Hypoxic Memory Mediates Prolonged Tumor-Intrinsic Type I Interferon Suppression to Promote Breast Cancer Progression.

Hypoxia is a common feature of many solid tumors due to aberrant proliferation and angiogenesis that is associated with tumor progression and metastasis. Most of the well-known hypoxia effects are mediated through hypoxia-inducible factors (HIF). Identification of the long-lasting effects of hypoxia beyond the immediate HIF-induced alterations could provide a better understanding of hypoxia-driven metastasis and potential strategies to circumvent it. Here, we uncovered a hypoxia-induced mechanism that exerts a prolonged effect to promote metastasis. In breast cancer patient-derived circulating tumor cell lines and common breast cancer cell lines, hypoxia downregulated tumor-intrinsic type I IFN signaling and its downstream antigen presentation (AP) machinery in luminal breast cancer cells, via both HIF-dependent and HIF-independent mechanisms. Hypoxia induced durable IFN/AP suppression in certain cell types that was sustained after returning to normoxic conditions, presenting a "hypoxic memory" phenotype. Hypoxic memory of IFN/AP downregulation was established by specific hypoxic priming, and cells with hypoxic memory had an enhanced ability for tumorigenesis and metastasis. Overexpression of IRF3 enhanced IFN signaling and reduced tumor growth in normoxic, but not hypoxic, conditions. The histone deacetylase inhibitor entinostat upregulated IFN targets and erased the hypoxic memory. These results point to a mechanism by which hypoxia facilitates tumor progression through a long-lasting memory that provides advantages for circulating tumor cells during the metastatic cascade. Significance: Long-term cellular memory of hypoxia leads to sustained suppression of tumor-intrinsic type I IFN signaling and the antigen presentation pathway that facilitates tumorigenesis and metastasis. See related commentary by Purdy and Ford, p. 3125.

Ectopic Expression of a Truncated Isoform of Hair Keratin 81 in Breast Cancer Alters Biophysical Characteristics to Promote Metastatic Propensity.

Keratins are an integral part of cell structure and function. Here, it is shown that ectopic expression of a truncated isoform of keratin 81 (tKRT81) in breast cancer is upregulated in metastatic lesions compared to primary tumors and patient-derived circulating tumor cells, and is associated with more aggressive subtypes. tKRT81 physically interacts with keratin 18 (KRT18) and leads to changes in the cytosolic keratin intermediate filament network and desmosomal plaque formation. These structural changes are associated with a softer, more elastically deformable cancer cell with enhanced adhesion and clustering ability leading to greater in vivo lung metastatic burden. This work describes a novel biomechanical mechanism by which tKRT81 promotes metastasis, highlighting the importance of the biophysical characteristics of tumor cells.

The Effect of Non-Pharmacological and Pharmacological Interventions on Measures Associated with Sarcopenia in End-Stage Kidney Disease: A Systematic Review and Meta-Analysis.

This systematic review and meta-analysis provides a synthesis of the available evidence for the effects of interventions on outcome measures associated with sarcopenia in end-stage kidney disease (ESKD). Thirteen databases were searched, supplemented with internet and hand searching. Randomised controlled trials of non-pharmacological or pharmacological interventions in adults with ESKD were eligible. Trials were restricted to those which had reported measures of sarcopenia. Primary outcome measures were hand grip strength and sit-to-stand tests. Sixty-four trials were eligible (with nineteen being included in meta-analyses). Synthesised data indicated that intradialytic exercise increased hand grip strength (standardised mean difference, 0.58; 0.24 to 0.91; p = 0.0007; I2 = 40%), and sit-to-stand (STS) 60 score (mean difference, 3.74 repetitions; 2.35 to 5.14; p < 0.001; I2 = 0%). Intradialytic exercise alone, and protein supplementation alone, resulted in no statistically significant change in STS5 (−0.78 s; −1.86 to 0.30; p = 0.16; I2 = 0%), and STS30 (MD, 0.97 repetitions; −0.16 to 2.10; p = 0.09; I2 = 0%) performance, respectively. For secondary outcomes, L-carnitine and nandrolone-decanoate resulted in significant increases in muscle quantity in the dialysis population. Intradialytic exercise modifies measures of sarcopenia in the haemodialysis population; however, the majority of trials were low in quality. There is limited evidence for efficacious interventions in the peritoneal dialysis and transplant recipient populations.

Associations between physical activity levels and renal recovery following acute kidney injury stage 3: a feasibility study.

BACKGROUND: Acute kidney injury (AKI) can lead to chronic kidney disease, which results in poor long-term outcomes. There is plausibility that increased levels of physical activity may promote renal recovery post-AKI. This study aimed to investigate associations between physical activity levels and renal recovery following stage 3 AKI, and to assess the feasibility of measuring physical activity levels in this population. METHODS: Forty One hospitalised patients with AKI stage 3 were enrolled. Serum creatinine and estimated glomerular filtration rate (eGFR) were collected at 12 months prior to the development of AKI, during the hospital admission when the episode of AKI stage 3 occurred, and at 1-, 3- and 6-months post discharge. All participants completed the General Practice Activity Questionnaire (GPPAQ) to assess physical activity levels. A pedometer was also worn for 7 days immediately following discharge and at 6-months post discharge to ascertain an average daily step count. Feasibility outcomes including eligibility, recruitment and retention rates, and losses to follow up were also assessed. RESULTS: The average (± SD) baseline eGFR and median (IQR) serum creatinine was 71 ± 20 mL/min/1.73m2 and 85 (49) µmol/L respectively. A threefold increase in creatinine occurred during hospitalisation 436 (265) µmol/L. Greatest renal recovery occurred prior to discharge, with recovery continuing for a further three months. Inactive individuals (low GPPAQ scores) had consistently higher serum creatinine values compared to those who were active: 1 months 122 (111) µmol/L vs 70 (0) µmol/L, 6 months 112 (57) µmol/L vs 68 (0) µmol/L. Individuals with higher step counts also displayed better renal recovery 6-months post discharge (r = -0.600, p = 0.208). CONCLUSIONS: Higher levels of physical activity are associated with improved renal recovery after 6- months following an episode of stage 3 AKI. A future randomised controlled trial is feasible and would be required to confirm these initial findings.

N-Acetyl-L-cysteine Promotes Ex Vivo Growth and Expansion of Single Circulating Tumor Cells by Mitigating Cellular Stress Responses.

Circulating tumor cells (CTC) can be isolated via a minimally invasive blood draw and are considered a "liquid biopsy" of their originating solid tumors. CTCs contain a small subset of metastatic precursors that can form metastases in secondary organs and provide a resource to identify mechanisms underlying metastasis-initiating properties. Despite technological advancements that allow for highly sensitive approaches of detection and isolation, CTCs are very rare and often present as single cells, posing an extreme challenge for ex vivo expansion after isolation. Here, using previously established patient-derived CTC lines, we performed a small-molecule drug screen to identify compounds that can improve ex vivo culture efficiency for single CTCs. We found that N-acetyl-L-cysteine (NAC) and other antioxidants can promote ex vivo expansion of single CTCs, by reducing oxidative and other stress particularly at the initial stage of single-cell expansion. RNA-seq analysis of growing clones and nongrowing clones confirmed the effect by NAC, but also indicates that NAC-induced decrease in oxidative stress is insufficient for promoting proliferation of a subset of cells with predominant senescent features. Despite the challenge in expanding all CTCs, NAC treatment led to establishment of single CTC clones that have similar tumorigenic features. IMPLICATIONS: Through a small molecule screen and validation study, we found that NAC could improve the success of ex vivo expansion of single CTCs by mitigating the initial stress, with the potential to facilitate the investigation of functional heterogeneity in CTCs.

Peripheral deficiency and antiallodynic effects of 2-arachidonoyl glycerol in a mouse model of paclitaxel-induced neuropathic pain.

BACKGROUND: Modulation of the endocannabinoid system has been shown to alleviate neuropathic pain. The aim of this study was to evaluate if treatment with paclitaxel, a chemotherapeutic agent that induces neuropathic pain, affects endocannabinoid levels at a time when mice develop paclitaxel-induced mechanical allodynia. We also evaluated the peripheral antiallodynic activity of the endocannabinoid 2-arachidonoyl glycerol (2-AG) and an inhibitor of monoacylglycerol lipase (MAGL), an enzyme responsible for 2-AG hydrolysis. METHODS: Female BALB/c mice were treated intraperitoneally with paclitaxel to induce mechanical allodynia. Levels of the endocannabinoids, N-arachidonoylethanolamine (anandamide, AEA), 2-AG, and the N-acylethanolamines (NAEs), N-palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA), which are structurally-related to AEA, in the brain, spinal cord and paw skin were measured using LC-MS/MS. Protein expression of MAGL in the paw skin was measured using Wes™. The effects of subcutaneous (s.c.) injection of 2-AG and JZL184 (a MAGL inhibitor) into the right hind paw of mice with paclitaxel-induced mechanical allodynia were assessed using the dynamic plantar aesthesiometer. The effects of pretreatment, s.c., into the right hind paw, with cannabinoid type 1 (CB1) receptor antagonist AM251 and CB2 receptor antagonist AM630 on the antiallodynic effects of 2-AG were also evaluated. RESULTS: The levels of 2-AG were reduced only in the paw skin of paclitaxel-treated mice, whilst the levels of AEA, PEA and OEA were not significantly altered. There was no change in the expression of MAGL in the paw skin. Administration of 2-AG and JZL184 produced antiallodynic effects against paclitaxel-induced mechanical allodynia in the injected right paw, but did not affect the uninjected left paw. The antiallodynic activity of 2-AG was antagonized by both AM251 and AM630. CONCLUSION: These results indicate that during paclitaxel-induced mechanical allodynia there is a deficiency of 2-AG in the periphery, but not in the CNS. Increasing 2-AG in the paw by local administration of 2-AG or a MAGL inhibitor, alleviates mechanical allodynia in a CB1 and CB2 receptor-dependent manner.

Circulating Tumor Cells Exhibit Metastatic Tropism and Reveal Brain Metastasis Drivers.

Hematogenous metastasis is initiated by a subset of circulating tumor cells (CTC) shed from primary or metastatic tumors into the blood circulation. Thus, CTCs provide a unique patient biopsy resource to decipher the cellular subpopulations that initiate metastasis and their molecular properties. However, one crucial question is whether CTCs derived and expanded ex vivo from patients recapitulate human metastatic disease in an animal model. Here, we show that CTC lines established from patients with breast cancer are capable of generating metastases in mice with a pattern recapitulating most major organs from corresponding patients. Genome-wide sequencing analyses of metastatic variants identified semaphorin 4D as a regulator of tumor cell transmigration through the blood-brain barrier and MYC as a crucial regulator for the adaptation of disseminated tumor cells to the activated brain microenvironment. These data provide the direct experimental evidence of the promising role of CTCs as a prognostic factor for site-specific metastasis. SIGNIFICANCE: Interests abound in gaining new knowledge of the physiopathology of brain metastasis. In a direct metastatic tropism analysis, we demonstrated that ex vivo-cultured CTCs from 4 patients with breast cancer showed organotropism, revealing molecular features that allow a subset of CTCs to enter and grow in the brain.This article is highlighted in the In This Issue feature, p. 1.

Dissecting macromolecular recognition sites in ribosome: implication to its self-assembly.

Interactions between macromolecules play a crucial role in ribosome assembly that follows a highly coordinated process involving RNA folding and binding of ribosomal proteins (r-proteins). Although extensive studies have been carried out to understand macromolecular interactions in ribosomes, most of them are confined to either large or small ribosomal-subunit of few species. A comparative analysis of macromolecular interactions across different domains is still missing. We have analyzed the structural and physicochemical properties of protein-protein (PP), protein-RNA (PR) and RNA-RNA (RR) interfaces in small and large subunits of ribosomes, as well as in between the two subunits. Additionally, we have also developed Random Forest (RF) classifier to catalog the r-proteins. We find significant differences as well as similarities in macromolecular recognition sites between ribosomal assemblies of prokaryotes and eukaryotes. PR interfaces are substantially larger and have more ionic interactions than PP and RR interfaces in both prokaryotes and eukaryotes. PP, PR and RR interfaces in eukaryotes are well packed compared to those in prokaryotes. However, the packing density between the large and the small subunit interfaces in the entire assembly is strikingly low in both prokaryotes and eukaryotes, indicating the periodic association and dissociation of the two subunits during the translation. The structural and physicochemical properties of PR interfaces are used to predict the r-proteins in the assembly pathway into early, intermediate and late binders using RF classifier with an accuracy of 80%. The results provide new insights into the classification of r-proteins in the assembly pathway.

Declining interest in general surgical training - Challenging misconceptions and improving access at undergraduate level.

BACKGROUND: In recent years, general surgery has faced a decline in applicants for postgraduate training. The St. George's Surgical Society hosted a national surgical conference with an aim to explore the reasons for the decline and to investigate the effectiveness of a one-day intervention on students' perceptions of general surgery and surgical skills. MATERIALS AND METHODS: The conference took place on 20th January 2018 at St. George's, University of London. During the conference, medical students received lectures on "careers in surgery" and small-group workshops introducing students to simulation-based laparoscopic machines. Delegates were invited to complete before and after questionnaires looking at various domains; (1) Perceptions of general surgery, (2) Simulation skills, and (3) Usefulness of the day. RESULTS: There were significant impacts on student perceptions of the speciality such as increases in the views that "general surgery contained the opportunities for personal and professional development" (26%, p = 0.04), and of "general surgery as a rewarding speciality" (26%, p = 0.05). There were also negative changes such as an increase in the perception that "general surgery is female unfriendly" (+32%, p = 0.01). There were positive findings in all aspects relating to the use of laparoscopic simulation. DISCUSSION: The challenges faced in improving access to general surgery has been highlighted. More importantly the benefits of a one day intervention in addressing misconceptions and improving knowledge was seen. This study also shows the benefits of simulation teaching in the undergraduate curriculum. CONCLUSION: The intervention has improved the perceptions on general surgery, surgical skills and knowledge and provided a platform for engaging students and clinicians.

Indomethacin plus minocycline coadministration relieves chemotherapy and antiretroviral drug-induced neuropathic pain in a cannabinoid receptors-dependent manner.

Neuropathic pain sometimes occurs during chemotherapy with paclitaxel or HIV/AIDS antiretroviral therapy with nucleoside reverse transcriptase inhibitors (NRTIs). We previously reported that coadministration of indomethacin plus minocycline (IPM) was antihyperalgesic in a cannabinoid type 1 (CB1) receptor-dependent manner in a mouse model of paclitaxel-induced neuropathic pain. We evaluated if IPM combination has antihyperalgesic and antiallodynic activities in animal models of paclitaxel or NRTI (ddC, zalcitabine)-induced neuropathic pain, and whether antagonists of CB1, CB2 receptors or G protein-coupled receptor 55 (GPR55) can inhibit these activities of IPM. IPM produced antihyperalgesic and antiallodynic effects against paclitaxel and ddC-induced thermal hyperalgesia and mechanical allodynia. WIN 55,212-2, a cannabinoid receptor agonist, also had antihyperalgesic activity. The antihyperalgesic and antiallodynic activities of IPM were antagonized by a CB1 receptor antagonist AM251 and a CB2 receptor antagonist AM630, but not a GPR55 antagonist ML193. IPM had no effects on the mean time spent on the rotarod, whereas WIN 55,212-2 reduced it in a dose-dependent manner. These results show that IPM at a fixed ratio produces antihyperalgesic and antiallodynic effects in mice models of both paclitaxel and NRTI-induced neuropathic pain which is dependent on both CB1 and CB2 receptors, without causing the typical cannabinoid receptor agonist-induced motor impairment.

Vascular surgery: What increases the flow of students to the speciality?

OBJECTIVES: Vascular surgery is in increasing demand due to the higher prevalence of cardiovascular disease in the general population. Meanwhile, applications to join the speciality have been in decline in the last few years. St George's Surgical Society held a one-day conference to expose undergraduates to the speciality in the hope that this will inspire more undergraduates to pursue it as a career. METHODS: The conference consisted of introductory lectures delivered by a consultant vascular surgeon, followed by practical skills workshops simulating aortic anastomosis on porcine aortas. Pre-course and post-course questionnaires assessed positive and negative perceptions of vascular surgery, knowledge of and self-reported confidence in practical surgical skills and utility of the course in their decision to potentially pursue a career in vascular surgery. RESULTS: There was a significant increase of 27% ( p = 0.03) in the positive perception that vascular surgery includes a diverse range of procedures and subspecialties. Attendees reported a 27% significant increase ( p = 0.02) in the perception that vascular surgery had a wide array of opportunity for academic/research work. Finally, there was an 18% decrease ( p = 0.03) in the negative perception that vascular surgery is somehow female-unfriendly or discriminatory against females. There was also a significant rise in both interest (33%) and understanding (73%) of vascular surgery. CONCLUSIONS: A one-day conference can significantly impact students' perception especially when there is a lack of exposure within the undergraduate curriculum. This course helped to increase positive perception and dispute negative misconceptions about the speciality. The simulation workshops held at this conference increased students' confidence and awareness of relevant surgical skills. This conference provided a unique experience that positively impacted and inspired students and thus served as an important supplement to core medical curriculum.

Reliability evaluation of inter-eminence line, Akagi and Dalury lines for intraoperative tibial rotation: An osteology-based study.

BACKGROUND: This large osteology study examined the reliability, reproducibility and correlation between previously described tibial tray rotation alignment lines (including Akagi and Dalury lines). In addition, it described a novel inter-eminence line utilising the tibial plateau inter-condylar eminences as a landmark. METHODS: A total of 214 post-medieval (18-19th centuries) skeletal tibia were examined. The inter/intra-observer variation and correlation between reference lines were measured. RESULTS: Inter-observer reproducibility was excellent and there were no differences between Akagi, Dalury, and inter-eminence lines. Similarly, intra-observer reliability was excellent for Akagi, Dalury, and inter-eminence lines. Qualitative review of tibial inter-condylar eminences suggested that these could be easily identifiable. When taking the medial angle from a medial-lateral reference line, the Akagi line showed a mean of 96.90° (±10.27), inter-eminence line 94.52° (±12.84), and Dalury line 88.06° (±11.75). The angle produced by the Dalury line was significantly different from both the Akagi and inter-eminence lines (P≤0.001). The Akagi line and inter-eminence line showed a strong correlation (r=0.74). The Dalury line showed a weaker correlation with both the Akagi line (r=0.69) and inter-eminence line (r=0.40). CONCLUSION: This study suggested that tibial rotation lines showed excellent intra/inter-observer reliability and reproducibility. The novel and easily drawn inter-eminence line showed strong correlation with the Akagi line and could be used for tibial tray rotational alignment in total knee arthroplasty.

Enhancing Medical Curricula: The Role of a 1-Day Plastic Surgery Course as an Educational Adjunct for Medical Students.

INTRODUCTION: Plastic surgery is underrepresented in medical school curricula. The St George׳s Surgical Society hosted a 1-day undergraduate course in plastic surgery. Our aim was to introduce students to plastic surgery and teach basic plastic surgical skills. METHODOLOGY: The skills day consisted of lectures from consultants and a core trainee followed by registrar-taught workshops in suturing, tendon repair, and local flap design. Precourse and postcourse questionnaires assessed perceptions of plastic surgery, confidence in performing basic plastic surgical skills, and usefulness of course components. RESULTS: Many perceptions of plastic surgery saw statistically significant changes. The belief that plastic surgery covers a wide range of specialties increased by 36% (p = 0.01). A -32% change (p = 0.00) was seen in the perception of plastic surgery only being pursued as a career for financial gain. Delegates reported greater confidence in all components of basic plastic surgical skills. The most useful activity was reported as performing surgical skills on real tissue. CONCLUSION: A 1-day course can significantly increase positive perceptions of plastic surgery, dispel preconceived false stereotypes, while improving student confidence in performing basic plastic surgery skills. It is important that university surgical societies provide opportunities for student education, to aid informed decisions about future careers.

Genome-wide identification of miRNAs and lncRNAs in Cajanus cajan.

BACKGROUND: Non-coding RNAs (ncRNAs) are important players in the post transcriptional regulation of gene expression (PTGR). On one hand, microRNAs (miRNAs) are an abundant class of small ncRNAs (~22nt long) that negatively regulate gene expression at the levels of messenger RNAs stability and translation inhibition, on the other hand, long ncRNAs (lncRNAs) are a large and diverse class of transcribed non-protein coding RNA molecules (> 200nt) that play both up-regulatory as well as down-regulatory roles at the transcriptional level. Cajanus cajan, a leguminosae pulse crop grown in tropical and subtropical areas of the world, is a source of high value protein to vegetarians or very poor populations globally. Hence, genome-wide identification of miRNAs and lncRNAs in C. cajan is extremely important to understand their role in PTGR with a possible implication to generate improve variety of crops. RESULTS: We have identified 616 mature miRNAs in C. cajan belonging to 118 families, of which 578 are novel and not reported in MirBase21. A total of 1373 target sequences were identified for 180 miRNAs. Of these, 298 targets were characterized at the protein level. Besides, we have also predicted 3919 lncRNAs. Additionally, we have identified 87 of the predicted lncRNAs to be targeted by 66 miRNAs. CONCLUSIONS: miRNA and lncRNAs in plants are known to control a variety of traits including yield, quality and stress tolerance. Owing to its agricultural importance and medicinal value, the identified miRNA, lncRNA and their targets in C. cajan may be useful for genome editing to improve better quality crop. A thorough understanding of ncRNA-based cellular regulatory networks will aid in the improvement of C. cajan agricultural traits.

Computational prediction of miRNAs and their targets in Phaseolus vulgaris using simple sequence repeat signatures.

BACKGROUND: MicroRNAs (miRNAs) are endogenous, noncoding, short RNAs directly involved in regulating gene expression at the post-transcriptional level. In spite of immense importance, limited information of P. vulgaris miRNAs and their expression patterns prompted us to identify new miRNAs in P. vulgaris by computational methods. Besides conventional approaches, we have used the simple sequence repeat (SSR) signatures as one of the prediction parameter. Moreover, for all other parameters including normalized Shannon entropy, normalized base pairing index and normalized base-pair distance, instead of taking a fixed cut-off value, we have used 99% probability range derived from the available data. RESULTS: We have identified 208 mature miRNAs in P. vulgaris belonging to 118 families, of which 201 are novel. 97 of the predicted miRNAs in P. vulgaris were validated with the sequencing data obtained from the small RNA sequencing of P. vulgaris. Randomly selected predicted miRNAs were also validated using qRT-PCR. A total of 1305 target sequences were identified for 130 predicted miRNAs. Using 80% sequence identity cut-off, proteins coded by 563 targets were identified. The computational method developed in this study was also validated by predicting 229 miRNAs of A. thaliana and 462 miRNAs of G. max, of which 213 for A. thaliana and 397 for G. max are existing in miRBase 20. CONCLUSIONS: There is no universal SSR that is conserved among all precursors of Viridiplantae, but conserved SSR exists within a miRNA family and is used as a signature in our prediction method. Prediction of known miRNAs of A. thaliana and G. max validates the accuracy of our method. Our findings will contribute to the present knowledge of miRNAs and their targets in P. vulgaris. This computational method can be applied to any species of Viridiplantae for the successful prediction of miRNAs and their targets.

In vivo antitumor activity of biosynthesized silver nanoparticles using Ficus religiosa as a nanofactory in DAL induced mice model.

Ficus religiosa leaf extract was chosen as a reducing agent to fabricate silver nanoparticles (AgNPs) by a simple, cost-effective and eco-friendly process with the aim of treating Dalton's ascites lymphoma (DAL) in mice model. The formation of synthesized nanoparticles were characterized by UV-visible analysis (UV-vis), Fourier transform infra-red (FT-IR), transmission electron microscopy (TEM), X-ray diffraction (XRD) and zeta potential analyses. A peak at 431nm indicated the surface plasmon resonance of AgNPs. FTIR studies indicated polyphenols and proteins as possible encapsulates. TEM analysis showed particles size in the range of 5-35nm. Healthy Swiss Albino mice (30-35g) were intraperitoneally induced with DAL cells and treated with F. religiosa derived AgNPs at a dose of 50µg/ml. Blood and liver tissues were collected subsequent to dissection and subjected to hematological, biochemical and anticancer assays. Hematological and biochemical analyses revealed revival after treating with F. religiosa derived AgNPs. Antioxidant activity results further proved supportive evidence. The apoptosis inducing effect of AgNPs was observed through acridine orange staining (AO and EB) and DNA fragmentation assay. Anti- angiogenic activity was confirmed by observing vessel development. All these observations indicate that the AgNPs were effective in treatment of DAL.