Automated Image Analysis for Characterization of Circulating Tumor Cells and Clusters Sorted by Magnetic Levitation.

Magnetic levitation-based sorting technologies have revolutionized the detection and isolation of rare cells, including circulating tumor cells (CTCs) and circulating tumor cell clusters (CTCCs). Manual counting and quantification of these cells are prone to time-consuming processes, human error, and inter-observer variability, particularly challenging when heterogeneous cell types in 3D clusters are present. To overcome these challenges, we developed "Fastcount," an in-house MATLAB-based algorithm for precise, automated quantification and phenotypic characterization of CTCs and CTCCs, in both 2D and 3D. Fastcount is 120 times faster than manual counting and produces reliable results with a ±7.3% deviation compared to a trained laboratory technician. By analyzing 400 GB of fluorescence imaging data, we showed that Fastcount outperforms manual counting and commercial software when cells are aggregated in 3D or staining artifacts are present, delivering more accurate results. We further employed Fastcount for automated analysis of 3D image stacks obtained from CTCCs isolated from colorectal adenocarcinoma and renal cell carcinoma blood samples. Interestingly, we observed a highly heterogeneous spatial cellular composition within CTCCs, even among clusters from the same patient. Overall, Fastcount can be employed for various applications with lab-chip devices, such as CTC detection, CTCC analysis in 3D and cell detection in biosensors.

BIO-INSPIRED MAGNETIC BEADS FOR ISOLATION OF SPERM FROM HETEROGENOUS SAMPLES IN FORENSIC APPLICATIONS.

Rapid and efficient processing of sexual assault evidence will accelerate forensic investigation and decrease casework backlogs. The standardized protocols currently used in forensic laboratories require the continued innovation to handle the increasing number and complexity of samples being submitted to forensic labs. Here, we present a new technique leveraging the integration of a bio-inspired oligosaccharide (i.e., Sialyl-LewisX) with magnetic beads that provides a rapid, inexpensive, and easy-to-use strategy that can potentially be adapted with current differential extraction practice in forensics labs. This platform (i) selectively captures sperm; (ii) is sensitive within the forensic cut-off; (iii) provides a cost effective solution that can be automated with existing laboratory platforms; and (iv) handles small volumes of sample (∼200 µL). This strategy can rapidly isolate sperm within 25 minutes of total processing that will prepare the extracted sample for downstream forensic analysis and ultimately help accelerate forensic investigation and reduce casework backlogs.

Levitating Cells to Sort the Fit and the Fat.

Density is a core material property and varies between different cell types, mainly based on differences in their lipid content. Sorting based on density enables various biomedical applications such as multi-omics in precision medicine and regenerative repair in medicine. However, a significant challenge is sorting cells of the same type based on density differences. Here, a new method for real-time monitoring and sorting of single cells based on their inherent levitation profiles driven by their lipid content is reported. As a model system, human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) from a patient with neutral lipid storage disease (NLSD) due to loss of function of adipose triglyceride lipase (ATGL) resulting in abnormal lipid storage in cardiac muscle are used. This levitation-based strategy detects subpopulations within ATGL-deficient hiPSC-CMs with heterogenous lipid content, equilibrating at different levitation heights due to small density differences. In addition, sorting of these differentially levitating subpopulations are monitored in real time. Using this approach, sorted healthy and diseased hiPSC-CMs maintain viability and function. Pixel-tracking technologies show differences in contraction between NLSD and healthy hiPSC-CMs. Overall, this is a unique approach to separate diseased cell populations based on their intracellular lipid content that cannot be achieved using traditional flow cytometry techniques.

Investigating circulating tumor cells and distant metastases in patient-derived orthotopic xenograft models of triple-negative breast cancer.

BACKGROUND: Circulating tumor cells (CTCs) represent a temporal "snapshot" of a patient's cancer and changes that occur during disease evolution. There is an extensive literature studying CTCs in breast cancer patients, and particularly in those with metastatic disease. In parallel, there is an increasing use of patient-derived models in preclinical investigations of human cancers. Yet studies are still limited demonstrating CTC shedding and metastasis formation in patient-derived models of breast cancer. METHODS: We used seven patient-derived orthotopic xenograft (PDOX) models generated from triple-negative breast cancer (TNBC) patients to study CTCs and distant metastases. Tumor fragments from PDOX tissue from each of the seven models were implanted into 57 NOD scid gamma (NSG) mice, and tumor growth and volume were monitored. Human CTC capture from mouse blood was first optimized on the marker-agnostic Vortex CTC isolation platform, and whole blood was processed from 37 PDOX tumor-bearing mice. RESULTS: Staining and imaging revealed the presence of CTCs in 32/37 (86%). The total number of CTCs varied between different PDOX tumor models and between individual mice bearing the same PDOX tumors. CTCs were heterogeneous and showed cytokeratin (CK) positive, vimentin (VIM) positive, and mixed CK/VIM phenotypes. Metastases were detected in the lung (20/57, 35%), liver (7/57, 12%), and brain (1/57, less than 2%). The seven different PDOX tumor models displayed varying degrees of metastatic potential, including one TNBC PDOX tumor model that failed to generate any detectable metastases (0/8 mice) despite having CTCs present in the blood of 5/5 tested, suggesting that CTCs from this particular PDOX tumor model may typify metastatic inefficiency. CONCLUSION: PDOX tumor models that shed CTCs and develop distant metastases represent an important tool for investigating TNBC.

Minimum Effective dose of Dexamethasone in Combination with Midazolam as Prophylaxis against Postoperative Nausea and Vomiting after Laparoscopic Cholecystectomy.

BACKGROUND: Postoperative nausea and vomiting (PONV) affects 20% and 30% of patients. As many as 60%-80% patients at high risk may be affected. Dexamethasone (D) and midazolam (M) are well studied as antiemetic. Use of D can be associated with certain undesirable side effects so minimum dose is preferred. M is a routinely used premedicant. Hence, this study was designed with both D and M in high risk patients for PONV to find minimum effective dose of D. AIMS: To determine the minimum dose of D that combined with M would provide effective prophylaxis of PONV after laparoscopic cholecystectomy (LC) in patients at high risk for PONV. SETTING AND DESIGN: This is a prospective, randomized double-blind trial. MATERIALS AND METHODS: One hundred and fifty-five patients scheduled for elective LC were randomized to 5 groups of 31 each. Group C was given normal saline, and the rest were administered D 1 mg (group MD1), 2 mg (group MD2), 4 mg (group MD4), or 8 mg (group MD8) in combination with 0.04 mg/kg M at induction. The incidence of nausea, vomiting, severity of nausea, and the use of rescue antiemetic and postoperative pain was analyzed. STATISTICAL ANALYSIS: Chi-square test was used to compare incidence of study variables. Independent Student's t-test was used for continuous variables. Demographic data were compared using ANOVA. P < 0.05 was considered statistically significant. RESULTS: The incidence of nausea was significantly lower in group MD4 (29%) and MD8 (6%) compared to placebo group (71%) (P < 0.001) and of vomiting was significantly lower in groups of MD2 (58%), MD4 (48%), and MD8 (6%) compared with placebo (90%) (P < 0.001) at 24 h. There was significant reduction in nausea, pain severity, and incidence of use of rescue antiemetic in MD4 and MD8 groups with no discernable side effects of the drugs. CONCLUSION: We conclude that 4 mg D with M and 2 mg D with M is effective for prevention of nausea and vomiting, respectively, in patients at high risk for PONV undergoing LC.

Double blind randomized control trial to evaluate the efficacy of ketoprofen patch to attenuate pain during venous cannulation.

BACKGROUND: Venipuncture pain is an uncomfortable suffering to the patient. It creates anxiety, fear and dissatisfaction. The ketoprofen transdermal patch is a proven treatment for musculoskeletal and arthritic pain. We planned this study to evaluate the efficacy of the ketoprofen patch to reduce venipuncture pain. METHODS: Two hundred adult patients, aged 18-60 years, of either sex, ASA grade I or II, were enrolled. Presuming that therapy would decrease venipuncture pain by 30%, a power calculation with α = 0.05 and ß = 0.80 required enrollment of at least 24 patients into each group. However, 100 patients in each group were recruited. Group I (Control) received a placebo patch; Group II (Ketoprofen) received a 20 mg ketoprofen patch. A selected vein on the dorsum of the patient's non-dominant hand was cannulated with 18 g intravenous cannula 1 h after the application of the respective patch. Assessment of pain was done by a 10 cm visual analogue scale (VAS) of 0-10, where 0 depicts "no pain" and 10 is "the worst imaginable pain". The venipuncture site was assessed for the presence of skin erythema, swelling and rashes at 12 h, 24 h and at the time of decannulation. RESULTS: Incidence of pain was 100% (94/94) in the control group as compared to 93% (85/91) in the ketoprofen group. The severity of the venipuncture pain was 6 (2) and 2 (2) for control and ketoprofen groups respectively (P < 0.05). CONCLUSIONS: Application of a ketoprofen patch at the proposed site of venipuncture one hour before the attempt is effective and safe for attenuating venipuncture pain.

Comparison of Immunohistochemical Expression of Antiapoptotic Protein Survivin in Normal Oral Mucosa, Oral Leukoplakia, and Oral Squamous Cell Carcinoma.

Background. Oral squamous cell carcinoma is the sixth most frequent malignant tumor worldwide and the third most common cancers in developing countries. Oral leukoplakia is the best-known precursor lesion of oral squamous cell carcinoma. The aim of the present study was to compare immunohistochemical expression of antiapoptotic protein survivin in normal oral mucosa, oral leukoplakia, and oral squamous cell carcinoma. Method. Total 45 specimens of formalin fixed paraffin embedded tissue blocks, 15 in each of the following: normal oral mucosa, leukoplakia, and oral squamous cell carcinoma were used for the study. Immunohistochemical reaction for survivin protein was performed for the 4 µm thick histological sections taken on positively charged slides. Results. 20% normal mucosa cases, 53.33% cases of leukoplakia, and 80% of oral squamous cell carcinoma were found out to be survivin positive. One way ANOVA test indicated statistically significant difference of survivin expression between the three different groups (p < 0.001). Conclusion. A high incidence of survivin protein expression in oral epithelial dysplasia and squamous cell carcinoma samples indicate that survivin protein expression may be an early event in initiation and progression of oral squamous cell carcinoma.

Determination of sexual dimorphism via maxillary first molar teeth in Himachali population.

CONTEXT: Sex determination of skeletal remains forms part of archaeological and medicolegal examinations. It is an aspect of forensic odontology. Forensic odontology primarily deals with identification, based on recognition of unique features present in an individual's dental structures. Correct sex determination limits the pool of missing persons to just one half of the population. AIM OF STUDY: Purpose of this study is to evaluate the existence of sexual dimorphism and variation in left and right maxillary first molars using bucco-lingual and mesio-distal dimensions in population of Sirmour District, H.P. MATERIALS AND METHODS: Base sample comprised 100 subjects (50 males and 50 females) of an age group ranging from 17 to 25 years. STATISTICAL ANALYSIS USED: Unpaired t-test. RESULTS: It was observed that the comparison of mean values of bucco-lingual and mesio-distal parameters showed highly statistically significant differences between males and females, measured both intraorally and on study casts. There were no significant differences between the mean values of both the parameters on the left side as compared to right side. CONCLUSION: The study concludes that sexual dimorphism is population specific. Among Himachali people, mesio-distal dimensions and bucco-lingual dimensions of first molar can aid in sex determination.

Identifying direct targets of transcription factor Rfx2 that coordinate ciliogenesis and cell movement.

Recently, using the frog Xenopus laevis as a model system, we showed that transcription factor Rfx2 coordinates many genes involved in ciliogenesis and cell movement in multiciliated cells (Chung et al., 2014). To our knowledge, it was the first paper to utilize the genomic resources, including genome sequences and interim gene annotations, from the ongoing Xenopus laevis genome project. For researchers who are interested in the application of genomics and systems biology approaches in Xenopus studies, here we provide additional details about our dataset (NCBI GEO accession number GSE50593) and describe how we analyzed RNA-seq and ChIP-seq data to identify direct targets of Rfx2.

Mandibular plasmacytoma of jaw - a case report.

The plasma cell neoplasm may present as Extramedullary Plasmacytoma (EMP) in soft tissues in bone as a Solitary Plasmacytoma of bone (SPB) or as a part of multi focal disseminated disease Multiple Myeloma (MM). The majority of 80% occurs in head and neck region. In our case, a 62-year-old male patient presented with a non tender swelling of short duration. The swelling was noted obliterating the vestibular depth in right lower mandibular region. The radiological features were non specific.

Developmental enhancers are marked independently of zygotic Nodal signals in Xenopus.

To determine the hierarchy of transcriptional regulation within the in vivo vertebrate embryo, we examined whether developmental enhancers were influenced by Nodal signaling during early embryogenesis in Xenopus tropicalis. We find that developmental enhancers, defined by the active enhancer chromatin marks H3K4me1 and H3K27ac, are established as early as blastula stage and that Smad2/3 only strongly associates with these regions at gastrula stages. Significantly, when we perturb Nodal signaling using the drug SB431542, most enhancers remain marked, including at genes known to be sensitive to Nodal signaling. Overall, as enhancers are in an active conformation prior to Nodal signaling and are established independently of Nodal signaling, we suggest that many developmental enhancers are marked maternally, prior to exposure to extrinsic signals.

H3K4me3 breadth is linked to cell identity and transcriptional consistency.

Trimethylation of histone H3 at lysine 4 (H3K4me3) is a chromatin modification known to mark the transcription start sites of active genes. Here, we show that H3K4me3 domains that spread more broadly over genes in a given cell type preferentially mark genes that are essential for the identity and function of that cell type. Using the broadest H3K4me3 domains as a discovery tool in neural progenitor cells, we identify novel regulators of these cells. Machine learning models reveal that the broadest H3K4me3 domains represent a distinct entity, characterized by increased marks of elongation. The broadest H3K4me3 domains also have more paused polymerase at their promoters, suggesting a unique transcriptional output. Indeed, genes marked by the broadest H3K4me3 domains exhibit enhanced transcriptional consistency and [corrected] increased transcriptional levels, and perturbation of H3K4me3 breadth leads to changes in transcriptional consistency. Thus, H3K4me3 breadth contains information that could ensure transcriptional precision at key cell identity/function genes.

Epithelioid hemangioma of lingual alveolar mucosa: an immunohistochemical case report.

Epithelioid hemangioma is a rare benign vascular lesion that presents as a nodular lesion in the skin of head and neck region. It is a superficial vascular entity which can either be due to tumor or reactive lesion, but the exact etiology is still unknown. We hereby present a rare case which has been reported with the history of small nodular-like growth on mandibular buccal and lingual area. The excisional biopsy was performed and tissue was submitted for histopathological diagnosis. The immunohistochemistry was performed to check the expression of CD31 marker which proved that origin of epithelioid cells was vascular.

Coordinated genomic control of ciliogenesis and cell movement by RFX2.

The mechanisms linking systems-level programs of gene expression to discrete cell biological processes in vivo remain poorly understood. In this study, we have defined such a program for multi-ciliated epithelial cells (MCCs), a cell type critical for proper development and homeostasis of the airway, brain and reproductive tracts. Starting from genomic analysis of the cilia-associated transcription factor Rfx2, we used bioinformatics and in vivo cell biological approaches to gain insights into the molecular basis of cilia assembly and function. Moreover, we discovered a previously un-recognized role for an Rfx factor in cell movement, finding that Rfx2 cell-autonomously controls apical surface expansion in nascent MCCs. Thus, Rfx2 coordinates multiple, distinct gene expression programs in MCCs, regulating genes that control cell movement, ciliogenesis, and cilia function. As such, the work serves as a paradigm for understanding genomic control of cell biological processes that span from early cell morphogenetic events to terminally differentiated cellular functions. DOI: http://dx.doi.org/10.7554/eLife.01439.001.

Chromatin immunoprecipitation and deep sequencing in Xenopus tropicalis and Xenopus laevis.

Chromatin immunoprecipitation and deep sequencing (ChIP-SEQ) represents a powerful tool for identifying the genomic targets of transcription factors, chromatin remodeling factors, and histone modifications. The frogs Xenopus laevis and Xenopus tropicalis have historically been outstanding model systems for embryology and cell biology, with emerging utility as highly accessible embryos for genome-wide studies. Here we focus on the particular strengths and limitations of Xenopus cell biology and genomics as they apply to ChIP-SEQ, and outline a methodology for ChIP-SEQ in both species, providing detailed strategies for sample preparation, antibody selection, quality control, sequencing library preparation, and basic analysis.

Biosafe alternative to xylene: A comparative study.

BACKGROUND: Xylene in one of the non-substitutable chemical used in histology laboratories. However, it is known to have many toxic effects. The toxic effects of xylene include heart and kidney injuries, some fatal blood dyscrasia and other less dangerous problems, such as skin erythema, drying, scaling and secondary infections. The exposure and handling of xylene is maximum during deparaffinizing tissue sections. AIMS: The aim of this study was to evaluate the efficacy of 1.7% dishwashing soap (DWS) solution as a deparaffinizing agent for hematoxylin and eosin (H and E) staining and compare it with xylene. MATERIALS AND METHODS: Sixty sections of 4 µm were obtained from 30 formalin-fixed paraffin-embedded (FFPE) tissues and were considered in two different groups, groups A and B. Slides in group A were stained with routine H and E staining procedure; whereas, slides in group B were stained using 1.7% DWS as a deparaffinizing agent. STATISTICAL ANALYSIS USED: Wilcoxon matched-pairs signed rank test was used to calculate the test of significance (P-value significant at ≤0.05). RESULTS AND CONCLUSION: 1.7% DWS was found to be an effective alternative deparaffinizing agent to xylene and meanwhile facilitating as less biohazardous, economical and a faster deparaffinizing agent.