Highly sensitive spatial transcriptomics using FISHnCHIPs of multiple co-expressed genes.

High-dimensional, spatially resolved analysis of intact tissue samples promises to transform biomedical research and diagnostics, but existing spatial omics technologies are costly and labor-intensive. We present Fluorescence In Situ Hybridization of Cellular HeterogeneIty and gene expression Programs (FISHnCHIPs) for highly sensitive in situ profiling of cell types and gene expression programs. FISHnCHIPs achieves this by simultaneously imaging ~2-35 co-expressed genes (clustered into modules) that are spatially co-localized in tissues, resulting in similar spatial information as single-gene Fluorescence In Situ Hybridization (FISH), but with ~2-20-fold higher sensitivity. Using FISHnCHIPs, we image up to 53 modules from the mouse kidney and mouse brain, and demonstrate high-speed, large field-of-view profiling of a whole tissue section. FISHnCHIPs also reveals spatially restricted localizations of cancer-associated fibroblasts in a human colorectal cancer biopsy. Overall, FISHnCHIPs enables fast, robust, and scalable cell typing of tissues with normal physiology or undergoing pathogenesis.

Mutations of 1p genes do not consistently abrogate tumor suppressor functions in 1p-intact neuroblastoma.

BACKGROUND: Deletion of 1p is associated with poor prognosis in neuroblastoma, however selected 1p-intact patients still experience poor outcomes. Since mutations of 1p genes may mimic the deleterious effects of chromosomal loss, we studied the incidence, spectrum and effects of mutational variants in 1p-intact neuroblastoma. METHODS: We characterized the 1p status of 325 neuroblastoma patients, and correlated the mutational status of 1p tumor suppressors and neuroblastoma candidate genes with survival outcomes among 100 1p-intact cases, then performed functional validation of selected novel variants of 1p36 genes identified from our patient cohort. RESULTS: Among patients with adverse disease characteristics, those who additionally had 1p deletion had significantly worse overall survival. Among 100 tumor-normal pairs sequenced, somatic mutations of 1p tumor suppressors KIF1Bß and CHD5 were most frequent (2%) after ALK and ATRX (8%), and BARD1 (3%). Mutations of neuroblastoma candidate genes were associated with other synchronous mutations and concurrent 11q deletion (P = 0.045). In total, 24 of 38 variants identified were novel and predicted to be deleterious or pathogenic. Functional validation identified novel KIF1Bß I1355M variant as a gain-of-function mutation with increased expression and tumor suppressive activity, correlating with indolent clinical behavior; another novel variant CHD5 E43Q was a loss-of-function mutation with decreased expression and increased long-term cell viability, corresponding with aggressive disease characteristics. CONCLUSIONS: Our study showed that chromosome 1 gene mutations occurred frequently in 1p-intact neuroblastoma, but may not consistently abrogate the function of bonafide 1p tumor suppressors. These findings may augment the evolving model of compounding contributions of 1p gene aberrations toward tumor suppressor inactivation in neuroblastoma.

Author Correction: Highly specific multiplexed RNA imaging in tissues with split-FISH.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Highly specific multiplexed RNA imaging in tissues with split-FISH.

We present split-FISH, a multiplexed fluorescence in situ hybridization method that leverages a split-probe design to achieve enhanced specificity. Split-FISH reduces off-target background fluorescence, decreases false positives and enables accurate RNA profiling in uncleared tissues. We demonstrate the efficacy of split-FISH on various mouse tissues by quantifying the distribution and abundance of 317 genes in single cells and reveal diverse localization patterns for spatial regulation of the transcriptome in complex tissues.

Primary paediatric epidural sarcomas: molecular exploration of three cases.

BACKGROUND: Primary paediatric epidural sarcomas are extremely rare. Overall, there remains a paucity of knowledge in paediatric epidural sarcomas owing to the infrequent number of cases. The Archer FusionPlex Sarcoma Kit (ArcherDX, Inc) is a next-generation sequencing assay that has been reported to be a useful technique to detect recurrent fusion in sarcomas. We report the molecular exploration of 3 primary paediatric epidural sarcomas-one in the cranium (mesenchymal chondrosarcoma) and 2 in the spine (mesenchymal chondrosarcoma and Ewing sarcoma respectively). CASE PRESENTATION: This is a study approved by the hospital ethics board. Clinico-pathological information from 3 consenting patients with primary epidural sarcomas was collected. These selected tumours are interrogated via Archer FusionPlex Sarcoma Kit (ArcherDX, Inc) for genomic aberrations. Results were validated with RT-PCR and Sanger sequencing. All findings are corroborated and discussed in concordance with current literature. Our findings show 2 variants of the HEY1-NCOA2 gene fusion: HEY1 (exon 4)-NCOA2 (exon 13) and HEY1 (exon 4)-NCOA2 (exon 14), in both mesenchymal chondrosarcoma patients. Next, the Ewing sarcoma tumour is found to have EWSR1 (exon 10)-FLI1 (exon 8) translocation based on NGS. This result is not detected via conventional fluorescence in situ testing. CONCLUSIONS: This is a molecularly-centered study based on 3 unique primary paediatric epidural sarcomas. Our findings to add to the growing body of literature for these exceptionally rare and malignant neoplasms. The authors advocate global collaborative efforts and in-depth studies for targeted therapy to benefit affected children.

Pericytoma With t(7;12): The First Ovarian Case Reported and a Review of the Literature.

We report the first case of an ovarian pericytoma with t(7;12). An 11-year-old child presented with abdominal pain and distension. A suprapubic mass was detected on examination and radiological investigations revealed a 16.5 cm solid-cystic ovarian mass. Histologically, the tumor was composed of spindle cells with S100-protein, Bcl-2, and CD10 reactivity on immunohistochemistry. Alpha fetoprotein, calretinin, alpha-inhibin, WT1, smooth muscle actin, caldesmon, desmin, cytokeratins, chromogranin, synaptophysin, EMA, Sox10, CD117, CD31, CD34, and CD68 were all negative. Molecular tests showed t(7;12)(p22;q13), resulting in the fusion of the ACTB with GLI1 genes and a diagnosis of pericytoma with t(7;12) of the ovary was made. We discuss the difficulties in diagnosing this lesion in the ovary and highlight the importance on molecular tests in characterizing challenging cases, especially primary ovarian spindle cell mesenchymal tumors.

Comparison of different treatment methods for protein solubilisation from waste activated sludge.

Biomaterials recovery from wasted activated sludge has become an increasing interesting research topic. The purpose of this study was to systemically evaluate different sludge disintegration methods (ultrasonic, alkaline, and thermal treatments) for protein solubilisation from waste activated sludge (WAS). Compared to control without treatment, the soluble protein concentration increased by 11, 23 and 12 times under the optimal treatment conditions (ultrasonic treatment of 1 W mL-1, alkaline treatment of pH 12 and thermal treatment at 80 °C). The increased soluble protein were significantly correlated with the release of total organic carbon (TOC), total dissolved nitrogen (TDN) and total organic nitrogen (TON) in soluble EPS, and the degradation of above parameters in tightly bound EPS. For all sludge samples treated by various methods, tyrosine-like protein with molecular weight less than 20 kDa predominated, and alkaline treatment at pH 12 showed the highest protein dominance. Further surface analysis of sludge by X-ray photoelectron spectroscopy indicated this might be related with the significant protein-N conversion occurred at pH 12. The economic analysis indicated alkaline treatment at pH 12 was economically feasible with a net saving of 25.57 USD per ton wet sludge compared to conventional sludge treatment and disposal method.

Variations in physical, chemical and biological properties in relation to sludge dewaterability under Fe (II) - Oxone conditioning.

The mechanism of Fe (II) - oxone conditioning to improve sludge dewaterability was investigated in this study. Five different types of sludge were tested, including raw sludge (Group 1: mixed primary and secondary sludge, waste activated sludge and anaerobic digested sludge) and pretreated sludge with prior solubilisation (Group 2: ultrasonic or thermal pretreated sludge). After Fe (II) - oxone conditioning, the concentrations of dissolved organic carbon, protein and polysaccharide of soluble extracellular polymeric substances (SB EPS) increased for Group 1, but decreased for Group 2. For all types of sludge investigated, the related organic compounds of loosely bound (LB) and tightly bound (TB) EPS decreased with Fe (II) - oxone conditioning, and increased sludge filterability showed strong and positive correlation with the removal of low molecular weight protein and neutrals in LB EPS. Fe (II) - oxone was very effective in disintegrating cell membrane and caused potential cell lysis, as indicated by increased percentage of damaged microbial cells. From this study, the mechanism of Fe (II) - oxone conditioning was proposed and can be divided into two steps: (1) Oxidation step - sulfate radicals degraded organic compounds in LB and TB EPS in sludge and transformed bound water to free water that was trapped in TB and LB EPS; It also damaged cells membrane and may help to release intracellular water content. Sludge flocs were broken into smaller particles; (2) Coagulation step - Fe (III), generated from the oxidation step can act as a coagulant to agglomerate smaller particles into larger ones and reduce the repulsive electrostatic interactions. Combined effects from above two steps can greatly improve sludge filterability.