Axillary sentinel lymph node identification using superparamagnetic iron oxide versus radioisotope in early stage breast cancer: The UK SentiMag trial (SMART study).

BACKGROUND & OBJECTIVES: Sentinel lymph node biopsy (SLNB) is an accurate and reliable method for staging the axilla in early breast cancer. The gold standard technique for localizing the sentinel lymph node (SLN) is the use of radioisotope with or without blue dye. However, this technique has its limitations. Various alternatives have been explored to overcome the disadvantages of the standard SLNB technique and superparamagnetic iron oxide mapping agents have garnered significant attention. The SMART study aims to compare the magnetic technique using the superparamagnetic iron oxide particles (SPIO, Sienna+®) to the radioisotope technique (Tc99) +/- blue dye, for SLN identification in patients with early breast cancer. METHODS: A prospective, multicenter study was done that recruited 109 clinically node-negative early-stage breast cancer patients from five centres in the United Kingdom (UK). The patients received radioisotope ± blue dye injections, followed by intraoperative injection of magnetic tracer prior to SLNB. The sentinel node identification rate was compared between the magnetic and standard techniques to evaluate detection rate (per patient and per node), non-inferiority and concordance. RESULTS: Data was analysed for 107 patients. The per patient detection rate was 98.13% (105/107) when using the magnetic tracer and 92.26% (103/107) when using the standard technique. The nodal detection rate was 93.07% (188/202 nodes) when using the magnetic tracer and 96.53% (195/202) when using the standard technique. Of the 31 patients with positive sentinel lymph nodes (SLNs), all 31 (100%) were detected by both techniques. CONCLUSION: Our study demonstrates that the magnetic technique is a feasible method for SLNB, with an identification rate that is not inferior to the standard technique. The magnetic technique offers a suitable alternative to the standard technique thereby avoiding the need for the complexities of nuclear medicine, the hazards of radiation and the anaphylaxis risk of blue dye.

IRF8 and BATF3 interaction enhances the cDC1 specific Pfkfb3 gene expression.

Dendritic cells (DCs) play central role in innate as well as adaptive immune responses regulated by diverse DC subtypes that vary in terms of surface markers, transcriptional profile and functional responses. Generation of DC diversity from progenitor stage is tightly regulated by complex molecular inter-play between transcription factors. We earlier demonstrated that Batf3 and Id2 expression have a synergistic effect on the Irf8 directed classical cDC1 development. In present study, Bi-molecular fluorescence complementation assay suggested that IRF8 interacts with BATF3, and ID2 may aid cDC1 development independently. Genome wide recruitment analysis of IRF8 and BATF3 from different DC subtypes led to identification of the overlapping regions of occupancy by these two transcription factors. Further analysis of overlapping peaks of IRF8 and BATF3 occupancy in promoter region within the cDC1 subtype specific transcriptional pattern identified a metabolically important Pfkfb3 gene. Among various immune cell types; splenic cDC1 subtype displayed enhanced expression of Pfkfb3. Analysis of Irf8-/-, Irf8R294C and Batf3DCKO DC confirmed direct regulation of Pfkfb3 enhanced expression specifically in cDC1 subtype. Further we show that inhibition of PFKFB3 enzymatic activity by a chemical agent PFK15 led to reduction in cDC1 subtype in both in vitro FLDC cultures as well as in vivo mouse spleens. Together, our study identified the direct regulation of cDC1 specific enhanced expression of Pfkfb3 in glycolysis and cDC1 biology.

RelB suppresses type I Interferon signaling in dendritic cells.

Type I Interferon (IFN) signaling plays a critical role in dendritic cell (DC) development and functions. Inhibition of hyper type I IFN signaling promotes cDC2 subtype development. Relb is essential to development of cDC2 subtype and here we analyzed its effect on type I IFN signaling in DCs. We show that Relb suppresses the homeostatic type I IFN signaling in cDC2 cultures. TLR stimulation of FL-DCs led to RelB induction coinciding with fall in IFN signatures; conforming with the observation Relb expression reduced TLR stimulated IFN induction along with decrease in ISGs. Towards understanding mechanism, we show that effects of RelB are mediated by increased levels of IκBα. We demonstrate that RelB dampened antiviral responses by lowering ISG levels and the defect in cDC2 development in RelB null mice can be rescued in Ifnar1-/- background. Overall, we propose a novel role of RelB as a negative regulator of the type I IFN signaling pathway; fine tuning development of cDC2 subtype.

miR-145 supports cancer cell survival and shows association with DDR genes, methylation pattern, and epithelial to mesenchymal transition.

BACKGROUND: Despite several reports describing the dual role of miR-145 as an oncogene and a tumor suppressor in cancer, not much has been resolved and understood. METHOD: In this study, the potential targets of miR-145 were identified bio-informatically using different target prediction tools. The identified target genes were validated in vitro by dual luciferase assay. Wound healing and soft agar colony assay assessed cell proliferation and migration. miR-145 expression level was measured quantitatively by RT-PCR at different stages of breast tumor. Western blot was used to verify the role of miR-145 in EMT transition using key marker proteins. RESULT: Wound healing and soft agar colony assays, using miR-145 over-expressing stably transfected MCF7 cells, unraveled its role as a pro-proliferation candidate in cancerous cells. The association between miR-145 over-expression and differential methylation patterns in representative target genes (DR5, BCL2, TP53, RNF8, TIP60, CHK2, and DCR2) supported the inference drawn. These in vitro observations were validated in a representative set of nodal positive tumors of stage 3 and 4 depicting higher miR-145 expression as compared to early stages. Further, the role of miR-145 in epithelial-mesenchymal (EMT) transition found support through the observation of two key markers, Vimentin and ALDL, where a positive correlation with Vimentin protein and a negative correlation with ALDL mRNA expression were observed. CONCLUSION: Our results demonstrate miR-145 as a pro-cancerous candidate, evident from the phenotypes of aggressive cellular proliferation, epithelial to mesenchymal transition, hypermethylation of CpG sites in DDR and apoptotic genes and upregulation of miR-145 in later stages of tumor tissues.

Cutting Edge: ACVRL1 Signaling Augments CD8α+ Dendritic Cell Development.

Dendritic cells (DCs) are a collection of different subtypes, each of which is characterized by specific surface markers, gene-expression patterns, and distinct functions. Members of the IFN regulatory factor family play critical roles in DC development and functions. Recently, Irf8 was shown to activate TGF-ß signaling, which led to exacerbated neuroinflammation in the experimental autoimmune encephalomyelitis mouse model. We analyzed the effect of Irf8 on TGF-ß/bone morphogenetic protein pathway-specific genes in DCs and identified Acvrl1, a type I TGF-ß superfamily receptor, as a gene strongly induced by Irf8 expression. Among various DC subtypes, Acvrl1 is differentially expressed in CD8α(+) DCs. ACVRL1 signaling augmented Irf8-directed classical CD8α(+) DC development. Irf8 expression is essential for plasmacytoid DC and CD8α(+) DC development, and this study demonstrates that ACVRL1 signaling plays a pivotal role whereby it suppresses plasmacytoid DC development while enhancing that of CD8α(+) DCs, thus contributing to DC diversity development.

Batf3 and Id2 have a synergistic effect on Irf8-directed classical CD8α+ dendritic cell development.

Dendritic cells (DCs) are heterogeneous cell populations represented by different subtypes, each varying in terms of gene expression patterns and specific functions. Recent studies identified transcription factors essential for the development of different DC subtypes, yet molecular mechanisms for the developmental program and functions remain poorly understood. In this study, we developed and characterized a mouse DC progenitor-like cell line, designated DC9, from Irf8(-/-) bone marrow cells as a model for DC development and function. Expression of Irf8 in DC9 cells led to plasmacytoid DCs and CD8α(+) DC-like cells, with a concomitant increase in plasmacytoid DC- and CD8α(+) DC-specific gene transcripts and induction of type I IFNs and IL12p40 following TLR ligand stimulation. Irf8 expression in DC9 cells led to an increase in Id2 and Batf3 transcript levels, transcription factors shown to be important for the development of CD8α(+) DCs. We show that, without Irf8, expression of Id2 and Batf3 was not sufficient for directing classical CD8α(+) DC development. When coexpressed with Irf8, Batf3 and Id2 had a synergistic effect on classical CD8α(+) DC development. We demonstrate that Irf8 is upstream of Batf3 and Id2 in the classical CD8α(+) DC developmental program and define the hierarchical relationship of transcription factors important for classical CD8α(+) DC development.

Role of miRNAs in Brain Development.

Non-coding RNAs that are small in size, called microRNAs (miRNAs), exert a conse-quence in neutralizing gene activity after transcription. The nervous system is a massively ex-pressed organ, and an expanding body of research reveals the vital functions that miRNAs play in the brain's growth and neural activity. The significant benefit of miRNAs on the development of the central nervous system is currently shown through new scientific methods that concentrate on targeting and eradicating vital miRNA biogenesis pathways the elements involving Dicer and DGCR8. Modulation of miRNA has been associated with numerous essential cellular processes on neural progenitors, like differentiation, proliferation, and destiny determination. Current re-search discoveries that emphasize the significance of miRNAs in the complex process of brain development are included in this book. The miRNA pathway plays a major role in brain devel-opment, its operational dynamics, and even diseases. Recent studies on miRNA-mediated gene regulation within neural discrepancy, the circadian period and synaptic remodeling are signs of this. We also discussed how these discoveries may affect our comprehension of the fundamental processes behind brain diseases, highlighting the novel therapeutic opportunities miRNAs pro-vide for treating various human illnesses.

An optimised patient-derived explant platform for breast cancer reflects clinical responses to chemotherapy and antibody-directed therapy.

Breast Cancer is the most common cancer among women globally. Despite significant improvements in overall survival, many tumours are refractory to therapy and so novel approaches are required to improve patient outcomes. We have evaluated patient-derived explants (PDEs) as a novel preclinical platform for breast cancer (BC) and implemented cutting-edge digital pathology and multi-immunofluorescent approaches for investigating biomarker changes in both tumour and stromal areas at endpoint. Short-term culture of intact fragments of BCs as PDEs retained an intact immune microenvironment, and tumour architecture was augmented by the inclusion of autologous serum in the culture media. Cell death/proliferation responses to FET chemotherapy in BC-PDEs correlated significantly with BC patient progression-free survival (p = 0.012 and p = 0.0041, respectively) and cell death responses to the HER2 antibody therapy trastuzumab correlated significantly with HER2 status (p = 0.018). These studies show that the PDE platform combined with digital pathology is a robust preclinical approach for informing clinical responses to chemotherapy and antibody-directed therapies in breast cancer. Furthermore, since BC-PDEs retain an intact tumour architecture over the short-term, they facilitate the preclinical testing of anti-cancer agents targeting the tumour microenvironment.

tREPs-A New Class of Functional tRNA-Encoded Peptides.

We asked if transfer RNA (tRNA) ever got an opportunity of translating its own sequence during evolution, what would have been the function of such tRNA-encoded peptides (tREPs)? If not, could one artificially synthesize tREPs to study the corresponding functional outcomes? Here, we report a novel, first-in-the-class, chemically synthesized tREP-18 molecule originating from the Escherichia coli tRNA sequence showing potent antileishmanial property. As a first step, E. coli tRNAs were computationally translated into peptide sequence equivalents and a database of full-length hypothetical tREPs was created. The tREP sequences were sent into sequence, structure, and energy filters to narrow down potential peptides for experimental validation. Based on the functional predictions, tREPs were screened against antiparasitic targets, leading to the identification of tREP-18 as a potential antiparasitic peptide. The in vitro assay of chemically synthesized tREP-18 on the Ag83 strain of Leishmania donovani showed its potent antileishmanial property (IC50 value of 22.13 nM). The atomic force microscopy and scanning electron microscopy images indicated significant alteration in the cytoskeletal architecture of tREP-18-treated parasites. Also, tREP-18 seems to destabilize the mitochondrial membrane potential of parasites, disrupting their cellular integrity and leading to parasitic death. The cellular assays of the tREP-18 peptide on the BS12 strain, a clinical isolate of post-kala azar dermal leishmaniasis, demonstrated its significant efficacy at an IC50 value of 15 nM. The tREP-18 peptide showed a toxic effect on the amastigote stage of the parasite, showing macrophage pathogen clearance at a concentration of 22.5 nM. This study provides the proof of the concept of making a new class of functional peptides from tRNA sequences. It also opens a huge untapped tRNA-peptide space toward novel discoveries and applications. In the future, it would be interesting to perform tREP edits and redesign tREPs toward specific applications.

Recent Progress in Development of Dressings Used for Diabetic Wounds with Special Emphasis on Scaffolds.

Diabetic wound (DW) is a secondary application of uncontrolled diabetes and affects about 42.2% of diabetics. If the disease is left untreated/uncontrolled, then it may further lead to amputation of organs. In recent years, huge research has been done in the area of wound dressing to have a better maintenance of DW. These include gauze, films, foams or, hydrocolloid-based dressings as well as polysaccharide- and polymer-based dressings. In recent years, scaffolds have played major role as biomaterial for wound dressing due to its tissue regeneration properties as well as fluid absorption capacity. These are three-dimensional polymeric structures formed from polymers that help in tissue rejuvenation. These offer a large surface area to volume ratio to allow cell adhesion and exudate absorbing capacity and antibacterial properties. They also offer a better retention as well as sustained release of drugs that are directly impregnated to the scaffolds or the ones that are loaded in nanocarriers that are impregnated onto scaffolds. The present review comprehensively describes the pathogenesis of DW, various dressings that are used so far for DW, the limitation of currently used wound dressings, role of scaffolds in topical delivery of drugs, materials used for scaffold fabrication, and application of various polymer-based scaffolds for treating DW.

Carbon monoxide protects against ischemia-reperfusion injury in an experimental model of controlled nonheartbeating donor kidney.

BACKGROUND: CO-releasing molecule-3 (CORM-3) is a transitional metal carbonyl that liberates carbon monoxide under appropriate conditions. Carbon monoxide exerts effects on intracellular apoptotic and inflammatory pathways, which suggest a role in reducing the effects of renal ischemia/reperfusion (I/R) injury. This study investigated the effects of CORM-3 administered at the time of reperfusion in a model of controlled nonheartbeating donor kidneys. METHODS: Porcine kidneys (n=4) were subjected to 10 min warm ischemia and 18 hr cold storage (CS) and then treated as follows: CORM-3 (50, 100, 200, and 400 microM doses), iCORM-3 (inactive carbon monoxide-releasing molecule, 50 microM), and control (no further intervention). Renal hemodynamics and function were then measured during 3-hr reperfusion with autologous blood using an isolated organ-perfusion system. RESULTS: CORM-3 at a concentration of 50 microM improved renal blood flow (RBF) compared with the iCORM and control groups (area under the curve 774+/-19 vs. 448+/-88 vs. 325+/-70, respectively, P=0.002). CO-releasing molecule-3 at a concentration of 50 microM also improved renal function during reperfusion with a greater area under the curve for creatinine clearance (CORM-3: 14+/-6 vs. iCORM: 3.3+/-0.1 vs. control: 2.2+/-2 mL/min, P=0.006) and higher urine output (CORM-3: 793+/-212 vs. iCORM: 368+/-72 vs. control: 302+/-211 mL, P=0.01). CO-releasing molecule-3 at a concentration of 100 microM exerted similar effects. Treatment with CORM-3 at higher doses (200 and 400 microM) led to poor renal hemodynamics and function after reperfusion. CONCLUSION: Low-dose CORM-3 significantly ameliorates the effects of ischemia/reperfusion in a porcine model of controlled nonheartbeating donor kidney transplantation.

Deciphering the role of microRNA - A step by step guide.

MicroRNAs (miRNAs), are small non-coding RNAs of approximately 22 nucleotides in length, playing an important role in regulating gene expression post-transcriptionally. Understanding the effect of miRNA regulation in a pathway-specific manner unravels the approaches adopted to apprehend biological mechanisms, the information, which is scanty for researchers, not primed already for miR related research. Here, we describe a quick perspective in 5 steps with probable approaches and assays at every level to unravel the specific role of a microRNA, miR-145a-5p, as an example. This perspective as a guide would help in identifying novel targets for a microRNA, as shown for miR-145a-5p, which down-regulated the mRNA expression of ADD3 and BRCA2, using bioinformatic tools and experimental assays.

Biomarkers of oxidative damage to predict ischaemia-reperfusion injury in an isolated organ perfusion model of the transplanted kidney.

Ischaemia-reperfusion (IR) injury is known to be a risk factor influencing both short and long-term graft function following transplantation. The pathophysiology of IR injury is suggested to involve elevated reactive oxygen species production resulting in oxidative damaged cellular macromolecules. The objective of this study was to evaluate oxidative damage following IR using an isolated organ perfusion model of the transplanted kidney, in order to determine a simple, preferably non-invasive biomarker for IR injury. Porcine kidneys were retrieved with 10 or 40 min warm ischaemic (WI) time and haemoperfused for 6 h on an isolated organ perfusion machine. ELISA was used to detect carbonyls, 8-isporostane and 8-hydroxy-2'-deoxyguanosine, representing protein, lipid and DNA damage respectively in pre and post reperfusion samples of plasma, urine and biopsy material. Plasma carbonyl and 8-isporostane and were significantly increased in the 40 min group compared to pre-perfusion (0.96 +/- 0.10 vs. 0.62 +/- 0.06, P < 0.001 and 1.57(1.28-4.9) vs. 0.36(0.09-0.59), P < 0.05). The levels also correlated with creatinine clearance used to determine renal function (r = - 0.6150, P < 0.01 and r = - 0.7727, P < 0.01). The results of this study suggest both plasma carbonyl and 8-isporostane to be reliable biomarkers to predict the level IR injury.

Function annotation of peptides generated from the non-coding regions of D. melanogaster genome.

De novo emergence of genes is the most fundamental form of genetic diversity that is attracting the attention of the scientific community. Identification of short open reading frames (sORFs) from the non-coding regions of different genomes has been leading this thought recently. The coding potential of these newly identified sORFs have been investigated through experimental and computational approaches in recent studies. In the present work we have tried to make peptides from intergenic sequences of D. melanogaster genome leading to therapeutic applications. Towards this goal of making novel peptides from non-coding genome, we have found strong computational evidence of 145 peptides with conformational stability from the intergenic sequences of D. melanogaster. The structure of these completely unique peptides was predicted using ab initio method. The function annotation of these peptides was carried out using this structural information. The newly generated proteins were categorised as DNA/Protein/ion binding proteins, electron transporters and a very few as enzymes too. Experimental studies can certainly provide validations to these preliminary findings. This work provides further evidence of untapped potential of non-coding genome.

Fish bone perforation of the terminal ileum presenting as acute appendicitis.

We report a case of a bowel perforation due to a fish bone that presented as an acute abdomen. This patient's gastrointestinal perforation was treated with laparoscopic and open technique. Diagnosis can be difficult as foreign body bowel perforation can mimic other causes of acute abdomen. Diagnosis is still most commonly made intraoperatively. Laparoscopy proved useful in this case as it allowed the most appropriate surgical approach to be made.

Health-related quality of life after living donor nephrectomy: a randomized controlled trial of laparoscopic versus open nephrectomy.

BACKGROUND: The aim of this study was to compare patient-reported health status and quality of life after randomization to laparoscopic donor nephrectomy (LDN) or short-incision open donor nephrectomy (ODN). METHODS: Live kidney donors were randomized in a 2:1 ratio to LDN (n=56) or ODN (n=28). Health-related quality of life was assessed using the Short Form 36 questionnaire preoperatively and at 6 weeks postdonation. RESULTS: Postoperative morphine requirement was lower in the LDN group (median [range], 59 [6-136]) versus ODN group (90 [35-312] mg; P=0.001). Norm adjusted physical components scores decreased significantly at 6 weeks in both the LDN and ODN groups. The bodily pain domain score of physical components score at 6 weeks returned to baseline in the laparoscopic group (86.4±19.8 vs. 81.8±15.9; P=0.2277) but not in the open group (87.3±18.3 vs. 69.0±25.0; P=0.05). The mental component score decreased in the ODN group (53.5±7.6 vs. 45.3±10.1; P=0.0084) but returned to baseline 6 weeks after LDN (53.8±6.5 vs. 51.9±7.2; P=0.2931). CONCLUSIONS: Donors undergoing laparoscopic nephrectomy reported less bodily pain in the first 6 weeks postdonation, and this was associated with an improved mental health component of quality of life compared with ODN (51.9±7.2 vs. 45.3±10.1; P=0.0009).

Effects of erythropoietin on ischaemia/reperfusion injury in a controlled nonheart beating donor kidney model.

Erythropoietin (EPO) has been shown to have an anti-apoptotic action and has the potential to protect against ischaemia/reperfusion injury. This study investigated the effect of high dose EPO (5000 U), administered as a bolus at the onset of reperfusion and at the onset of cold storage in a model of controlled nonheart beating donors kidneys. Porcine kidneys(n = 6) were subjected to 10min warm ischaemia and preserved as follows: Group 1:16 h Cold storage +2 h Normothermic perfusion (16 h CS + 2 h NP) Group 2:16 h CS + 2 h NP + EPO given at the onset of reperfusion Group 3:18 h CS (static hypothermic storage) Group 4:18 h CS + EPO given at the onset of cold storage Haemodynamic and functional parameters were assessed during 3-h reperfusion using autologous blood. Renal blood flow improved in Groups 1 and 2 vs. Groups 3 and 4 though no difference was noted between Groups 3 and 4 (563 +/- 119 vs. 491 +/- 95 vs. 325 +/- 70 vs. 418 +/- 112, respectively; P = 0.012). Total urine output showed no difference between Groups (271 +/- 172 vs. 359 +/- 184 vs. 302 +/- 21 vs. 421 +/- 88; P = 0.576). Percentage serum creatinine fall at 3 h was significantly better in Groups 1 and 2 vs. Group 3 (64 +/- 17 vs. 60 +/- 11 vs. 44 +/- 13 vs. 52 +/- 8; P = 0.04). Fractional-excretion of sodium was significantly lower for Groups 1 and 2 vs. Group 3 and 4 (17 +/- 14 vs. 18 +/- 9 vs. 49 +/- 21 vs. 45 +/- 16 respectively; P = 0.002). There was significant improvement in oxygen consumption in Groups 2 vs. Groups 3 and 4 (P = 0.037) (39 +/- 10 vs. 46 +/- 10 vs. 24 +/- 12 vs. 24 +/- 7 respectively). EPO added at the time of reperfusion improved oxygen consumption when added to NP in comparison to static hypothermic storage but did not exert any other major benefits.

Differential expression of cytoprotective and apoptotic genes in an ischaemia-reperfusion isolated organ perfusion model of the transplanted kidney.

The optimal kidney preservation system and methods to ameliorate reperfusion injury are major factors in accomplishing successful graft function following transplantation. Ischaemia and reperfusion lead to cellular stress and the adaptive response may include the activation of genes involved in cellular protection and/or cell death by apoptosis. We investigated the expression of cytoprotective heme oxygenase-1 (HO-1), anti-apoptotic Bcl-2 and pro-apoptotic Bax after 6 h isolated organ perfusion in porcine kidneys that had been given 10 and 40 min warm ischaemic time. The level of HO-1 was shown to be significantly higher in the 10-min warm ischaemic group compared with 40-min group (0.90 +/- 0.03 vs. 0.83 +/- 0.03; P = 0.002). The levels of HO-1 showed a significant positive correlated with parameters of renal function, creatinine clearance, and renal blood flow and urine output (AUC; r = 0.8042, P = 0.03; r = 0.6028, P = 0.04; r = 0.6055, P = 0.04), demonstrating a possible protective role of this gene in this model of renal transplantation.