= 0.95. As such, further analyses combined N1M1 and N0M1 into one M1 group. There was a significant difference in 5- and 10-year OS of N1M0 patients (34.4% and 27.3%, respectively) relative to (N1/0)M1 patients (10% and 5.4%, respectively); p<0.0001. Using the NCDB, we demonstrate a significant separation in OS between N1M0 and M1 adult patients with STS. We confirm that N0 patients fare much better than their N1 counterparts in OS. The data also demonstrate that N1 patients have significantly improved 10-year OS (27.3%) compared to that of M1 patients (5.4%). These results suggest that the new AJCC 8th edition grouping of N1 and M1 patients into stage IV may obscure the more favorable prognosis of patients with N1M0 disease.

Evaluation of the perfusion index according to the visual analog scale in postoperative patients.

Objectives:To evaluate the possible value of the perfusion index (PI) as a tool for pain assessment.Methods:This prospective, observational study was performed with 89 patients underwent surgery with general anesthesia. The patients with visual analog scale (VAS)>3 were grouped as M1, and patients with VAS≤3 and performed morphine were grouped as M2. After surgery patients with VAS>3 were given 2mg morphine. Patients with VAS>3 were given increments of intravenous morphine (2 mg) at 20 minute intervals until VAS<3. The correlation and difference between PI and VAS score values were evaluated before and after analgesic administration.Results:Significant changes were found in both PI values and VAS scores between M1 and M2 groups (2.80±0.77, 3.97±0.94, p<0.001; 6.60±1.20, 2.74±0.46, p<0.001) Despite no correlation was found between PI values and VAS scores of M1 and M2 groups, weak negative correlation was detected between differences in PI values and VAS scores among groups (r=-0.255, p=0.016).Conclusion:Perfusion index is a parameter that can be used in the assessment of postoperative pain and responses to analgesics.

The Impact of Diffusion Tensor Imaging Fiber Tracking of the Corticospinal Tract Based on Navigated Transcranial Magnetic Stimulation on Surgery of Motor-Eloquent Brain Lesions.

Navigated transcranial magnetic stimulation (nTMS) enables preoperative mapping of the motor cortex (M1). The combination of nTMS with diffusion tensor imaging fiber tracking (DTI-FT) of the corticospinal tract (CST) has been described; however, its impact on surgery of motor-eloquent lesions has not been addressed. To analyze the impact of nTMS-based mapping on surgery of motor-eloquent lesions. In this retrospective case-control study, we reviewed the data of patients operated for suspected motor-eloquent lesions between 2012 and 2015. The patients underwent nTMS mapping of M1 and, from 2014, nTMS-based DTI-FT of the CST. The impact on the preoperative risk/benefit analysis, surgical strategy, craniotomy size, extent of resection (EOR), and outcome were compared with a control group. We included 35 patients who underwent nTMS mapping of M1 (group A), 35 patients who also underwent nTMS-based DTI-FT of the CST (group B), and a control group composed of 35 patients treated without nTMS (group C). The patients in groups A and B received smaller craniotomies (P = .01; P = .001), had less postoperative seizures (P = .02), and a better postoperative motor performance (P = .04) and Karnofsky Performance Status (P = .009) than the controls. Group B exhibited an improved risk/benefit analysis (P = .006), an increased EOR of nTMS-negative lesions in absence of preoperative motor deficits (P = .01), and less motor and Karnofsky Performance Status worsening in case of preoperative motor deficits (P = .02, P = .03) than group A. nTMS-based mapping enables a tailored surgical approach for motor-eloquent lesions. It may improve the risk/benefit analysis, EOR and outcome, particularly when nTMS-based DTI-FT is performed.

Microcystin and pyriproxyfen are toxic to early stages of development in Rhamdia quelen: An experimental and modelling study

The recent increase of freshwater eutrophication has favored cyanobacteria blooms and consequently the increase of toxins such as microcystin-LR in aquatic environments, but few is know about the associated effect of toxin and other compounds. Pyriproxyfen is an insecticide indicated by WHO (World Health Organization) to control Aedes aegypti mosquito (vector of Dengue, Chikungunya and Zika diseases), however, the effects are not well described to non-target species, such as fish. The early life stages (ELS) of fish are more sensitive to chemical stress due to higher metabolic rate, immature immune system and high superficial area/volume ratio. In the current study, ELS of R. quelen a Neotropical fish were exposed to environmentally realistic concentrations of microcystin (1, 10 and 100 µg L−1; M1, M2 and M3 groups, respectively) from an algal extract, pyriproxyfen (1 and 10 µg L−1, P1 and P2) and their association (co-exposure). The hatching, survival and larvae deformities were analyzed, and applied a mathematical model to evaluate the effects on the population size along further generations. Both compounds were toxic to embryos/larvae of fish, but the effects were more pronounced in M2, P1M2 and P2M1 for hatching and M2, P1M2, P2M1 and P1 for survival. Deformities prevailed in groups exposed to the chemicals at 48 hpf (hours post-fertilization) were suggestions of toxicological interaction in P1M2, P2M1 and P2M2 at 48 and 72 hpf. In 96 hpf, the levels of deformities were lower than in previous times. Model predicted population density over 100 years decreased to lower than 0.5 (50%) in all groups, except for P1M1, indicating risk of extinction. P1M2 had the worse results, followed by M2, P1M3 and P2M1. Cyanobacterial blooms can lead to microcystin-LR levels higher than M2 (10 µg L−1), and the suggestion of toxicological interaction with pyriproxyfen is relevant because both compounds may potentially coexist in aquatic environments. Finally, mathematical models may provide an ecological interpretation of the risk of exposure of fish.

Random Mutagenesis Analysis of the Influenza A M2 Proton Channel Reveals Novel Resistance Mutants

The influenza M2 proton channel is a major drug target, but unfortunately, the acquisition of resistance mutations greatly reduces the functional life span of a drug in influenza treatment. New M2 inhibitors that inhibit mutant M2 channels otherwise resistant to the early adamantine-based drugs have been reported, but it remains unclear whether and how easy resistance could arise to such inhibitors. We have combined a newly developed proton conduction assay with an established method for selection and screening, both Escherichia coli-based, to enable the study of M2 function and inhibition. Combining this platform with two groups of structurally different M2 inhibitors allowed us to isolate drug resistant M2 channels from a mutant library. Two groups of M2 variants emerged from this analysis. A first group appeared almost unaffected by the inhibitor, M_089 (N13I, I35L, and F47L) and M_272 (G16C and D44H), and the single-substitution variants derived from these (I35L, L43P, D44H, and L46P). Functionally, these resemble the known drug resistant M2 channels V27A, S31N, and swine flu. In addition, a second group of tested M2 variants were all still inhibited by drugs but to a lesser extent than wild type M2. Molecular dynamics simulations aided in distinguishing the two groups where drug binding to the wild type and the less resistant M2 group showed a stable positioning of the ligand in the canonical binding pose, as opposed to the drug resistant group in which the ligand rapidly dissociated from the complex during the simulations.

Expression of the intrarenal angiotensin receptor and the role of renin-angiotensin system inhibitors in IgA nephropathy.

The critical role of the intrarenal renin-angiotensin system (RAS) in the development of kidney disease has been well demonstrated in animal and cell-culture experiments, but evidence from human kidney tissues is lacking. In this study, we screened 438 patients with IgA nephropathy (IgAN) and analyzed their clinical characteristics. Renal biopsy revealed the expression of angiotensin II type 1 receptor (AT1R), angiotensin II type 2 receptor (AT2R), and MAS receptor (MASR) in the tissues of 260 patients not treated with RAS inhibitors, 32 patients treated with angiotensin-converting enzyme inhibitors (ACEIs), and 89 patients treated with angiotensin receptor blockers (ARBs). The correlations in expression among these three receptors and the results of Oxford typing were analyzed, together with the ability of ACEIs and ARBs to reduce proteinuria and the effects of ARBs on AT1R and AT2R expression. The results showed significantly higher AT1R, AT2R, and MASR expression in the M1 group (mesangial score > 0.5) than in the M0 group (mesangial score < 0.5), significantly higher AT1R expression in the S1 group (presence of segmental glomerulosclerosis) than in the S0 group (absence of segmental glomerulosclerosis); AT1R expression in the C2 group (crescent formation > 25%) was significantly higher than in the C0 (crescent formation = 0) and C1 (crescent formation < 25%) groups. Patients treated with an ARB for < 6 months had significantly lower urinary protein levels than those taking these drugs for > 6 months. These findings imply that overexpression of AT1R on the mesangial cells of IgAN patients is associated with mesangial cell proliferation, glomerular segmental sclerosis, and crescent formation. In addition, long-term administration of ARB may decrease the efficacy of these medications in terms of reducing proteinuria.

Effect of methylprednisolone on endoplasmic reticulum stress in rats with ventilator-induced lung injury: the relationship with PI3K/Akt signaling pathway

Objective To evaluate the effect of methylprednisolone on endoplasmic reticulum stress in rats with ventilator-induced lung injury (VILI) and the relationship with phosphatidylinositol 3-kinase/serine-threonine protein kinase (PI3K/Akt) signaling pathway. Methods One hundred clean-grade male Sprague-Dawley rats, aged 4-5 months, weighing 270-320 g, were divided into 5 groups (n=20 each) using a random number table method: control group (C group), VILI group (V group) and different doses of methylprednisolone groups (M1-3 groups). Group C received no mechanical ventilation and kept spontaneous breathing for 4 h. Rats were mechanically ventilated (tidal volume 40 ml/kg, respiratory rate 15-17 breaths/min, inspiratory/expiratory ratio 1∶1, positive end-expiratory pressure 0, fraction of inspired oxygen 21% during OLV) in group V. Methylprednisolone 2, 10 and 30 mg/kg were intravenously injected at 20 min before mechanical ventilation in M1-3 groups, respectively, and the equal volume of normal saline was given in group V. Blood samples and lung tissues were taken at 4 h of ventilation for measurement of the lung permeability index (LPI) and wet/dry lung weight ratio (W/D ratio), for examination of pathological changes, and for determination of apoptosis index (AI) in lung tissues (by TUNEL), expression of Akt, phosphorylated Akt (p-Akt), glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP) and caspase-12 in lung tissues (by Western blot). Injured alveoli rate (IAR) was calculated. Results Compared with group C, the W/D ratio, LPI, IAR and AI were significantly increased, the expression of p-Akt was down-regulated, and the expression of GRP78, CHOP and caspase-12 was up-regulated in V and M1 groups (P 0.05). Compared with group V, the W/D ratio, LPI, IAR and AI were significantly decreased, p-Akt expression was up-regulated, and the expression of GRP78, CHOP and caspase-12 was down-regulated in M2 and M3 groups (P<0.05). Conclusion Methylprednisolone inhibits endoplasmic reticulum stress, thus inhibiting cell apoptosis, and the mechanism is related to activating PI3K/Akt signaling pathway in rats with VILI. Key words: Methylprednisolone; Respiration, artificial; Respiratory distress syndrome, adult; Endoplasmic reticulum; Stress; 1-Phosphatidylinositol 3-kinase; Protein-serine-threonine kinases

Abstract B094: Quantitative digital image analysis and machine learning for staging of prostate cancer at diagnosis

Abstract Introduction and Objective: Prostate cancer (PC) with de novo bone metastases at diagnosis (M1) carries a 5-year survival rate of 28% and requires early, aggressive treatment. Clinical assays and the pathology of prostate needle biopsies (PNBX) cannot distinguish primary M1 tumors from high-grade localized (M0) cases. We hypothesized that digital image analysis can be applied to obtain morphologic biomarkers, not recognizable by pathologists. Here we demonstrate how novel software tools that involve deep learning frameworks can be used to systematically extract handcrafted and autoencoder features and to build models to predict M1 stage at the time of diagnosis. Methods: A study cohort, nested within a biorepository of 2150 PC patients at the Greater LA VA, consisted of 86 high-grade M0 and 85 M1 cases. Slides were digitized at 40X and 2 pathologists annotated all cancer foci. Approximately 30 image tiles were selected from each case. 62 handcrafted (HC) and 64 autoencoder (AE) features were extracted from nuclei. Feature values normalized. The normalized profile of each primary feature gave rise to 11 secondary features, representing the distribution of the feature within a case. We separated cases into training + testing versus validation groups at an 80:20 ratio. Using a bootstrapping method, we selected the best GLMNET models predicting M0 versus M1 status in the training + testing set and applied them to an independent validation set of cases. Results: After successful conversion of M0 and M1 image tiles to digital nuclear masks and color normalization, ~ 400,000 nuclei were isolated using parameters that enriched for nuclei from cancer cells. A denoising autoencoding neural network was used to generate AE biomarkers for each nucleus. HC features quantified nuclear shape, size, color, and texture. A systematic pipeline of preprocessing, normalization, and conversion to case-level secondary features was applied to AE and HC features. For both feature types, the average of 50,000 bootstrapping models resulted in an AUC of 0.8 for the training and an average accuracy of 0.7 for the test cohort. The best 38 AE models or 16 HC models were applied to the independent validation cohort of 24 cases and assigned each case to the M0 versus M1 groups by majority voting. At a threshold of 0.5, this resulted in an accuracy of 70% for M0 versus M1 distinction. Conclusion: We applied digital imaging technology and machine learning software to AE and HC features in order to predict M0 versus M1 stage from the tumor in PNBXs at diagnosis. Unexpectedly, hidden features in nuclei differed between M0 and M1 cases and succeeded in predicting metastatic disease with 70% accuracy. The ultimate goal is to combine the inexpensive risk prediction from quantitative imaging with clinical parameters and RNA sequencing data to develop accurate prediction models of occult metastases and risk of future metastatic progression at the time of diagnosis in all patients with high-grade PC. Funding: DOD PC131996, PCF-Movember GAP1 Unique TMAs Project, Prostate Cancer Foundation (PCF) Creativity Award, Jean Perkins Foundation, NIH/NCI P01 CA098912-09, NIH R01CA131255 and P50CA092131, Stephen Spielberg Team Science Award. Citation Format: Fangjin Huang, Nathan Ing, Miller Eric, Hootan Salemi, Michael Lewis, Isla Garraway, Arkadiusz Gertych, Beatrice Knudsen. Quantitative digital image analysis and machine learning for staging of prostate cancer at diagnosis [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B094.

Relationship between Early Treatment Response and Prognosis in Children with Acute Lymphoblastic Leukemia

To analyze the relationship between the early treatment response and the pregnosis in children with acute lymphoblastic leukemia(ALL). Two hundred and Seventy-eight ALL children diagnosed and treated in Hainan general hospital from March 2013 to March 2017 were collected. All ALL children received therapy with CCLg-ALL-2008 regimen. The 3 year event-free survival (EFS) rate of ALL children in different groups was analyzed in terms of 4 indexes including sensitivity response to prednison at day 8 (D8-SRP), bone marrow remission at day 15 (D15-BMR) and at day 33 (D33-BMR), and minimal residual disease at day 33 (D33-BMR), and minimal residual disease at day 33(D33-MRD). These 4 indexes and other indexes possibly affecting the prognosis of ALL children were enrolled in Cox regression model for analysis of independent factors affecting the prognosis of ALL children. The D8-SRP test showed that among 269 ALL children, 240(89.22%) cases displayed prednisone poor response (PPR); the 3-year EFS rate in predrisone good response(PGR) group was significantly higher than that in PPR group(P<0.05). The D15-BMR detection showed that among 262 ALL children, the bone marrow remission(BMR) as M1 was observed in 230 cases (87.79%), M2 in 20 cases (7.63%) and M3 in 9 cases (4.58%); the 3-year EFS rate showed as follows:M1 group >M2 group >M3 group(P<0.05). The D33-BMR detection showed that among 257 ALL children, the BMR as M1 was observed in 227 cases (88.33%), M2 in 21 cses(8.17%) and M3 in 9 caes (3.51%); the 3-year EFS rate in 3 groups showed as follows: M1 group >M2 group >M3 group(P<0.05). The D33-MRD detection showed that among 185 ALL children, MRD<10-10 was found in 128 cases (69.19%), MRD≥10-4-10-2 in 43 cases (23.24%), MRD ≥10-2 in 14 cases (7.57%); the 3-year EFS rate in 3 groups showed as follows: MRD <10-4 group > MRD≥ 10-4-10-2 group>MRD≥10-2 group. The Cox regression analysis showed that PPR in D8-SRP test, M2 and M3 in D15 and D33 BMR detection, and MRD≥10-2 in D33 MRD detection as well as T-ALL typing were independent risk factors affecting the prognosis of ALL children. The early treatment response can predict the prognosis of ALL children, which is an independent prognostic factor for ALL children.

Survival analysis and seasonal patterns of pregnancy outcomes in Egyptian buffaloes

The objective of this study was to analyze the survival patterns of pregnancy outcomes in Egyptian buffaloes under subtropical environmental conditions. The overall reproductive and production indices were performed on a total of 1224 records. The means of days at first AI postpartum, days open and calving interval of Egyptian buffaloes are 62.29, 83.13 and 395.1 days, respectively. The survival analysis of pregnancy outcomes in the present study was restricted to the first AI (576 first AI records). Compared to the low temperature-humidity index (THI), the conception rate (P/AI at 30 days) was significantly decreased at high THI level (OR = 0.45; p = 0.029), however, no significant variations were recorded between the referent (low) THI level and the moderate one (OR = 0.86; p = 0.673). Additionally, the pregnancy loss rate was significantly increased at a high THI level (OR = 2.43; p = 0.008). In comparison with the referent (primiparous) group, the conception rates were significantly increased in the M1 (2–5 parities; OR = 2.84) and M2 (>5 parities; OR = 4.06) multiparous groups (p = 0.007 and 0.004, respectively). The Egyptian buffaloes produce a relatively higher daily milk yield (>9 kg) have significantly lower conception rate compared with the referent (<7 kg) group (OR = 0.46; p = 0.046). However, the daylight length (continuous variable) had no significant effects on the conception (OR = 0.88; p = 0.298) and pregnancy loss (OR = 0.92; p = 0.608) rates. The Cox Proportional-hazards model for the hazard of pregnancy outcome (P/AI at 30 days) revealed that multiparous buffaloes had a higher hazard of pregnancy than primiparous buffaloes (HR = 2.37, p = 0.001). Moreover, buffalo cows calving in the summer and spring have a lower hazard of pregnancy than buffaloes calve in the winter season (HR = 0.57 and 0.76, p = 0.001 and 0.002, respectively). The current study indicates that Egyptian buffaloes have the ability to tolerate the thermal stress up to 80 THI values without deterioration of pregnancy outcomes. Moreover, primiparous or high producing buffalo cows may require more effective reproductive strategies as modulation of breeding plans to get calving in the winter and autumn seasons.