Technical optimization of spatially resolved single-cell transcriptomic datasets to study clinical liver disease.

Single cell and spatially resolved 'omic' techniques have enabled deep characterization of clinical pathologies that remain poorly understood, providing unprecedented insights into molecular mechanisms of disease. However, transcriptomic platforms are costly, limiting sample size, which increases the possibility of pre-analytical variables such as tissue processing and storage procedures impacting RNA quality and downstream analyses. Furthermore, spatial transcriptomics have not yet reached single cell resolution, leading to the development of multiple deconvolution methods to predict individual cell types within each transcriptome 'spot' on tissue sections. In this study, we performed spatial transcriptomics and single nucleus RNA sequencing (snRNAseq) on matched specimens from patients with either histologically normal or advanced fibrosis to establish important aspects of tissue handling, data processing, and downstream analyses of biobanked liver samples. We observed that tissue preservation technique impacts transcriptomic data, especially in fibrotic liver. Single cell mapping of the spatial transcriptome using paired snRNAseq data generated a spatially resolved, single cell dataset with 24 unique liver cell phenotypes. We determined that cell-cell interactions predicted using ligand-receptor analysis of snRNAseq data poorly correlated with cellular relationships identified using spatial transcriptomics. Our study provides a framework for generating spatially resolved, single cell datasets to study gene expression and cell-cell interactions in biobanked clinical samples with advanced liver disease.

Technical optimization of spatially resolved single-cell transcriptomic datasets to study clinical liver disease.

Single cell and spatially resolved 'omic' techniques have enabled deep characterization of clinical pathologies that remain poorly understood, providing unprecedented insights into molecular mechanisms of disease. However, transcriptomic platforms are costly, limiting sample size, which increases the possibility of pre-analytical variables such as tissue processing and storage procedures impacting RNA quality and downstream analyses. Furthermore, spatial transcriptomics have not yet reached single cell resolution, leading to the development of multiple deconvolution methods to predict individual cell types within each transcriptome 'spot' on tissue sections. In this study, we performed spatial transcriptomics and single nucleus RNA sequencing (snRNASeq) on matched specimens from patients with either histologically normal or advanced fibrosis to establish important aspects of tissue handling, data processing, and downstream analyses of biobanked liver samples. We observed that tissue preservation technique impacts transcriptomic data, especially in fibrotic liver. Deconvolution of the spatial transcriptome using paired snRNASeq data generated a spatially resolved, single cell dataset with 24 unique liver cell phenotypes. We determined that cell-cell interactions predicted using ligand-receptor analysis of snRNASeq data poorly correlated with celullar relationships identified using spatial transcriptomics. Our study provides a framework for generating spatially resolved, single cell datasets to study gene expression and cell-cell interactions in biobanked clinical samples with advanced liver disease.

Creation of a Single Cell RNASeq Meta-Atlas to Define Human Liver Immune Homeostasis.

The liver is unique in both its ability to maintain immune homeostasis and in its potential for immune tolerance following solid organ transplantation. Single-cell RNA sequencing (scRNA seq) is a powerful approach to generate highly dimensional transcriptome data to understand cellular phenotypes. However, when scRNA data is produced by different groups, with different data models, different standards, and samples processed in different ways, it can be challenging to draw meaningful conclusions from the aggregated data. The goal of this study was to establish a method to combine 'human liver' scRNA seq datasets by 1) characterizing the heterogeneity between studies and 2) using the meta-atlas to define the dominant phenotypes across immune cell subpopulations in healthy human liver. Publicly available scRNA seq data generated from liver samples obtained from a combined total of 17 patients and ~32,000 cells were analyzed. Liver-specific immune cells (CD45+) were extracted from each dataset, and immune cell subpopulations (myeloid cells, NK and T cells, plasma cells, and B cells) were examined using dimensionality reduction (UMAP), differential gene expression, and ingenuity pathway analysis. All datasets co-clustered, but cell proportions differed between studies. Gene expression correlation demonstrated similarity across all studies, and canonical pathways that differed between datasets were related to cell stress and oxidative phosphorylation rather than immune-related function. Next, a meta-atlas was generated via data integration and compared against PBMC data to define gene signatures for each hepatic immune subpopulation. This analysis defined key features of hepatic immune homeostasis, with decreased expression across immunologic pathways and enhancement of pathways involved with cell death. This method for meta-analysis of scRNA seq data provides a novel approach to broadly define the features of human liver immune homeostasis. Specific pathways and cellular phenotypes described in this human liver immune meta-atlas provide a critical reference point for further study of immune mediated disease processes within the liver.

Assessment of the global practice of living donor liver transplantation.

Criteria that drive the selection and utilization of living liver donors are limited. Herein, the global availability of living donor liver transplantation (LDLT) and components of donor selection and utilization were assessed via an international survey. There were 124 respondents representing 41 countries, including 47 from Asia/Middle East (A/ME), 20 from Europe, and 57 from the Americas. Responses were obtained from 94.9% of countries with ≥10 LDLT cases/year. Most centers (82.3%) have defined donor age criteria (median 18-60 years), while preset recipient MELD cutoffs (median 18-30) were only reported in 54.8% of programs. Overall, 67.5% of programs have preset donor BMI (body mass index) ranges (median 18-30), and the mean acceptable macrosteatosis was highest for A/ME (20.2 ± 9.2%) and lowest for Americas (16.5 ± 8.4%, P = 0.04). Americas (56.1%) and European (60.0%) programs were more likely to consider anonymous donors versus A/ME programs (27.7%, P = 0.01). There were no differences in consideration of complex anatomical variations. Most programs (75.9%) perform donor surgery via an open approach, and A/ME programs are more likely to use microscopic arterial reconstruction. Despite variations in practice, key aspects of living donor selection were identified. These findings provide a contemporary reference point as LDLT continues to expand into areas with limited access to liver transplantation.

A rare cause of conductive hearing loss: High lateralized jugular bulb with bony dehiscence.

We present a rare case of pediatric conductive hearing loss due to a high lateralized jugular bulb. An 8-year-old boy with a right-sided conductive hearing loss of 40 dB was found to have a pink bulge toward the inferior part of the right eardrum. Computed tomography showed a high, lateralized right jugular bulb that had a superolaterally pointing diverticulum that bulged into the lower mesotympanum and posterior external auditory meatus. It was explained to the child's parents that it is important never to put any sharp objects into the ears because of the risk of injury to the jugular vein. A high, lateralized jugular bulb with a diverticulum is a rare anatomic abnormality. Correct diagnosis of this abnormality is important so that inappropriate intervention does not occur.

Obstruction of the external auditory meatus secondary to a giant pyogenic granuloma.

Pyogenic granuloma is a benign lesion of the skin and mucosa commonly known to occur in the head and neck region. The current literature has not yet identified its occurrence within the conchal bowl, a condition that leads to obstruction of the external auditory meatus. We present the case of a 28-year-old man who presented with a history of 3-4 weeks of a rapidly enlarging pedunculated lesion within the conchal bowl of the right ear and conductive hearing loss. Initial management included excision under local anaesthesia. The histological report concluded that it was a pyogenic granuloma. Later, reoccurrence was treated with a more definitive excision under general anaesthesia. During follow-up, the operative site was seen to have healed by secondary intention without reoccurrence. Although a pyogenic granuloma within the conchal bowl is benign, early therapeutic excision is important for histological diagnosis as much as to relieve consequential secondary obstruction and conductive hearing loss.

Reverse pedicle-based greater saphenous neuro-veno-fasciocutaneous flap for reconstruction of lower leg and foot.

INTRODUCTION: Paucity of soft tissue available locally for reconstruction of defects in leg and foot presents a challenge for reconstructive surgeon. The use of reverse pedicle-based greater saphenous neuro-veno-fasciocutaneous flap in reconstruction of lower leg and foot presents a viable alternative to free flap and cross-leg flap reconstruction. The vascular axis of the flap is formed by the vessels accompanying the saphenous nerve and the greater saphenous vein. We present here our experience with reverse saphenous neurocutaneous flap which provides a stable cover without the need to sacrifice any important vessel of leg. PATIENTS AND METHODS: The study is conducted from March 2003 through Dec 2009 and included a total of 96 patients with defects in lower two-thirds of leg and foot. There are 74 males and 22 females. Distal pivot point was kept approximately 5-6 cm from tip of medial malleolus, thus preserving the distal most perforator, and the flap is turned and inserted into the defect. Donor site is covered with a split thickness skin graft. Postoperative follow-up period was 6 weeks to 6 months. RESULT: The procedure is uneventful in 77 cases. Infection is observed in 14 cases. Partial flap necrosis occurs in 2 cases. Total flap necrosis is noted in 3 cases. CONCLUSION: Reverse pedicle saphenous flap can be used to reconstruct defects of lower one-third leg and foot with a reliable blood supply with a large arc of rotation while having minimal donor site morbidity.

Evaluation of Fermentation Efficiency of Yeast Strains and their Effect on Quality of Young Wines.

The yeast has important role in fermentation of wine grapes and wine quality. The fermentation of wine grapes affect by efficiency of particular yeast strain, sugar content, pH, available temperature, etc. To evaluate the efficiency of yeast strains (Premier Cuvee, RS-1, RS-2, RS-3 and natural), present study was conducted on two wine grape varieties viz.; Sauvignon Blanc (White) and Cabernet Sauvignon (Red). Efficiency of yeast strains was evaluated in terms of conversion rate of sugar into alcohol. As per recorded data, strain RS-3 (Pichia kudriavzevii) was found more efficient than other strains in fermentation of Cabernet Sauvignon with efficiency of 84.4 per cent but in case of Sauvignon Blanc, the commercial culture Premier Cuevee was found superior over RS-3. The quality parameters of young wines of both the varieties were also affected by the used strains. Considering the efficiency and impact on various parameters of wines, local strain, i.e., RS-3 was found at par with commercial culture (Premier Cuvee). The RS-3 strain has potential to produce quality wines. However, studies on effects of RS-3 strain on some specific quality parameters of wines like varietal aroma compounds, flavours etc. are needed.

Skull base presentation of multiple myeloma.

Pathologic proliferation of the plasma cell population can produce a wide spectrum of disorders, ranging from benign solitary plasmacytoma to malignant multiple myeloma. The presentation of the resulting disease can be either localized or systemic, depending on the affected area. Multiple myeloma typically presents with systemic symptoms secondary to skeletal lytic lesions, anemia, renal failure, infection, and hyperviscosity syndrome; a diagnosis of multiple myeloma is not suspected in the absence of these features. Multiple myeloma of the skull base is very rare. We present the case of a 66-year-old man who came to us with a 2-year history of disequilibrium and who was found to have multiple myeloma with extensive involvement of the skull base.