Regulation of Stem Cell Differentiation Into Cardiomyocytes by Lysophosphatidic Acid

The mechanisms that regulate the differentiation of stem cells (SCs) into cardiomyocytes are still unclear and the role of endogenous molecules on this process remains unexplored. One such molecule is the bioactive phospholipid lysophosphatidic acid (LPA) which accumulates in the myocardium following acute infarction and exerts multiple biological functions, including the regulation of cell growth and differentiation as well as cell survival (Tigyi et al., 2003; Sengupta, et al., 2004). Experiments were therefore carried out in this thesis to reveal whether LPA can induce the differentiation of stem cells into cardiomyocytes and to identify the signalling mechanisms that mediate this effect. All experiments were carried out in the mouse P19 carcinoma stem cell line. Treatments with LPA in the absence and presence of various pharmacological compounds were conducted in embryoid bodies (EBs) formed from the P19 cells in sterile Petri dishes over 4 days. The EBs were subsequently transferred into 6-well cell culture plates and cultured for specific time points. Lysates were generated and subjected to western blotting for expression of cardiac- specific myosin light chain -1v (MLC-1v). To look at the expression of LPA receptors (LPAR1-LPAR5) experiments were carried out by RT-PCR using specific primers for each LPA receptor and the role of the latter in mediated responses to LPA were examined in the presence of the LPAR 1/3 antagonist, Ki16425, or the LPAR 4 receptor blocker suramin. In addition, experiments were carried out investigating the role of Gαi and specific signalling pathways that may be involved in the differentiation of P19 cells. These were carried out using potent inhibitors/antagonists of Gi inhibitor (Pertussis toxin), PI3K inhibitor (LY294002), Akt inhibitor (Akt inhibitor XIII), PKC inhibitor (Bisindolylmaleimide I BIM-I), ROCK inhibitor (Y-27632), p38-MAPK inhibitor (SB203580) and ERK1/2 inhibitor (PD98059). Further experiments were carried out to establish whether the presence of LPA results in the phosphorylation of the targeted kinases. These studies were however limited to Akt, p38 MAPK and ERK1/2. Incubation of cells with LPA resulted in the differentiation of P19 cells into cardiomyocytes as reflected by the induction of MLC-1v. The latter increased significantly above basal in a time-dependent manner, reaching a maximum 10 days after plating EBs in 6-well plates. The induction of MLC-1v was more pronounced in cells incubated with 5 μM LPA at 6 days but showed little concentration differences at day 12. RT-PCR analysis confirmed the expression of LPA receptors 1 to 4 but not 5. Pre-incubating cells with suramin and Ki16425 concentration-dependently inhibited MLC-1v expression with 0.05 mg/ml and 10 M respectively, virtually abolishing the expression of MLC-1v. Additionally, inhibitors of LPAR1/3 and LPAR4 receptors and all the signalling inhibitors except SB203580 abolished the phosphorylation of ERK1/2. Similarly, p38 MAPK activation was completely abolished by LPAR1/3 and LPAR4 receptor antagonists, Interestingly, only LY294002 (5 M) and Y27632 (10 M) abolished the LPA induced activation of p38 MAPK while SB203580, BIM-I, Akt inhibitor XIII and PD95080 caused no significant changes to the phosphorylation of p38 MAPK. In conclusion, the studies carried out in this thesis have shown that LPA can induce P19 stem cells to differentiate into cardiomyocytes and they are linked to the well characterised LPA receptors (LPAR1/3 and 4). These receptors are coupled to downstream signalling pathways of which those involving the ROCK, PI3K, PKC and/or Akt may be critical, and may converge on ERK1/2. Inhibition of any of these pathways has the potential to suppress differentiation. In contrast, signalling leading to p38 activation may potentially suppress differentiation but this needs further clarification

Molecular pathology of Usher 1B patient-derived retinal organoids at single cell resolution

Usher syndrome-associated retinitis pigmentosa (RP) causes progressive retinal degeneration, which has no cure. The pathomechanism of Usher type 1B (USH1B)-RP caused by MYO7A mutation remains elusive because of the lack of faithful animal models and limited knowledge of MYO7A function. Here, we analyzed 3D retinal organoids generated from USH1B patient-derived induced pluripotent stem cells. Increased differential gene expression occurred over time without excessive photoreceptor cell death in USH1B organoids compared with controls. Dysregulated genes were enriched first for mitochondrial functions and then proteasomal ubiquitin-dependent protein catabolic processes and RNA splicing. Single-cell RNA sequencing revealed MYO7A expression in rod photoreceptor and Müller glial cells corresponding to upregulation of stress responses in NRL+ rods and apoptotic signaling pathways in VIM+ Müller cells, pointing to the defensive mechanisms that mitigate photoreceptor cell death. This first human model for USH1B-RP provides a representation of patient retina in vivo relevant for development of therapeutic strategies

Generation of 3D retinal tissue from human pluripotent stem cells using a directed small molecule-based serum-free microwell platform

Retinal degenerative diseases are a leading cause of blindness worldwide with debilitating life-long consequences for the affected individuals. Cell therapy is considered a potential future clinical intervention to restore and preserve sight by replacing lost photoreceptors and/or retinal pigment epithelium. Development of protocols to generate retinal tissue from human pluripotent stem cells (hPSC), reliably and at scale, can provide a platform to generate photoreceptors for cell therapy and to model retinal disease in vitro. Here, we describe an improved differentiation platform to generate retinal organoids from hPSC at scale and free from time-consuming manual microdissection steps. The scale up was achieved using an agarose mould platform enabling generation of uniform self-assembled 3D spheres from dissociated hPSC in microwells. Subsequent retinal differentiation was efficiently achieved via a stepwise differentiation protocol using a number of small molecules. To facilitate clinical translation, xeno-free approaches were developed by substituting Matrigel™ and foetal bovine serum with recombinant laminin and human platelet lysate, respectively. Generated retinal organoids exhibited important features reminiscent of retinal tissue including correct site-specific localisation of proteins involved in phototransduction

Decoding the X-ray flare from MAXI J0709-159 using optical spectroscopy and multi-epoch photometry

We present a follow-up study on the recent detection of two X-ray flaring events by MAXI/GSC observations in soft and hard X-rays from MAXI J0709-159 in the direction of HD 54786 (LY CMa), on 2022 January 25. The X-ray luminosity during the flare was around 10^(37) erg/s (MAXI), which got reduced to 10^(32) erg/s (NuSTAR) after the flare. We took low-resolution spectra of HD 54786 from HCT and VBT facilities in India, on 2022 February 1 and 2. In addition to H-alpha emission, we found emission lines of He I in the optical spectrum of this star. By comparing our spectrum of the object with those from literature we found that He I lines show variability. Using photometric study we estimate that the star is having effective temperature of 20000 K. Although HD 54786 is reported as a supergiant in previous studies, our analysis favours it to be evolving off the main sequence in the Color-Magnitude Diagram. We could not detect any infrared excess, ruling out the possibility of IR emission from a dusty circumstellar disc. Our present study suggests that HD 54786 is a Be/X-ray binary system with a compact object companion, possibly a neutron star.Comment: 9 pages, 5 figures, 1 table, accepted for publication in ApJ

Analysis of serum copper and iron levels in oral submucous fibrosis patients: A case–control study

Background: Oral submucous fibrosis (OSF) is a chronic debilitating disease and a potentially malignant disorder of the oral cavity. The pathogenesis of the disease is not well established. Trace elements such as copper and iron play an important role in the pathogenesis of OSF. Estimation of these elements in serum of the patients may be helpful in understanding the pathologic mechanism. Therefore, a study was carried out to analyze the level of serum copper and iron in the population of Central India. Materials and Methods: A case–control study was carried out on 35 patients with clinically diagnosed of OSF and 35 healthy controls. OSF patients were categorized by clinical staging. Serum copper and iron concentrations were measured by atomic absorption spectrophotometry. Results: Results of the study shows that the mean serum copper concentration was greater in study group (133.3 ± 19.2) compared to control group (113.9 ± 22.1) and the mean serum iron was lower in study group (116.0 ± 24.1) compared to control group (128.2 ± 23.4). The result obtained was statistically significant. The serum copper level increases as the clinical staging of OSF progresses, whereas serum iron level decreases as clinical staging progresses. Conclusion: There was an increase in copper level and decrease in iron level in study group compared to control group; this suggests that there is an increase in copper level with the advancement of clinical staging of OSF

Subcutaneous Panniculitis- like T-Cell Lymphoma: Report of two cases

Subcutaneous panniculitis-like T-cell lymphoma is a distinct variant of cutaneous T-cell lymphoma, characterized by primary involvement of the subcutaneous fat in a manner mimicking panniculitis. It accounts for less than one percent of all non Hodgkin lymphoma. We describe two such patients who presented with cutaneous nodules. A 28 year old male presented with a one and a half month history of multiple subcutaneous nodules over the thighs, abdominal wall and chest. A clinical diagnosis of panniculitis was made. An excision biopsy of one the nodules was performed and the histopathology revealed subcutaneous panniculitis-like T-cell lymphoma. The other patient was a 44 year old male who underwent excision of a subcutaneous mass in the right thigh and on histopathological examination a diagnosis of subcutaneous panniculitis-like T-cell lymphoma was made. The patients received one cycle of CHOP (cyclophoshamide, vincristine, doxorubicin and prednisolone) regimen, followed by systemic steroids and were advised follow up

Paleoproductivity shifts since the last 130 ka off Lakshadweep, Southeastern Arabian Sea

Marine sediment deposited on the ocean floor and near coastal areas, the western coastal regions of India provide records of monsoonal shifts and productivity. To understand the paleoproductivity in the northern Indian Ocean, we analyzed carbon, hydrogen, nitrogen, sulfur, total organic carbon (TOC) and calcium carbonate (CaCO3) in a deep-sea sediment core collected using gravity corer (GC-01) off Lakshadweep, Southeastern Arabian Sea. The results were then compared with the previous data generated from the surrounding area sediment cores to understand the productivity variations since the last 130 ka. The CaCO3 content in the sediment core varies from 40.82% to 62.48% (with a mean value of 51.96%) and it is noted that these values were lower during the glacial episodes (Marine Isotope Stages-2 and 4) than the interglacial episodes (MIS-1, 3 and 5). The C/N ratio varied from 0.14 to 34.25, but was less than 9, since ∼74 ka to recent, suggesting a marine origin for the organic carbon. The C/N ratio fluctuated significantly during MIS-5, and relatively the highest C/N ratio was observed at 5e ∼127, 5d ∼110 and 5b ∼85 ka, corresponding to stadials 5b and 5d, (except 5e) indicating terrestrial OC from C3 plants. The low C/N ratios during ∼128, ∼102, ∼76 and ∼32 ka match with the interstadials especially during MIS 5 (5a, 5c and 5e), correspondingly, and are marine OC in the source. This suggests that the MIS-5 stadial was interrupted via land source signifying higher productivity owing to the strong southwest monsoon during these periods. Further, high productivity was also observed during the Last Glacial Maximum (LGM) and Holocene in the Southeastern Arabian Sea since the 130 ka.Ministry of Earth Science (MoES), Govt. of India with reference number MoES/16/07/11(i)-RDEAS and MoES/P.O.(Seismic)8(09)-Geochron/201