A semiconductor 96-microplate platform for electrical-imaging based high-throughput phenotypic screening.

High-content imaging for compound and genetic profiling is popular for drug discovery but limited to endpoint images of fixed cells. Conversely, electronic-based devices offer label-free, live cell functional information but suffer from limited spatial resolution or throughput. Here, we introduce a semiconductor 96-microplate platform for high-resolution, real-time impedance imaging. Each well features 4096 electrodes at 25 µm spatial resolution and a miniaturized data interface allows 8× parallel plate operation (768 total wells) for increased throughput. Electric field impedance measurements capture >20 parameter images including cell barrier, attachment, flatness, and motility every 15 min during experiments. We apply this technology to characterize 16 cell types, from primary epithelial to suspension cells, and quantify heterogeneity in mixed co-cultures. Screening 904 compounds across 13 semiconductor microplates reveals 25 distinct responses, demonstrating the platform's potential for mechanism of action profiling. The scalability and translatability of this semiconductor platform expands high-throughput mechanism of action profiling and phenotypic drug discovery applications.

p16 expression in urothelial carcinoma: Experience from a tertiary care center in coastal South India.

Background: Urothelial carcinomas (UC) account for 6 and 2% of all cancers in men and women, respectively. Human papillomavirus (HPV) is one of the causative agents in cancers of the uterine cervix and head and neck. The role of HPV is also being studied in cancers of the urinary bladder, penis, and prostate. As p16-INK4a is a surrogate marker for high-risk HPVE7 oncoprotein, this study aims to highlight the utility of p16 immunohistochemistry (IHC) in the evaluation of HPV-associated UC. Materials and Methods: A retrospective study was conducted on UC of the bladder received in the Pathology department between January 2013 and December 2018. Bladder biopsies from non-neoplastic lesions served as controls. IHC was done for the detection of the p16 antigen. The p16 staining was recorded as positive, when there was strong staining in >50% of tumor nuclei. The p16 positive and negative tumors were compared based on age, gender, tumor size, grade, and muscle invasion. P value <0.05 was considered statistically significant. Results: The expression of p16 was analyzed in 72 UC and compared with 20 non-neoplastic cases, of which 26.4% of the cases showed p16 expression. The p16 expression was absent in the non-neoplastic lesions. While the majority (87.5%) of the low-grade tumors were negative for p16 expression, 43.8% high-grade tumors were positive. Similarly, a larger proportion of invasive carcinomas (38.8%) expressed p16 as compared to non-invasive carcinomas (13.8%). Thus, p16 expression showed a significant association with grade and stage in these malignancies (P < 0.05). Conclusion: The p16 expression was associated with high-grade and muscle-invasive UC. The p16 was absent in all non-neoplastic and precursor lesions. Thus, it can provide essential information not only about HPV association but also on the prognostic implications for the patients.

Effect of resveratrol and combination of resveratrol and donepezil on the expression of microglial cells and astrocytes in Wistar albino rats of colchicine-induced Alzheimer's disease.

Aim: The goal was to evaluate the effect of resveratrol (RS) and combination therapy of RS and donepezil (DPZ), on the numerical expression of microglial cells and astrocytes, in the frontal cortex, regions of the hippocampus in colchicine-induced Alzheimer's disease (AD) model. Methods: The study involved male albino Wistar rats of three months, age and consisted of 6 groups, with six animals each. The immunohistochemical staining with mouse monoclonal anti-human CD 68 and mouse monoclonal anti-GFAP was performed to assess the number of microglial cells and astrocytes, respectively. Results: AD group showed an increase in the number of microglia, and the numbers declined in the treatment groups, RS 10, RS 20, RS10/10 and DPZ + RS (p < 0.001). Astrocyte count was increased in the treatment groups in contrast to the AD group (p < 0.05). The DPZ + RS combination group revealed substantial elevation in the number of astrocytes and decreased microglial number among all the groups (p < 0.001). Conclusion: RS administration has diminished the microglial number and elevated the number of astrocytes. The elevated reactive astrocytes have decreased the microglial population. However, the limitation of our study is utilizing the colchicine for the induction of neurodegeneration. Using the transgenic models of AD may give a better insight into the pathogenesis and effect of RS. Another limitation of this study is the administration of RS and DPZ through different routes. The prospects of this research include studying the probiotic nature of RS and the effect of RS in other neurodegenerative disorders.

A semiconductor 96-microplate platform for electrical-imaging based high-throughput phenotypic screening.

Profiling compounds and genetic perturbations via high-content imaging has become increasingly popular for drug discovery, but the technique is limited to endpoint images of fixed cells. In contrast, electronic-based devices offer label-free, functional information of live cells, yet current approaches suffer from low-spatial resolution or single-well throughput. Here, we report a semiconductor 96-microplate platform designed for high-resolution real-time impedance "imaging" at scale. Each well features 4,096 electrodes at 25 µm spatial resolution while a miniaturized data interface allows 8× parallel plate operation (768 total wells) within each incubator for enhanced throughputs. New electric field-based, multi-frequency measurement techniques capture >20 parameter images including tissue barrier, cell-surface attachment, cell flatness, and motility every 15 min throughout experiments. Using these real-time readouts, we characterized 16 cell types, ranging from primary epithelial to suspension, and quantified heterogeneity in mixed epithelial and mesenchymal co-cultures. A proof-of-concept screen of 904 diverse compounds using 13 semiconductor microplates demonstrates the platform's capability for mechanism of action (MOA) profiling with 25 distinct responses identified. The scalability of the semiconductor platform combined with the translatability of the high dimensional live-cell functional parameters expands high-throughput MOA profiling and phenotypic drug discovery applications.

Web in the Neck - An Interesting Case Report.

Lateral neck masses are common in children, ranging from simple benign diseases to pathologies with malignant potential. Plexiform neurofibromas are extremely rare peripheral nerve sheath tumours involving multiple nerve sheath fascicles. They are typically seen in the paediatric population, with the majority affecting the craniofacial area and neck. Due to the close clinical and histological resemblance with other benign neck lesions such as lymphadenitis and branchial cysts, these cases can often go misdiagnosed. We describe a lesion in a young girl who presented with a progressive lateral neck swelling and how it was managed.

Diagnostic conundrum in an ambiguous posterior mandibular osseous lesion: Case report and radiological review.

INTRODUCTION AND IMPORTANCE: Non-odontogenic osseous lesions of the mandible are relatively uncommon entities compared to odontogenic lesions. Although the posterior mandible is not a usual site, it is not rare either for these osseous lesions, making the diagnosis ambiguous, and if misdiagnosed leading to different treatment protocols. CASE PRESENTATION: A 43-year-old female presented with a hard tissue lesion of the posterior mandible, misdiagnosed as a sialolith of the submandibular salivary gland in two other centers due to overlapping of symptoms, anatomic complexity, and inadequate investigations. The lesion was later diagnosed to be an osteoma of the posterior mandible with added investigations, and surgically excised. Histopathology confirmed the diagnosis. CLINICAL DISCUSSION: A variety of hard tissue lesions are known to occur in the posterior mandible like Submandibular sialolith, Osteomas, Calcified Submandibular lymph nodes, Phlebolith, and Tonsillolith. However, due to the region's structural complexity, localization of a hard tissue lesion may not always be forthright, even with radiographs. Moreover, in cases with conflicting symptoms, as was in this case there are more chances of misdiagnosis. The reasons for such diagnostic challenges are deliberated with radiological review of posterior mandibular osseous lesions. Recommendations are also suggested for proper investigations, thereby management of these posterior mandibular osseous lesions. CONCLUSION: Misdiagnosis of these posterior mandibular lesions may lead to the patient undergoing unnecessary surgical procedures as different lesions require different management. Differential diagnosis and adequate protocol for investigations are required.

Study of pre-operative neutrophil-lymphocyte ratio in urothelial carcinoma.

OBJECTIVES: Neutrophil-lymphocyte ratio (NLR), as an indicator of heightened systemic inflammatory response, predicts increased disease burden and poor oncological outcomes in urothelial carcinoma (UC). The study was undertaken with an aim to evaluate the association of NLR with clinicopathological variables and survival outcomes. METHODS: A total of 80 patients of UC were enrolled in the current retrospective study. Pre-operative NLR (within one month prior to the procedure), patient age, sex, tumour grade, pathological stage, recurrence free survival (RFS), progression free survival (PFS) and cancer specific survival (CSS) were recorded. We chose a cut-off value of 2.7 for NLR and patients were divide into two groups (NLR <2.7 and ≥2.7). RESULTS: NLR ≥2.7 was significantly associated with advanced tumour stage (p=0.001), but not with tumour grade (p=0.116). Progression (p=0.032) and death rates (p=0.026) were high in patients with NLR ≥2.7. Mean RFS (p=0.03), PFS (p=0.04) and CSS (p=0.04) were reduced in patients with NLR ≥2.7. On univariate analysis, NLR ≥2.7 predicted worse RFS (HR=2.928, p=0.007), PFS (HR=3.180, p=0.006) and CSS (HR=3.109, p=0.016). However, it was not an independent predictor of outcomes on multivariate analysis. CONCLUSIONS: Tumour stage and grade are the only independent predictors of RFS, PFS and CSS. High NLR at a cut-off value of ≥2.7 is associated with advanced pathological stage, but does not have an independent predictive value for RFS, PFS and CSS.

Immunohistochemical expression of androgen receptors in urothelial carcinoma of urinary bladder. Is it significant? Experience from coastal India.

BACKGROUND: Bladder carcinoma (BC) ranks second among the genitourinary cancers worldwide. Influence of androgens and expression of androgen receptors in neoplasms are recent findings which were implicated in the development of BC. We aimed to study androgen receptor (AR) expression in bladder urothelial neoplasms and correlate its expression with grade and stage of the tumor. METHODS: Immunohistochemistry (IHC) was done on samples collected in a tertiary care hospital over one year consisting of 71 urothelial BC and 20 non-neoplastic urothelial conditions. Two pathologists graded the IHC and nuclear staining was considered as positive expression. RESULTS: AR was expressed in 23.9% (17/71) of bladder urothelial neoplasms. AR was expressed in 25.7% and 22.3% of high and low-grade tumors and 25% and 22.3% of non muscle-invasive and muscle-invasive BC. AR expression had no significant correlation with gender, age (> 50 years), muscle invasion or grade. AR expression was significantly absent in non-neoplastic conditions (p = 0.018). CONCLUSIONS: AR has varied expression in BC and it is relatively lower in this study population.

Tumor Budding Assessment with Cytokeratin and Its Significance in Laryngeal Squamous Cell Carcinomas.

There is a need for novel prognostic parameters in assessing laryngeal squamous cell carcinoma (LSCC). Tumor budding is an instrumental parameter, which has hardly been studied before in this organ. This study aimed to assess tumor budding in LSCCs with routine hematoxylin and eosin (H&E) stain as well as cytokeratin (CK) immunohistochemistry (IHC). Objectives were to compare the effectiveness of both these methods to assess tumor budding, to investigate the association of tumor budding and clinicopathologic features, and to determine the prognostic significance of tumor budding in LSCCs. Fifty laryngectomy specimens were included. Tumor budding was counted (20x) on slides stained with IHC-CK, and highest count per slide was noted. The cases were classified as positive (> 1 buds) or negative (no buds present). The budding index was categorized as low (< 5 buds) or high (> 5 buds). Tumor budding on H&E was absent, low and high grade in 28%, 30% and 42% cases respectively, when compared to CK-IHC in 17%, 24% and 59% of cases, respectively. Presence of lymphoplasmacytic infiltration significantly correlated with tumor budding and higher grade. Transglottic location of tumor and pT stage was associated with high budding. Presence of lymphoplasmacytic infiltrate significantly correlated with worse prognosis. Tumor budding, an easily assessable, inexpensive histopathologic parameter has seldom been studied in LSCCs. Presence of lymphoplasmacytic infiltrate in routine preoperative biopsy reporting could be useful in prognostication. CK-IHC is helpful to detect especially cases with low-grade tumor budding.

Modeling and mechanical perturbations reveal how spatially regulated anchorage gives rise to spatially distinct mechanics across the mammalian spindle.

During cell division, the spindle generates force to move chromosomes. In mammals, microtubule bundles called kinetochore-fibers (k-fibers) attach to and segregate chromosomes. To do so, k-fibers must be robustly anchored to the dynamic spindle. We previously developed microneedle manipulation to mechanically challenge k-fiber anchorage, and observed spatially distinct response features revealing the presence of heterogeneous anchorage (Suresh et al., 2020). How anchorage is precisely spatially regulated, and what forces are necessary and sufficient to recapitulate the k-fiber's response to force remain unclear. Here, we develop a coarse-grained k-fiber model and combine with manipulation experiments to infer underlying anchorage using shape analysis. By systematically testing different anchorage schemes, we find that forces solely at k-fiber ends are sufficient to recapitulate unmanipulated k-fiber shapes, but not manipulated ones for which lateral anchorage over a 3 µm length scale near chromosomes is also essential. Such anchorage robustly preserves k-fiber orientation near chromosomes while allowing pivoting around poles. Anchorage over a shorter length scale cannot robustly restrict pivoting near chromosomes, while anchorage throughout the spindle obstructs pivoting at poles. Together, this work reveals how spatially regulated anchorage gives rise to spatially distinct mechanics in the mammalian spindle, which we propose are key for function.

Semantic segmentation of low magnification effusion cytology images: A semi-supervised approach.

Cytopathologists examine microscopic images obtained at various magnifications to identify malignancy in effusions. They locate the malignant cell clusters at a low magnification and then zoom in to investigate cell-level features at a high magnification. This study predicts the malignancy at low magnification levels such as 4X and 10X in effusion cytology images to reduce scanning time. However, the most challenging problem is annotating the low magnification images, particularly the 4X images. This paper extends two semi-supervised learning (SSL) models, MixMatch and FixMatch, for semantic segmentation. The original FixMatch and MixMatch algorithms are designed for classification tasks. While performing image augmentation, the generated pseudo labels are spatially altered. We introduce reverse augmentation to compensate for the effect of the spatial alterations. The extended models are trained using labelled 10X and unlabelled 4X images. The average F-score of benign and malignant pixels on the predictions of 4X images is improved approximately by 9% for both Extended MixMatch and Extended FixMatch respectively compared with the baseline model. In the Extended MixMatch, 62% sub-regions of low magnification images are eliminated from scanning at a higher magnification, thereby saving scanning time.

Wolf in sheep's clothing: Primary orbital Ewing sarcoma in a child masquerading as internal angular dermoid.

Ewing sarcoma is one of the rare, highly malignant neuroectodermal tumors frequently involving bones. Primary orbital Ewing sarcoma is extremely uncommon. We report a rare case of a 5-year-old boy who presented to us with a painless, slow-growing mass above the medial canthus of the left eye, the clinical appearance of which was masquerading as an internal angular dermoid. The child was subsequently diagnosed to have primary orbital Ewing sarcoma. Primary orbital Ewing sarcoma is a rare tumor with poor prognosis, poses diagnostic challenges, and demands a high index of clinical suspicion.

The Astrin-SKAP complex reduces friction at the kinetochore-microtubule interface.

The kinetochore links chromosomes to spindle microtubules to drive chromosome segregation at cell division. While we know nearly all mammalian kinetochore proteins, how these give rise to the strong yet dynamic microtubule attachments required for function remains poorly understood. Here, we focus on the Astrin-SKAP complex, which localizes to bioriented kinetochores and is essential for chromosome segregation but whose mechanical role is unclear. Live imaging reveals that SKAP depletion dampens the movement and decreases the coordination of metaphase sister kinetochores and increases the tension between them. Using laser ablation to isolate kinetochores bound to polymerizing versus depolymerizing microtubules, we show that without SKAP, kinetochores move slower on both polymerizing and depolymerizing microtubules and that more force is needed to rescue microtubules to polymerize. Thus, in contrast to the previously described kinetochore proteins that increase the grip on microtubules under force, Astrin-SKAP reduces the grip, increasing attachment dynamics and force responsiveness and reducing friction. Together, our findings suggest a model where the Astrin-SKAP complex effectively "lubricates" correct, bioriented attachments to help preserve them.

Immunohistochemical comparison of p63 and p40 in head and neck Spindle Cell Carcinoma.

BACKGROUND: Spindle Cell Carcinoma (SpCC) is a rare type of squamous cell carcinoma (SCC) with prominent malignant spindle cell component. This unique biphasic feature on histopathological examination makes its diagnosis problematic. Detection of p63 antigens in SpCC can be helpful however its expression in variousb proliferating soft tissue lesions demands for better marker. METHODS: In this study, histopathologically diagnosed SpCC of head and neck region were considered as cases, and 22 soft tissue sarcomas, reactive lesions and spindle cell lesions of the body were taken as controls. Immunohistochemistry (IHC) was done using Anti-p63 and p40 clone and the results were compared. CK was done for negative cases to prove their epithelial origin. P. value < 0.05 considered statistically significant. RESULTS: Among 22 cases of SpCC, 19 cases showed positive immunoreactivity to p63, and 18 cases for p40. IHC of controls showed no immunoreactivity in any of the sarcomas, reactive lesions or spindle cell lesions. The sensitivity of p63 is 86% while that of p40 is 82%. Specificity of both the markers was 100% CONCLUSION: Though p63 is a slightly (4%) more sensitive marker than p40, percentage of cell positivity for p40 is higher compared to p63. Both of these markers are 100% specific for SpCC.

Discriminant value of automated leucocyte VCS parameters in the detection of tropical infections.

INTRODUCTION: In India, infectious diseases are a leading treatable cause of morbidity and mortality. Mangalore being endemic to many vector-borne diseases, their incidence is known to show seasonal variations with sharp increase during monsoon. Leucocytes have substantial role in the immunological pathogenesis of infections. METHODS: The present series was a hospital-based cross-sectional study performed in a tertiary care hospital for a period of three months from June-August wherein the cell population data of cases of malaria, dengue, leptospirosis, typhoid and rickettsial infections along with equal number of healthy controls were collected and analysed. Effectiveness of leucocyte-related volume (V), conductivity (C) and scatter (S) parameters by Coulter®DXH800 haematology analyser in predicting these infections was appraised. RESULTS: A total of 324 cases comprising of malaria (50%), dengue (30.9%), leptospirosis (13.9%), typhoid (4.0%) and rickettsial infections (1.2%) were included. There was statistically significant differences (P < 0.05) in the mean values of complete blood count parameters-haemoglobin, total leucocyte count, red blood cell count, haematocrit, red cell distribution width, differential leucocyte count, platelet count and plateletcrit between cases and controls and also between specific infections. The mean volumes of neutrophil, monocyte and lymphocyte were considerably increased in malaria and dengue fever compared to leptospirosis, typhoid and rickettsial infections. VCS parameters were the least altered in typhoid fever, except for a strikingly high conductivity and scatter of eosinophils. CONCLUSIONS: Haematological analysis is a part of routine evaluation of any case of febrile illness. This study showed that there are specific alterations in VCS parameters in different types of infections such as malaria, dengue, leptospira, typhoid and rickettsia, the information and analysis of which comes without any additional cost.

Deep Learning Model based Ki-67 Index estimation with Automatically Labelled Data.

Ki-67 labelling index is a biomarker which is used across the world to predict the aggressiveness of cancer. To compute the Ki-67 index, pathologists normally count the tumour nuclei from the slide images manually; hence it is timeconsuming and is subject to inter pathologist variability. With the development of image processing and machine learning, many methods have been introduced for automatic Ki-67 estimation. But most of them require manual annotations and are restricted to one type of cancer. In this work, we propose a pooled Otsu's method to generate labels and train a semantic segmentation deep neural network (DNN). The output is postprocessed to find the Ki-67 index. Evaluation of two different types of cancer (bladder and breast cancer) results in a mean absolute error of 3.52%. The performance of the DNN trained with automatic labels is better than DNN trained with ground truth by an absolute value of 1.25%.

Diagnostic dilemma of a thumb swelling: An unpropitious turn of events.

Benign and malignant bone tumors arise in small bones of the hands and feet. Nevertheless, secondary deposits at these sites are extremely rare. We report a peculiar case of an adult man who presented with thumb swelling, which was later discovered to be a metastasis from renal cell carcinoma. Such cases have a sinister prognosis with a survival rate of 6-12 months from the time of diagnosis. We intend to discuss the diagnostic dilemma and treatment of acrometastases.

Individual kinetochore-fibers locally dissipate force to maintain robust mammalian spindle structure.

At cell division, the mammalian kinetochore binds many spindle microtubules that make up the kinetochore-fiber. To segregate chromosomes, the kinetochore-fiber must be dynamic and generate and respond to force. Yet, how it remodels under force remains poorly understood. Kinetochore-fibers cannot be reconstituted in vitro, and exerting controlled forces in vivo remains challenging. Here, we use microneedles to pull on mammalian kinetochore-fibers and probe how sustained force regulates their dynamics and structure. We show that force lengthens kinetochore-fibers by persistently favoring plus-end polymerization, not by increasing polymerization rate. We demonstrate that force suppresses depolymerization at both plus and minus ends, rather than sliding microtubules within the kinetochore-fiber. Finally, we observe that kinetochore-fibers break but do not detach from kinetochores or poles. Together, this work suggests an engineering principle for spindle structural homeostasis: different physical mechanisms of local force dissipation by the k-fiber limit force transmission to preserve robust spindle structure. These findings may inform how other dynamic, force-generating cellular machines achieve mechanical robustness.

Myomatous Erythrocytosis Syndrome: Case Report and Review of the Literature.

Myomatous Erythrocytosis Syndrome is defined as erythrocytosis, myomatous uterus, and the return of normal hematologic values following surgical resection. The exact role of erythropoietin in disease pathogenesis is unknown. In this study we report the case of a 49 year old premenopausal woman who was found to have an enlarged heterogeneous mass arising from the uterus concerning for malignancy. Her RBC count was 5.75 T/L, hemoglobin was 17.6 g/dL and hematocrit was 54.3%. Pre-operative erythropoietin levels were 24.6 mIU/mL and JAK2 mutation was not detected. She underwent Total Abdominal Hysterectomy and Bilateral Salpingo-Oophorectomy. The pathology was consistent with a uterine leiomyoma. Laboratory evaluation performed eight weeks after surgery showed a RBC count of 4.5 T/L, hemoglobin of 13.6 g/dL, hematocrit of 40.5%. Post-operative erythropoietin level was 5.4 mIU/mL. The tissue showed diffuse moderate to strong cytoplasmic immunopositive for Erythropoietin. Erythropoietin plays an important role in this condition, however the exact mechanism is still under investigation. The theory of erythropoietin secreting tumor autonomously without negative feedback is the most credible so far. However, further studies with use of blood erythropoietin level, tissue erythropoietin detection using immune-stain and new molecular biology techniques need to be done and compared to uterine myoma patients with no erythrocytosis. Usually, no further treatment is required following surgical removal.