Probes for INS

Background and Aims Patients with inflammatory bowel disease (IBD) demonstrate nutritional selenium deficiencies and are at greater risk for colon cancer. Previously, we determined that global reduction of the secreted antioxidant selenium-containing protein, Selenoprotein P (SELENOP), substantially increased tumor development in an experimental colitis-associated cancer (CAC) model. We next sought to delineate tissue-specific contributions of SELENOP to intestinal inflammatory carcinogenesis and define clinical context. Methods Selenop floxed mice crossed with Cre driver lines to delete Selenop from the liver, myeloid lineages, or intestinal epithelium were placed on an azoxymethane/dextran sodium sulfate (AOM/DSS) experimental CAC protocol. SELENOP loss was assessed in human ulcerative colitis (UC) organoids, and expression was queried in human and adult UC samples. Results Although large sources of SELENOP, both liver- and myeloid-specific Selenop deletion failed to modify AOM/DSS-mediated tumorigenesis. Instead, epithelial-specific deletion increased CAC tumorigenesis, likely due to elevated oxidative stress with a resulting increase in genomic instability and augmented tumor initiation. SELENOP was downregulated in UC colon biopsies and levels were inversely correlated with endoscopic disease severity and tissue S100A8 (calprotectin) gene expression. Conclusions While global selenium status is typically assessed by measuring liver-derived plasma SELENOP levels, our results indicate that the peripheral SELENOP pool is dispensable for CAC. Colonic epithelial SELENOP is the main contributor to local antioxidant capabilities. Thus, colonic SELENOP is the most informative means to assess selenium levels and activity in IBD patients and may serve as a novel biomarker for UC disease severity and identify patients most predisposed to CAC development.

Objective: Identification of the developmental steps leading to somatosensory neuron development. Background: Our sense of touch is essential for life and relies on Low-Threshold Mechanoreceptors (LTMRs). LTMR subtypes characterized by early embryonic expression of Ntrk2 (TrkB) and Ret exhibit distinct properties depending on the skin region they innervate - hairy skin or glabrous(hairless) skin. In glabrous skin, TrkB+ and Ret+ LTMRs form Meissner corpuscles, while in hairy skin they form longitudinal lanceolate endings around hair follicles. These morphological features reflect the physiological properties and specialized functions of these neurons. The developmental steps leading to glabrous and hairy skin LTMR properties are largely unknown, in particular whether they are genetically pre-specified or whether interactions with different target skin regions define their unique features. Design/Methods: Sparse genetic labeling experiments demonstrate that morphological specialization of glabrous- and hairy paw skin-innervating TrkB+ and Ret+ LTMRs arise at nearly identical times during postnatal development. Interestingly, we find that individual neurons that terminate along the border of glabrous and hairy skin, termed “border neurons”, exhibit branches that form both lanceolate endings and Meissner corpuscle endings. Additionally, transcriptomic profiling and RNAscope experiments show that neonatal glabrous skin-and hairy skin-innervating TrkB+ and Ret+ neurons are transcriptionally similar, although distinct from other DRG neuron types. Lastly, using mouse mutants that have either ectopic glabrous skin or ectopic hairy skin we find that neurons that innervate ectopic skin regions of these mutants form ending types (either lanceolate or Meissner corpuscle endings) in accordance with the ectopic skin type. Results: These findings support a model in which embryonic TrkB+ and Ret+ LTMRs are able to form either Meissner corpuscle or lanceolate endings, and that the skin target region differentially instructs morphological maturation of these LTMR types. Conclusions: This model implies that neuronal identity in the peripheral nervous system is flexibly determined by target tissue.

How parasites develop and survive, and how they stimulate or modulate host immune responses are important in understanding disease pathology and for the design of new control strategies. Microarray analysis and bulk RNA sequencing have provided a wealth of data on gene expression as parasites develop through different life-cycle stages and on host cell responses to infection. These techniques have enabled gene expression in the whole organism or host tissue to be detailed, but do not take account of the heterogeneity between cells of different types or developmental stages, nor the spatial organisation of these cells. Single-cell RNA-seq (scRNA-seq) adds a new dimension to studying parasite biology and host immunity by enabling gene profiling at the individual cell level. Here we review the application of scRNA-seq to establish gene expression cell atlases for multicellular helminths and to explore the expansion and molecular profile of individual host cell types involved in parasite immunity and tissue repair. Studying host-parasite interactions in vivo is challenging and we conclude this review by briefly discussing the applications of organoids (stem-cell derived mini-tissues) to examine host-parasite interactions at the local level, and as a potential system to study parasite development in vitro. Organoid technology and its applications have developed rapidly, and the elegant studies performed to date support the use of organoids as an alternative in vitro system for research on helminth parasites.

The increased awareness surrounding health is a significant factor contributing to the trend of health awareness. People are showing extra care with changing lifestylesleading to more proactive care toward their health. There was an immense need to fill this gap. The founders of Healthians, India’s most trusted diagnostics, sensed this need and converted that into a successful business model. This article analyses Healthians governance and brand strategy making it one of the largest players in the Indian market. The article begins by describing the initial journey of Healthians and its founder. The article also highlights the financial strategy of the company along with the funding details. The industry analysis had also been done along with an analysis of major players in the diagnostic industry, followed by a discussion on the expansion strategy of the company. The business model, corporate governance, and marketing strategy of the company have been discussed in detail, followed by the brand strategy, in order to derive useful learning from the journey of this company. Adequate discussion on the products of the company had been done, along with the mentioning opportunities waiting to be explored by the company. With the highest competitive and volatile market of the healthcare industry, this company ensures that the highest standards in corporate governance and business ethics are being followed in the company. The article concludes with some dilemmas being faced by the company which may decide its future course of action and the various alternatives available to the company.

Congenital disorders of glycosylation (CDG) are a group of clinically and genetically heterogeneous diseases caused by disorders of glycoproteins synthesis. Patients manifest a wide range of symptoms, phenotypes, and severity, usually with neurological compromise. The conserved oligomeric Golgi (COG) complex plays an important role in vesicular tethering in retrograde Golgi transport. Mutation in this complex is considered a multiple-pathway CDG. Only 6 cases of pathogenic variants of COG1 have been reported in the literature. We present a 10 year-old-female born at term to healthy non-consanguineous Chilean parents. At birth, the main findings were weak suction, hypotonia, and high creatine kinase (CK). Due to development delay, hypotonia, and persistently elevated CK levels, sometimes over 10 times normal values, electromyography was performed, suggestive of a predominantly proximal myopathic compromise. Muscle biopsy revealed dystrophic changes and abnormal alpha-dystroglycan immunohistochemistry. The patient's symptoms progressed, and she currently continues with motor difficulties, muscle weakness, joint hypermobility, recurrent patellar dislocation, and severe progressive kyphoscoliosis. A lower limb muscular magnetic resonance image revealed mild fat replacement mainly on soleus and gastrocnemius muscles. No cognitive impairment or additional neurological symptoms have appeared, but persistent thrombocytopenia and intermittent leukopenia appeared after age 6 years. A neuromuscular NGS panel was negative, and exome sequencing revealed a homozygous frameshift mutation in COG1 gene (c.2665dupC, p.Arg889Profs*12). This mutation has been previously reported and is considered pathogenic. However, this is the first report of a COG1 mutation manifesting mainly as congenital muscular dystrophy with a musculoskeletal phenotype and without the intellectual phenotype expected due to the COG1 mutation. This communication expands the COG1 clinical spectrum, including muscle compromise and COG1 mutations as a potential gene candidate in the differential diagnosis of congenital muscular dystrophies.

BACKGROUND: Medulloblastoma with extensive nodularity (MBEN) represents a rare type of cerebellar tumors of infancy comprising two histologically distinct components that differ in cell differentiation and mitotic activity. Whereas some children suffering from MBEN experience disease recurrence, MBEN can also spontaneously differentiate and discontinue to grow. The underlying mechanisms of this variable biological behavior may offer insight into how embryonal tumors develop. METHODS: Fresh frozen and FFPE tumor tissue from nine MBEN-patients was subjected to multi-omics characterization including bulk sequencing, microdissection followed by RNA sequencing, single nucleus RNA-sequencing using the 10X Genomics- and SMART Seq. V2-protocols and spatial transcriptomics via RNAscope. RESULTS: All cases were molecularly classified as Sonic Hedgehog (SHH)-MB, and harbored somatic mutations within the SHH-pathway. After quality control, a total of ~30.000 cells were subjected to downstream analysis. Several non-malignant cell types, such as glial cells, were identified. In accordance with previous studies, we found only sparse immune infiltration. Unsupervised clustering identified cell clusters that differed in differentiation state and represented a continuum from embryonal-like cells with SHH-upregulation over intermediate cell states, to neuronal-like, postmitotic cells. Mapping to a single nucleus sequencing atlas of cerebellar development indicated that tumor cells reflected various stages of normally developing cerebellar granular precursors. Interestingly, one cluster of malignant cells with tumor-specific copy number alterations showed both transcriptomic features of astrocytes and embryonal cells. Using spatial transcriptomics, we were able to correlate different clusters of MBEN cells with distinct histologic MBEN compartments, with astrocyte-like tumor cells being located in the internodular compartment and in close proximity to mitotically active cancer cells. CONCLUSION: MBEN is formed by a continuum of malignant cell differentiation along the granular precursor lineage, with a subset of cells developing into cells that may represent tumor astrocytes. This differentiation process is reflected in the bicompartmental structure of MBEN.

Background: Smooth muscle cells (SMC) transition into a number of different phenotypes during atherosclerosis, including those that resemble fibroblasts and chondrocytes, and make up the majority of cells in the atherosclerotic plaque. To better understand the epigenetic and transcriptional mechanisms that mediate these cell state changes, and how they relate to risk for coronary artery disease (CAD), we have investigated the causality and function of transcription factors (TFs) at genome wide associated loci. Methods: We employed CRISPR-Cas 9 genome and epigenome editing to identify the causal gene and cell(s) for a complex CAD GWAS signal at 2q22.3. Subsequently, single-cell epigenetic and transcriptomic profiling in murine models and human coronary artery smooth muscle cells were employed to understand the cellular and molecular mechanism by which this CAD risk gene exerts its function. Results: CRISPR-Cas 9 genome and epigenome editing showed that the complex CAD genetic signals within a genomic region at 2q22.3 lie within smooth muscle long-distance enhancers for ZEB2, a TF extensively studied in the context of epithelial mesenchymal transition (EMT) in development and cancer. ZEB2 regulates SMC phenotypic transition through chromatin remodeling that obviates accessibility and disrupts both Notch and TGFβ signaling, thus altering the epigenetic trajectory of SMC transitions. SMC specific loss of ZEB2 resulted in an inability of transitioning SMCs to turn off contractile programing and take on a fibroblast-like phenotype, but accelerated the formation of chondromyocytes, mirroring features of high-risk atherosclerotic plaques in human coronary arteries. Conclusions: These studies identify ZEB2 as a new CAD GWAS gene that affects features of plaque vulnerability through direct effects on the epigenome, providing a new thereapeutic approach to target vascular disease.

Circular RNAs (circRNAs) are regulatory RNAs which have recently been shown to have clinical significance in several diseases, including, but not limited to, various cancers, neurological diseases and cardiovascular diseases. The function of such regulatory RNAs is largely dependent on their subcellular localization. Several circRNAs have been shown to conduct antagonistic roles compared to the products of the linear isoforms, and thus need to be characterized distinctly from the linear RNAs. However, conventional fluorescent in situ hybridization (FISH) techniques cannot be employed directly to distinguish the signals from linear and circular isoforms because most circRNAs share the same sequence with the linear RNAs. In order to address this unmet need, we adapted the well-established method of single-molecule FISH by designing two sets of probes to differentiate the linear and circular RNA isoforms by virtue of signal colocalization. We call this method 'circular fluorescent in situ hybridization' (circFISH). Linear and circular RNAs were successfully visualized and quantified at a single-molecule resolution in fixed cells. RNase R treatment during the circFISH reduced the levels of linear RNAs while the circRNA levels remain unaltered. Furthermore, cells with shRNAs specific to circRNA showed the loss of circRNA levels, whereas the linear RNA levels were unaffected. The optimization of the in-situ RNase R treatment allowed the multiplexing of circFISH to combine it with organelle staining. CircFISH was found to be compatible with multiple sample types, including cultured cells and fresh-frozen and formalin-fixed tissue sections. Thus, we present circFISH as a versatile method for the simultaneous visualization and quantification of the distribution and localization of linear and circular RNA in fixed cells and tissue samples.

Following stroke, the survival of neurons and their ability to reestablish connections is critical to functional recovery. This is strongly influenced by the balance between neuronal excitation and inhibition. In the acute phase of experimental stroke, lethal hyperexcitability can be attenuated by positive allosteric modulation of GABAA receptors (GABAARs). Conversely, in the late phase, negative allosteric modulation of GABAAR can correct the suboptimal excitability and improves both sensory and motor recovery. Here, we hypothesized that octadecaneuropeptide (ODN), an endogenous allosteric modulator of the GABAAR synthesized by astrocytes, influences the outcome of ischemic brain tissue and subsequent functional recovery. We show that ODN boosts the excitability of cortical neurons, which makes it deleterious in the acute phase of stroke. However, if delivered after day 3, ODN is safe and improves motor recovery over the following month in two different paradigms of experimental stroke in mice. Furthermore, we bring evidence that, during the subacute period after stroke, the repairing cortex can be treated with ODN by means of a single hydrogel deposit into the stroke cavity.SIGNIFICANCE STATEMENT Stroke remains a devastating clinical challenge because there is no efficient therapy to either minimize neuronal death with neuroprotective drugs or to enhance spontaneous recovery with neurorepair drugs. Around the brain damage, the peri-infarct cortex can be viewed as a reservoir of plasticity. However, the potential of wiring new circuits in these areas is restrained by a chronic excess of GABAergic inhibition. Here we show that an astrocyte-derived peptide, can be used as a delayed treatment, to safely correct cortical excitability and facilitate sensorimotor recovery after stroke.

Murine astrovirus 2 (MuAstV2) is a novel murine astrovirus recently identified in laboratory and wild mice. MuAstV2 readily transmits between immunocompetent mice yet fails to transmit to highly immunocompromised mouse strains-a unique characteristic when contrasted with other murine viruses including other astroviruses. We characterized the viral shedding kinetics and tissue tropism of MuAstV2 in immunocompetent C57BL/6NCrl mice and evaluated the apparent resistance of highly immunocompromised NOD- Prkdcem26Cd52Il2rgem26Cd22 /NjuCrl mice to MuAstV2 after oral inoculation. Temporal patterns of viral shedding were determined by serially measuring fecal viral RNA. Tissue tropism and viral load were characterized and quantified by using in-situ hybridization (ISH) targeting viral RNA. Cellular tropism was characterized by evaluating fluorescent colocalization of viral ISH with various immunohistochemical markers. We found a rapid increase of fecal viral RNA in B6 mice, which peaked at 5 d after inoculation (dpi) followed by cessation of shedding by 168 dpi. The small intestine had the highest percentage of hybridization (3.09% of tissue area) of all tissues in which hybridization occurred at 5 dpi. The thymus displayed the next highest degree of hybridization (2.3%) at 7 dpi, indicating extraintestinal viral spread. MuAstV2 RNA hybridization was found to colocalize with only 3 of the markers evaluated: CD3 (T cells), Iba1 (macrophages), and cytokeratin (enterocytes). A higher percentage of CD3 cells and Iba1 cells hybridized with MuAstV2 as compared with cytokeratin at 2 dpi (CD3, 59%; Iba1, 46%; cytokeratin, 6%) and 35 dpi (CD3, 14%; Iba1, 55%; cytokeratin, 3%). Neither fecal viral RNA nor viral hybridization was noted in NCG mice at the time points examined. In addition, mice of mixed genetic background were inoculated, and only those with a functioning Il2rg gene shed MuAstV2. Results from this study suggest that infection of, or interaction with, the immune system is required for infection by or replication of MuAstV2.

Introduction Medulloblastoma is the most common tumor of the brain of neuroendocrine origin in children and typically demonstrates an aggressive, relentless clinical behavior and high recurrence rate. Extraneuraxial metastasis of medulloblastoma to the jaw is extremely rare, with fewer than 10 cases reported in the literature. Case Report A 10-year-old male patient presented with a swelling in the right side of the face for 15 days. The patient had a past history of medulloblastoma diagnosed a few years earlier. Clinical examination revealed a diffuse painless mass in the right mandible. Multiplanar reconstruction demonstrated a 1.5 × 1.5 cm well-defined round lesion with extensive necrosis involving mostly the ramus of right mandible with resultant bone erosion on the lingual aspect. The lesion involved the regional structures including maxillary sinus, pterygoid process, and carotid sheath. Microscopic examination of the biopsy specimen exhibited sheets of poorly differentiated malignant small round blue cells with extensive necrosis. The tumor cells revealed molding of nuclei with speckled chromatin and inconspicuous cytoplasm. Evidence of vascular and perineural invasion was noted. Immunohistochemical studies demonstrated weak granular positivity for synaptophysin, but staining for AE1/3, desmin, CD99, and LCA was negative in the lesion tissue. A diagnosis of round blue cell malignancy of neuroendocrine origin compatible with medulloblastoma metastatic to the jaw was rendered. Our case represents only the 10th such as case in the English-language literature. Owing to the rarity of this entity, a more accurate understanding of the prognosis and treatment of metastatic medulloblastoma is not well known.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is known for its aggressive biology and lethality. Due to a low success rate of current diagnostic and therapeutic approaches in clinic, there is an urgent need for preclinical research studies to investigate the underlying biology of this malignancy. This knowledge is indispensable to facilitate the development and validation of potential new therapeutic compounds. Superior to conventional biomedical research models, the focus of this study is on the development and use of a well-established patient-derived 3D model, mimicking the tumor as it is present in a human body. Aims: The development and characterization of patient-derived organoids (PDO) and patient-derived xenografts (PDX) of PDAC. Materials and Methods: The models are extensively analysed using advanced histological methods such as BaseScope^®, 3D imaging and DNA hotspot sequencing. Results: 10 established PDAC-PDO and their corresponding parental tumors are already validated using immunostainings and DNA hotspot sequencing. The latter confirms presence of tumor cells in the organoids. In addition, this study is the first to show in situ detection of important driver mutations of pancreatic cancer, like KrasG12D, both in parental tumor and PDO. Thus far, 5 PDX have been generated that will undergo similar analysis. Conclusion: We have successfully started a pre-clinical screening platform for PDAC based on PDO and PDX. Altogether, spatial-omics analysis of both models can substantiate (1) resemblance to parental tissue and (2) spatial genomic characteristics associated with the type of model used. Ultimately, the screening platform can be used by pharmaceutical companies to facilitate oncological drug testing.