RapidET: a MEMS-based platform for label-free and rapid demarcation of tumors from normal breast biopsy tissues

The rapid and label-free diagnosis of malignancies in ex vivo breast biopsy tissues has significant utility in pathology laboratories and operating rooms. We report a MEMS-based platform integrated with microchips that performs phenotyping of breast biopsy tissues using electrothermal sensing. The microchip, fabricated on a silicon substrate, incorporates a platinum microheater, interdigitated electrodes (IDEs), and resistance temperature detectors (RTDs) as on-chip sensing elements. The microchips are integrated onto the platform using a slide-fit contact enabling quick replacement for biological measurements. The bulk resistivity (ρB), surface resistivity (ρS), and thermal conductivity (k) of deparaffinized and formalin-fixed paired tumor and adjacent normal breast biopsy samples from N = 8 patients were measured. For formalin-fixed samples, the mean ρB for tumors showed a statistically significant fold change of 4.42 (P = 0.014) when the tissue was heated from 25 °C to 37 °C compared to the adjacent normal tissue, which showed a fold change of 3.47. The mean ρS measurements also showed a similar trend. The mean k of the formalin-fixed tumor tissues was 0.309 ± 0.02 W m−1 K−1 compared to a significantly higher k of 0.563 ± 0.028 W m−1 K−1 for the adjacent normal tissues. A similar trend was observed in ρB,ρS, and k for the deparaffinized tissue samples. An analysis of a combination of ρB, ρS, and k using Fisher’s combined probability test and linear regression suggests the advantage of using all three parameters simultaneously for distinguishing tumors from adjacent normal tissues with higher statistical significance.

Integrating RNA-Seq With GWAS Reveals a Novel SNP in Immune-Related HLA-DQB1 Gene Associated With Occupational Pulmonary Fibrosis Risk: A Multi-Stage Study

ObjectiveTo evaluate the association between single-nucleotide polymorphisms (SNPs) in RNA-seq identified mRNAs and silicosis susceptibility.MethodsA comprehensive RNA-seq was performed to screen for differently expressed mRNAs in the peripheral blood lymphocytes of eight subjects exposed to silica dust (four silicosis cases and four healthy controls). Following this, the SNPs located on the shortlisted mRNAs, which may affect silicosis susceptibility, were screened through silicosis-related genome-wide association studies (GWAS) (155 silicosis cases and 141 healthy controls), whereas functional expression quantitative trait locus (eQTL)-SNPs were identified using the GTEx database. Finally, the association between functional eQTL-SNPs and silicosis susceptibility (194 silicosis cases and 235 healthy controls) was validated.ResultsA total of 70 differentially expressed mRNAs (fold change > 2 or fold change < 0.5, P < 0.05) was obtained using RNA-seq. Furthermore, 476 SNPs located on the shortlisted mRNAs, which may affect silicosis susceptibility (P < 0.05) were obtained using GWAS, whereas subsequent six functional eQTL-SNPs were identified. The mutant A allele of rs9273410 in HLA-DQB1 indicated a potential increase in silicosis susceptibility in the validation stage (additive model: odds ratio (OR)= 1.31, 95% confidence interval (CI) = 0.99–1.74, P = 0.061), whereas the combination of GWAS and the validation results indicated that the mutant A allele of rs9273410 was associated with increased silicosis susceptibility (additive model: OR = 1.35, 95% CI =1.09–1.68, P = 0.006).ConclusionThe mutant A allele of rs9273410 was associated with increased silicosis susceptibility by modulating the expression of HLA-DQB1.

Down-regulation of the brain-specific cell-adhesion molecule contactin-3 in tuberous sclerosis complex during the early postnatal period

Background The genetic disorder tuberous sclerosis complex (TSC) is frequently accompanied by the development of neuropsychiatric disorders, including autism spectrum disorder and intellectual disability, with varying degrees of impairment. These co-morbidities in TSC have been linked to the structural brain abnormalities, such as cortical tubers, and recurrent epileptic seizures (in 70–80% cases). Previous transcriptomic analysis of cortical tubers revealed dysregulation of genes involved in cell adhesion in the brain, which may be associated with the neurodevelopmental deficits in TSC. In this study we aimed to investigate the expression of one of these genes – cell-adhesion molecule contactin-3. Methods Reverse transcription quantitative polymerase chain reaction for the contactin-3 gene (CNTN3) was performed in resected cortical tubers from TSC patients with drug-resistant epilepsy (n = 35, age range: 1–48 years) and compared to autopsy-derived cortical control tissue (n = 27, age range: 0–44 years), as well as by western blot analysis of contactin-3 (n = 7 vs n = 7, age range: 0–3 years for both TSC and controls) and immunohistochemistry (n = 5 TSC vs n = 4 controls). The expression of contactin-3 was further analyzed in fetal and postnatal control tissue by western blotting and in-situ hybridization, as well as in the SH-SY5Y neuroblastoma cell line differentiation model in vitro. Results CNTN3 gene expression was lower in cortical tubers from patients across a wide range of ages (fold change = − 0.5, p < 0.001) as compared to controls. Contactin-3 protein expression was lower in the age range of 0–3 years old (fold change = − 3.8, p < 0.001) as compared to the age-matched controls. In control brain tissue, contactin-3 gene and protein expression could be detected during fetal development, peaked around birth and during infancy and declined in the adult brain. CNTN3 expression was induced in the differentiated SH-SY5Y neuroblastoma cells in vitro (fold change = 6.2, p < 0.01). Conclusions Our data show a lower expression of contactin-3 in cortical tubers of TSC patients during early postnatal period as compared to controls, which may affect normal brain development and might contribute to neuropsychiatric co-morbidities observed in patients with TSC.

Maternal immune activation induces methylation changes in schizophrenia genes

Susceptibility to schizophrenia is mediated by genetic and environmental risk factors. Infection driven maternal immune activation (MIA) during pregnancy is a key environmental risk factor. However, little is known about how MIA during pregnancy could contribute to adult-onset schizophrenia. In this study, we investigated if maternal immune activation induces changes in methylation of genes linked to schizophrenia. We found that differentially expressed genes in schizophrenia brain were significantly enriched among MIA induced differentially methylated genes in the foetal brain in a cell-type-specific manner. Upregulated genes in layer V pyramidal neurons were enriched among hypomethylated genes at gestational day 9 (fold change = 1.57 , FDR = 0.049) and gestational day 17 (fold change = 1.97 , FDR = 0.0006). We also found that downregulated genes in GABAergic Rosehip interneurons were enriched among hypermethylated genes at gestational day 17 (fold change = 1.62, FDR= 0.03). Collectively, our results highlight a connection between MIA driven methylation changes during gestation and schizophrenia gene expression signatures in the adult brain. These findings carry important implications for early preventative strategies in schizophrenia.

Impact of Integrase Sequences From HIV-1 Subtypes A6/A1 on the In Vitro Potency of Cabotegravir or Rilpivirine

The FLAIR study demonstrated noninferiority of monthly long-acting cabotegravir + rilpivirine vs daily oral dolutegravir/abacavir/lamivudine for maintaining virologic suppression. Three participants who received long-acting therapy had confirmed virologic failure (CVF) at Week 48, and all had HIV-1 that was originally classified as subtype A1 and contained the baseline integrase polymorphism L74I; updated classification algorithms reclassified all 3 as HIV-1 subtype A6. Retrospectively, the impact of L74I on in vitro sensitivity and durability of response to cabotegravir in HIV-1 subtype B and A6 backgrounds was studied. Site-directed L74I and mutations observed in participants with CVF were generated in HIV-1 subtype B and a consensus integrase derived from 3 subtype A6 CVF baseline sequences. Rilpivirine susceptibility was assessed in HIV-1 subtype B and A1 containing reverse transcriptase mutations observed in participants with CVF. HIV-1 subtype B L74I and L74I/G140R mutants and HIV-1 subtype A6 I74L and I74/G140R mutants remained susceptible to cabotegravir; L74I/Q148R double mutants exhibited reduced susceptibility in HIV-1 subtypes B and A6 (half maximal effective capacity fold change, 4.4 and 4.1, respectively). Reduced rilpivirine susceptibility was observed across HIV-1 subtypes B and A1 with resistance-associated mutations K101E or E138K (half maximal effective capacity fold change, 2.21 to 3.09). In cabotegravir breakthrough experiments, time to breakthrough was similar between L74 and I74 viruses across HIV-1 subtypes B and A6; Q148R was selected at low cabotegravir concentrations. Therefore, the L74I integrase polymorphism did not differentially impact in vitro sensitivity to cabotegravir across HIV-1 subtype B and A6 integrase genes (ClinicalTrials.gov identifier: NCT02938520).

The Clinical Impact of Neoadjuvant Endocrine Treatment On Luminal-Like Breast Cancers and Its Prognostic Significance: Results From a Single-institution Prospective Cohort Study

Purpose: Neoadjuvant endocrine treatment (NET) has become a useful tool for the downstaging of luminal-like breast cancers in postmenopausal patients. It enables us to increase breast conserving surgery (BCS) rates, and provides an opportunity for assessing in vivo NET effectiveness and studying any biological changes that may act as valid biomarkers. The purpose of this study was to evaluate the safety and effectiveness of NET, and to assess the role of Ki67 proliferation rate changes as an indicator of endocrine responsiveness.Methods: From 2016 to 2020, a single-institution cohort of patients treated with NET and further surgery was evaluated. In patients with Ki67≥10%, a second core biopsy was performed after four weeks. Information regarding histopathological and clinical changes was gathered.Results: A total of 115 estrogen receptor positive (ER+)/HER2 negative patients were included. The median treatment duration was 5.0 months (IQR: 2.0-6.0). Median maximum size in the surgical sample was 40% smaller than pretreatment size measured by ultrasound (p<.0001). Median pretreatment Ki67 expression was 20.0% (IQR: 12.0-30.0), and was reduced to 5.0% (IQR: 1.8-10.0) after four weeks, and to 2.0% (IQR: 1.0-8.0) in the surgical sample (p<.0001). BCS was performed on 98 patients (85.2%). No pathological complete responses were recorded. A larger Ki67 fold-change after four weeks was significantly related to a PEPI score of 0 (p<.002). No differences were observed between luminal A- and B-like tumors with regard to fold-change and PEPI score.Conclusions: In our cohort, NET has proven effective for tumor size and Ki67 downstaging. This results in a higher rate of conservative surgery, aids in therapeutic decision-making, provides prognostic information, and constitutes a safe and well-tolerated approach

Reproducible and Opposing Microbiome Signatures Distinguish Autoimmune Diseases and Cancers: A Systematic Review and Meta-Analysis

Background: The gut microbiome promotes specific immune responses, and in turn the immune system has a hand in shaping the microbiome. Cancer and autoimmune diseases are two major disease families that result from the contrasting manifestations of immune dysfunction. We hypothesized that the opposing immunological profiles between cancer and autoimmunity yield analogously inverted gut microbiome signatures. To test this, we conducted a systematic review and meta-analysis on gut microbiome signatures and their directionality in cancers and autoimmune conditionsMethodology: We searched PubMed, Web of Science, and EMBAS to identify relevant articles to be included in this study. The study was conducted in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statements and PRISMA 2009 checklist. Study estimates were pooled by a generic inverse variance random-effects meta-analysis model. The relative abundance of microbiome features was converted to log fold-change and the standard error was calculated from the p-values, sample size and fold-change. Results: We screened 3,874 potentially relevant publications. A total of 82 eligible studies comprising 37 autoimmune and 45 cancer studies with 4,208 healthy human controls and 5,957 disease cases from 27 countries were included in this study. We identified a set of microbiome features that show consistent, opposite directionality between cancers and autoimmune diseases in multiple studies. Fusobacterium and Peptostreptococcus were the most consistently increased genera among the cancer cases which were found to be associated in a remarkable 13 (+0.54 log fold-change in 5 studies) and 11 studies (+3.75 log fold-change in 5 studies), respectively. Conversely, Bacteroides was the most prominent genus, which was found to be increased in 12 autoimmune studies (+0.24 log fold-change in 6 studies) and decreased in six cancer studies (-0.32 log fold-change in 4 studies). Sulfur-metabolism pathways were found to be the most frequent pathways among the member of cancer-increased genus and species.Conclusions: The surprising reproducibility of these associations across studies and geographies suggests a shared underlying mechanism shaping the microbiome across cancers and autoimmune diseases.

Comparative Proteomic Analysis of Leptospira Interrogans Serogroup Icterohaemorrhagiae Human Vaccine Strain and Epidemic Isolate of China

Background: Leptospira interrogans serogroup Icterohaemorrhagiae is the predominant pathogen causing leptospirosis in China and is still used as the vaccine strain for the current human inactivated vaccine. Unlike the clade ST17, which is distributed worldwide, ST1 is the most prevalent in serogroup Icterohaemorrhagiae in China. Purpose and Methods: To further characterize leptospiral pathogens, isobaric tags for relative and absolute quantitation and parallel reaction monitoring were used to analyze differences at the proteomic level between serogroup Icterohaemorrhagiae vaccine strain 56001 (ST1) and circulating isolate 200502 (ST17) from different periods. Results: Two hundred and eighty-one proteins were differentially expressed between ST17 and ST1, of which 166 were upregulated (>1.2 fold change, P < 0.05) and 115 (>1.2-fold change, P < 0.05) were downregulated. Function prediction revealed that nine upregulated proteins were outer membrane proteins, including several known immunogenic and/or virulence-related proteins, such as ompL1, LipL71 and LipL41. Furthermore, important expression differences in carbohydrate, amino acid, and energy metabolism and transport proteins were identified between ST1 and ST17, suggesting that these differences may reflect metabolic diversity and the potential of the pathogens to adapt to different environments. Conclusion: In summary, our findings provide insights into better understanding the component strains of the Chinese human leptospirosis vaccine at the proteomic level. Additionally, these data facilitate evaluating the mechanisms by which pathogenic Leptospira species adapt to the host environment.

Identification of Circulating Exosomal miR-101 and miR-125b Panel Act as a Potential Biomarker for Hepatocellular Carcinoma

Background. Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide with high mortality, and there is an urgent need of new diagnosis measures. This study is aimed at investigating whether circulating exosomal miRNAs could act as biomarkers for the diagnosis of HCC. Methods. A four-stage strategy was adopted in this study. Candidate miRNA was selected by comprehensive analysis of four GEO datasets and TCGA database. The expression of candidate miRNAs in serum exosomal samples were examined through qRT-PCR. The diagnostic utility of the final validated miRNAs was examined by receiver operating characteristic (ROC) curve analysis. Results. After synthetical analysis of four GEO datasets, six miRNAs were selected as candidates due to their higher differential fold change. miR-101 and miR-125b were selected as candidate miRNAs to further investigate their potential as biomarkers for HCC due to their differential fold change and their influence on overall survival based on the TCGA database. As a result, miR-101 and miR-125b expressions were remarkably downregulated in both tissues and serum exosomes of patients with HCC. The area under the ROC curves (AUCs) of circulating exosomal miR-101 and miR-125b were 0.894 (95% CI, 0.793–0.994) and 0.812 (95% CI, 0.675–0.950), respectively. The combination of the two miRNAs presented higher diagnostic utility for HCC ( AUC = 0.953 ). Conclusion. The exosomal miR-101 and miR-125b panel in the serum may act as a noninvasive biomarker for HCC detection.

MAFFIN: Metabolomics Sample Normalization Using Maximal Density Fold Change with High-Quality Metabolic Features and Corrected Signal Intensities

Sample normalization is a critical step in metabolomics to remove differences in total sample amount or concentration of metabolites between biological samples. Here, we present MAFFIN, an accurate and robust post-acquisition sample normalization workflow that works universally for metabolomics data collected by mass spectrometry (MS)-based platforms. The most important design of MAFFIN is the calculation of normalization factor using maximal density fold change (MDFC) value computed by a kernel density-based approach. MDFC is more accurate than traditional median FC-based normalization, especially when the numbers of up- and down-regulated metabolic features are different. In addition, we showcase two essential steps that are overlooked by conventional normalization methods, and incorporated them into MAFFIN. First, instead of using all detected metabolic features, MAFFIN automatically extracts and uses only the high-quality features to calculate FCs and determine the normalization factor. In particular, multiple orthogonal criteria are proposed to pick up the high-quality features. Second, to guarantee the accuracy of the FCs, the MS signal intensities of the high-quality features are corrected using serial quality control (QC) samples. Using simulated data and urine metabolomics datasets, we demonstrated the critical need of high-quality feature selection, MS signal correction, and MDFC. We also show the superior performance of MAFFIN over other commonly used post-acquisition sample normalization methods. Finally, a biological application on a human saliva metabolomics study shows that MAFFIN provides robust sample normalization, leading to better data separation in principal component analysis (PCA) and the identification of more significantly altered metabolic features.