C1QA and COMP: plasma-based biomarkers for early diagnosis of pancreatic neuroendocrine tumors.

Pancreatic Neuroendocrine tumors (PanNET) are challenging to diagnose and often detected at advanced stages due to a lack of specific and sensitive biomarkers. This study utilized proteomics as a valuable approach for cancer biomarker discovery; therefore, mass spectrometry-based proteomic profiling was conducted on plasma samples from 12 subjects (3 controls; 5 Grade I, 4 Grade II PanNET patients) to identify potential proteins capable of effectively distinguishing PanNET from healthy controls. Data are available via ProteomeXchange with the identifier PXD045045. 13.2% of proteins were uniquely identified in PanNET, while 60% were commonly expressed in PanNET and controls. 17 proteins exhibiting significant differential expression between PanNET and controls were identified with downstream analysis. Further, 5 proteins (C1QA, COMP, HSP90B1, ITGA2B, and FN1) were selected by pathway analysis and were validated using Western blot analysis. Significant downregulation of C1QA (p = 0.001: within groups, 0.03: control vs. grade I, 0.0013: grade I vs. grade II) and COMP (p = 0.011: within groups, 0.019: control vs grade I) were observed in PanNET Grade I & II than in controls. Subsequently, ELISA on 38 samples revealed significant downregulation of C1QA and COMP with increasing disease severity. This study shows the potential of C1QA and COMP in the early detection of PanNET, highlighting their role in the search for early-stage (Grade-I and Grade-II) diagnostic markers and therapeutic targets for PanNET.

Hydrogen sulfide promotes migration of trophoblast cells by a Rho GTPase mediated actin cytoskeleton reorganization.

INTRODUCTION: Preeclampsia (PE) arises due to defective spiral artery remodelling which may be due to deficient migration of trophoblast cells. Migration of human endothelial cells has been shown to be promoted via Hydrogen sulphide(H2S)/Rho GTPase Rac1 axis. This novel role of H2S and its downstream processes have not yet been studied in the development and function of the placental trophoblast cells. METHODS: Placental tissues were obtained post-delivery from consented preeclamptic and normotensive mothers (n = 60). The protein expression levels of cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthase (CBS) along with its downstream migratory molecules were compared in both the arms. The pro-migratory role of H2S was investigated in a first trimester placental cell line. RESULTS: H2S promoted the migration of trophoblast cells in a Rho GTPase dependent manner mediated by actin cytoskeleton reorganization. The reduced levels of H2S producing enzymes in the PE placentae along with decreased levels of Rho GTPases (Rac1 and Rho A) corroborate the results of PAG and AOAA treatment in down regulating the Rho GTPases in the in vitro grown placental cultures. Reduction of the migratory potential of trophoblastic cells caused due to hypoxia/reoxygenation was rescued by upregulating the H2S expression with the use of NaHS as a H2S donor. DISCUSSION: Exogenous H2S increases the migratory potential of the placental cells in culture conditions and also post hypoxia/reoxygenation injury. H2S as a gaso-transmitter holds a great potential as a therapeutic agent. Its long-term effects need to be investigated using model systems (rat/mouse) of PE following it up with clinical regulatory trials.

Mechanism of apoptosis activation by Curcumin rescued mutant p53Y220C in human pancreatic cancer.

The mutant p53Y220C (mutp53Y220C) is frequently observed in numerous tumors, including pancreatic cancer. The mutation creates a crevice in the DNA binding core domain and makes p53 a thermally unstable non-functional protein that assists tumor progression and confers resistance to chemotherapeutic drugs. Restoring mutp53 function to its wild type by selectively targeting this crevice with small molecules is a pivotal strategy to promote apoptosis. In this study, we have shown through different biophysical and cell-based studies that curcumin binds and rescues mutp53Y220C to an active wild-type conformation and restores its apoptotic transcription function in BxPC-3-pancreatic cancer cells. In addition, the curcumin-rescued-p53Y220C (CRp53) showed significant hyperphosphorylation at Ser15, Ser20, and acetylation at Lys382 with an 8-fold increase in transcription activity in the BxPC-3 cell lines. We also observed that the active CRp53 escapes Mdm2-mediated proteasomal degradation and the majority of the proteins were localized inside the nucleus with an increased half-life and transcription restoration compared to untreated BxPC-3 cells. By label-free proteomics analysis, we observed that upon curcumin treatment almost 227 proteins were dysregulated with the majority of them being transcriptional targets of p53. Based on our studies, it reflects that apoptosis in pancreatic cancer cells is mediated by curcumin-rescued mutant p53Y220C.

Status of VEGF in preeclampsia and its effect on endoplasmic reticulum stress in placental trophoblast cells.

OBJECTIVE: To explore the role of VEGF in attenuating endoplasmic reticulum stress in placental trophoblast cells. STUDY DESIGN: Study was divided into following parts: 1. Serum Analysis of GRP78 and VEGF using sandwich ELISA. 2. Expression of VEGF and GRP78 in placentae by immunohistochemistry (IHC). 3. In Vitro experiments. Status of ER stress markers (GRP78, eIF2α, XBP1, ATF6 and CHOP) was assessed at various time points (8 h, 14 h, 24 h) when trophoblast cells were treated with varying concentration(s) of VEGF and also by adding recombinant VEGF at protein (Immunofluorescence, Western blot) and transcript levels (qRT-PCR). RESULTS: Increased GRP78 and decreased VEGF protein levels in sera and placentae of preeclamptic pregnant women and reduced expression of various ER stress markers at both transcript and protein levels was observed in trophoblast cells when they were exposed to recombinant VEGF thereby indicating positive role of VEGF in alleviating ER stress. CONCLUSIONS: Reduced expression of ER stress markers in trophoblast cells against increased VEGF highlighted a new window to explore prospective drugs that can be designed to modulate the activities of various ER stress sensors in order to alleviate ER stress in pregnant women with preeclampsia.

Performance of a novel, manually operated intraosseous device in adult human cadavers.

AIM: Intraosseous (IO) access in adults is preferred using semi-automatic devices as it is difficult to penetrate the thick cortical layer of long bones using manual needles. The authors have developed an IO device which generates both rotational and axial thrust using a manual driver. This drilling mechanism addresses certain pain-points of current IO devices. The objective of this study was to evaluate the performance of this device in human cadavers. METHODS: The authors tested the ability of this device for IO access at proximal and distal tibia in 10 adult cadavers. Needle position was confirmed by fluoroscopy after contrast injection. Needle penetration time-defined as the time required for manual drilling of bone-and the total procedure time were calculated from video analysis. A successful IO procedure was defined as an appropriate needle position without any contrast extravasation, device, or procedure-related complication. After each procedure, the authors recorded damage to the device or fracture of the bone. RESULTS: A single physician performed 40 IO procedures. The IO access was successful in 35 (87.5 percent) and was accomplished in first attempt in 33 (82.5 percent) insertions. Reasons for failure were undershooting of needle (2/40, 5 percent), trocar damage (1/40, 2.5 percent), and detachment of plastic hub of the needle during removal in (2/40, 5 percent) procedures. There were no bone fractures. In all but one instance, needle penetration time was <3 seconds. The mean total procedure time was 40 ± 13 seconds. CONCLUSION: In this pilot study, the authors have demonstrated the efficacy of a novel, manually introduced IO device in adult cadavers.

Standardization of the technique of silicon injection of human cadaveric heads for opacification of cerebral vasculature in Indian conditions.

A surgeon's understanding of the surgical anatomy can be greatly enhanced by the dissection of preserved cadaveric specimens. A reliable and inexpensive biological model for testing and standardization of dye injection concentrations is proposed utilizing the goat's head as a biological model. The first phase was concerned with standardization of the dye by titrating its concentration and injecting various amounts into cerebral vessels of a goat's head until an optimal concentration had been ascertained. In the second phase, this optimum concentration of the dye was injected into four human cadaveric heads following the same technique standardized using the goat's head. Upon dissecting the four cadaveric human heads which were injected with silicon dyes and preserved in 10% formalin, the vessels were all well-opacified and the brain was of near normal consistency and good for dissection, without showing any features of putrefaction. The goat model, having similar color, texture, and the handling as the cadaveric head, offers an opportunity to test indigenously manufactured polymerizing dyes in the future. This biological model, therefore, has the potential to considerably reduce the cost of cadaver preparation.

Feasibility study of a novel intraosseous device in adult human cadavers.

BACKGROUND & OBJECTIVES: Intraosseous (IO) access is an alternative to difficult intravenous (iv) access during emergency clinical situations. Existing IO solutions are expensive, require power supply and trained manpower; limiting their use in resource constrained settings. To address these limitations, a novel IO device has been developed. The objectives of this study were to evaluate functionality and safety of this device in adult human cadavers. METHODS: The ability of the IO device to penetrate the proximal and/or distal tibia was evaluated in three adult cadavers. Subjective parameters of loss of resistance, stable needle hold, easy needle withdrawal and any damage to the device were evaluated during the study. The insertion time was the objective parameter measured. Four sets of radiographs per insertion confirmed the position of the needle and identified complications. RESULTS: A single physician performed 12 IO access procedures using the same device. Penetration of proximal and/or distal tibia was achieved in all instances. It was successful in the first attempt in eight (66.7%) and during second attempt in the remaining. The mean time to insertion was 4.1 ± 3.1 sec. Appropriate insertion of needle in the intra-medullary space of bone was confirmed with radiological examination in 10 (83.3%) insertions. In two occasions after penetrating the cortical layer of bone, the device overshot the intra-medullary space, as detected by radiological examination. Device got bent during insertion in one instance. There was no evidence of needle breakage or bone fracture. The needle could be withdrawn effortlessly in all instances. INTERPRETATION & CONCLUSIONS: The novel IO device could successfully penetrate the adult cadaver bones in most cases. Further studies are needed to confirm these results on a large sample.

Practical guidelines for setting up neurosurgery skills training cadaver laboratory in India.

Though the necessity of cadaver dissection is felt by the medical fraternity, and described as early as 600 BC, in India, there are no practical guidelines available in the world literature for setting up a basic cadaver dissection laboratory for neurosurgery skills training. Hands-on dissection practice on microscopic and endoscopic procedures is essential in technologically demanding modern neurosurgery training where ethical issues, cost constraints, medico-legal pitfalls, and resident duty time restrictions have resulted in lesser opportunities to learn. Collaboration of anatomy, forensic medicine, and neurosurgery is essential for development of a workflow of cadaver procurement, preservation, storage, dissection, and disposal along with setting up the guidelines for ethical and legal concerns.

Plastinated knee specimens: a novel educational tool.

INTRODUCTION: During the routine dissection of knee joints in an anatomy dissection hall, it was observed that the specimens had deteriorated overtime, due to their repeated handling and the use of high concentrations of formalin for their fixation. In order to stop their further deterioration, we decided to plastinate these specimens. Thus, the present study was undertaken to prepare plastinated knee specimens from old embalmed cadavers and to compare them with freshly fixed, plastinated specimens. OBJECTIVES: 1. To plastinate old embalmed and fresh formalin fixed knee regions.2. To demonstrate the extra and the intracapsular structures around both the plastinated knee regions.3. To compare their morphological features in terms of their colours, dilatation and flexibility. METHODS: A total of 15 knee joint specimens from among fresh formalin (5-8%) fixed (group I) and old embalmed bodies (group II) were collected, washed and plastinated by using the standard S-10 silicon technique and they were compared for their colours, dilatation and flexibility. RESULTS: All the plastinated specimens showed an accurate reproduction of the tissue details that were comparable to those of the natural unfixed specimens. A comparison among the two groups showed a significant difference in terms of the colour, dilatation and the flexibility of the specimens. The plastinated knee joint specimens from group I were of good quality, with negligible shrinkage, more flexibility and well preserved morphologies. CONCLUSION: Plastinated knee specimens can serve as an excellent educational tool for the undergraduate and postgraduate students of anatomy, radiology and orthopaedics, as they are dry, odourless and nontoxic, with a good structural preservation and a higher instructional value. The fresh knee regions, when they were plastinated, were aesthetically superior in terms of their colours, dilatation and flexibility, thus making them ideal for teaching and hands-on experiences.

A comparative study of apoptosis in placentas of normal and preeclamptic Indian pregnant women by TUNEL assay and M30 immunostaining.

BACKGROUND: Preeclampsia is a unique life-threatening disorder of human pregnancy associated with the abnormal placentation caused by the inadequate trophoblastic invasion due to altered apoptosis of these cells. The aim of the present study was to assess and compare the apoptosis in trophoblastic cells in various zones (villous and extravillous) of placentas of preeclamptic and normotensive nonproteinuric pregnant women. METHODS: Hematoxylin eosin staining, TUNEL assay and M30 immunostaining techniques were used for studying apoptosis in trophoblastic cells of placentas of two groups. The results of apoptotic indices by these techniques were compared in preeclamptic group. RESULTS: The TUNEL apoptotic indices were higher in all the zones of placentas of preeclamptic group as compared to control group though the results were not statistically significant. M30 immunostaining also gave higher apoptotic indices in all the zones of preeclamptic placentas as compared to the normal group but the result of apoptotic index of basal plate was not statistically significant. M30 immunostaining was absent in stromal cells. CONCLUSION: In our study, the trophoblastic apoptotic rates were more in villous as well as extravillous zones of preeclamptic placentas as compared to normal placentas and M30 immunostaining was found to be more sensitive and specific for the apoptotic trophoblastic cells as compared to TUNEL assay.

Role of oxidative stress markers and antioxidants in the placenta of preeclamptic patients.

AIM: The present study aimed to evaluate and compare the placental variables of oxidative stress markers in preeclamptic women. METHODS: A total of 60 placentas were collected. Of these, 30 were obtained from normotensive pregnancies, and 30 from pregnancies with preeclampsia as per International Society for the Study of Hypertension in Pregnancy (ISSHP) criteria. Each placental tissue was analyzed for levels of pro-oxidant (malondialdehyde) and antioxidants (glutathione and superoxide dismutase) using the standard enzymatic assays. RESULTS: Malondialdehyde levels were significantly higher (12.21 ± 4.1 versus 4.7 ± 2.1 nmol/g tissue, P < 0.0001) and glutathione (GSH) levels were significantly lower (0.46 ± 0.37 versus 1.03 ± 0.43 µmol/g tissue, P < 0.0001) in the placentas of preeclamptic women when compared to those of normal pregnancies. Though not statistically significant, decreases in superoxide dismutase levels were observed in placentas of preeclamptic women (4.14 ± 2.25 versus 5.22 ± 2.0 units/mg tissue protein, P < 0.055). Receiver operator characteristic curve analysis of malondialdehyde revealed a sensitivity of 87% and specificity of 87%, at a cutoff value 6.5 nmol/g. Similarly, GSH had a sensitivity of 77% and a specificity of 77% at a cutoff value 0.62 µmol/g. CONCLUSION: The present study demonstrated that increased placental lipid peroxidation and decreased levels of antioxidants may play an important role in the pathogenesis of preeclampsia. These findings are suggestive of involvement of oxidative stress markers in preeclamptic patients.

Circulating angiogenic factors in pregnancies complicated by pre-eclampsia.

BACKGROUND: Pre-eclampsia is an inflammatory disorder characterized by diffuse endothelial dysfunction possibly secondary to impaired trophoblast invasion of the spiral arteries during implantation. It is associated with alterations in maternal serum concentrations of vascular endothelial growth factor (VEGF), placental growth factor (PIGF) and soluble fms-like tyrosine kinase-1 (sFlt-1). We did a case-control study to ascertain whether pre-eclampsia is associated with changes in serum concentrations of VEGF, PIGF and sFlt-1 in Indian patients. METHODS: Serum samples were obtained from 40 women with pre-eclampsia and 40 normotensive, non-proteinuric pregnant women. The levels of VEGF, PIGF and sFlt-1 were analysed using ELISA. RESULTS: In the sera of pregnant women with pre-eclampsia, the levels of sFlt-1 were significantly higher than those in the sera of normotensive, non-proteinuric pregnantwomen (median 11295.25 v. 2936.2 pg/ml, p < 0.0001), whereas there was a significant reduction in the levels of free VEGF (mean [SD] 170.53 [36.56] pg/ml v. 254.61 [47.39] pg/ml, p < 0.0001) and PIGF (mean [SD] 236.77 [93.70] pg/ml v. 744.98 [168.55] pg/ml, p < 0.0001). CONCLUSION: An increase in sFlt-1 levels and a simultaneous decrease in free VEGF and PIGF levels in the sera of women with pre-eclampsia as compared with normotensive, nonproteinuric pregnant women suggest that an imbalance between the levels of these pro- and anti-angiogenic factors'may have a role to play in the pathogenesis of pre-eclampsia.