Exploring RECQ Helicase landscape in breast cancer

Background Breast cancer is the second most common cause of cancer death in females in the UK. Approximately 20 percent of breast cancers diagnosed are triple negative (TNBC) intrinsic subtype, which are aggressive high-grade tumours carrying a poor prognosis. About 20% of TNBC harbour a mutation in breast cancer gene (BRCA). BRCA plays important role in homologous recombination. There is evidence to suggest that non-familial breast cancers harbour homologous recombination repair defects that may be useful targets for therapy. The RECQ protein family is a highly conserved group of DNA helicases, which play diverse roles in various DNA metabolic processes such as DNA replication and recombination repair. In humans, five RECQ helicases (RECQL1, BLM, WRN, RECQL4, and RECQL5) have been identified and three of them namely, WRN, BLM, and RECQL4 have been associated with autosomal recessive disorders which are characterized by genomic instability, premature aging, and predisposition to various cancers. The role of RECQ helicase expression in sporadic breast cancer pathogenesis is unknown. We hypothesized that dysregulation in RECQ helicase expression may have an impact on breast cancer pathogenesis and survival. The aim of this thesis was to have a comprehensive assessment of the prognostic, predictive and clinicopathological significance of five RECQ helicases at the transcriptome and protein level. Methods Investigation of the protein expression of RECQ helicases in breast cancer was performed in two patient cohorts. The first cohort was a series of 1650 primary invasive breast cancer patients who were diagnosed between 1986 and 1999 and entered into the Nottingham tenovus series. The second cohort used for evaluation was an independent series of 252 ER-negative invasive breast cancers diagnosed and managed at Nottingham University Hospitals between 1999 and 2007. Standard immunohistochemistry technique was used for staining the breast TMA (tissue microarray) and assessment of staining was done using H-scoring. Evaluation of the mRNA expression was done in the METABRIC dataset (1977 cases). Data analysis was performed using SPSS (SPSS, version 22 Chicago, IL). Frequency histogram distributions and X tile (Version 3.6.1) were used to establish the cut-offs for expression values such that the resulting subgroups have significantly different survival courses. Cumulative survival probabilities were estimated using the Kaplan–Meier method. Statistical significant differences between survival outcomes were tested using the log-rank test. P values for each test were adjusted with (Benjamini & Hochberg, 1995) multiple P-value adjustments and an adjusted p-value of <0.05 was considered significant. Multivariate analysis with survival Cox proportional hazard model was performed where a statistically significant survival outcome was seen in the univariate analysis. Cytotoxicity of Cisplatin in BLM Ctr and BLM KD Hela cells was assessed using clonogenic and MTS cell survival assays. Functional consequences of Cytotoxicity in BLM KD Hela cell lines was determined using flow cytometry technique. Weasel (Victoria, Australia) flow cytometry analysis software was utilized for data analysis. Graphical visualisation and statistical analysis were carried out in GraphPad Prism 6 (GraphPad, La Jolla, USA). Two way ANOVA was used to determine the statistically significant differences between the variables. Results Low RECQL1 expression showed a statistically significant association with aggressive breast cancer phenotypes. Furthermore, low RECQL1 was linked to poor breast cancer survival (p<0.05) in the whole cohort and ER-positive patients including those who had adjuvant endocrine therapy, suggesting the prognostic and predictive significance of RECQL1 in sporadic breast cancers. High RECQL5 mRNA expression and high RECQL5/low RAD51 protein expression revealed a statistically significant association with aggressive breast cancer phenotypes and poor survival which was more pronounced in ER-positive subgroup. This suggests the predictive and prognostic significance of RECQL5 in breast cancer. RECQL5 remained an independent predictor of survival on multivariate analysis. RECQL4 amplification/gain in copy numbers, high RECQL4 mRNA, expression and low nuclear/high cytoplasmic RECQL4 protein expression were associated with aggressive breast cancer phenotypes, and poor survival (p<0.05). In tumours with high nuclear RECQL4 protein expression, c-MYC could stratify patients into prognostic groups (p<0.05).This confirms the clinicopathological, predictive and prognostic significance of RECQL4 in breast cancer. Low WRN mRNA levels were significantly associated with poor breast cancer-specific survival in the whole cohort and ER-positive patients including those who had adjuvant endocrine therapy, informing the prognostic and predictive significance. At the protein level, WRN protein expression remained an independent predictor of survival on multivariate analysis. ER-positive tumours, including those who had endocrine therapy, with high TOPO1/high WRN expression showed poor breast cancer-specific survival (p<0.05). High BLM mRNA levels were significantly associated with aggressive breast cancer phenotypes and poor breast cancer-specific survival in the whole cohort and ER-positive tumour including those who had endocrine therapy, showing predictive and prognostic significance. Low nuclear BLM/low nuclear RAD51 protein levels were associated with poor survival(p<0.05) in the whole cohort and ER-negative patients including those who had chemotherapy showing resistance to chemotherapy. BLM mRNA expression remained an independent predictor of survival on multivariate analysis. Moreover, preliminary cell line work showed that BLM-KD HeLa cells were sensitive to platinum chemotherapy.  Conclusion RECQ helicases expression in breast cancer clearly has predictive, prognostic, and clinicopathological significance. RECQ helicases could have a synthetic lethality relationship with PARP inhibitors or other DNA repair inhibitors. It is important to do further cell line work in breast cancer cell lines through BLM knockdown with siRNA and Knock out with CRISPR cas-9

Digital leisure for development: Reframing new media practice in the global south.

Photoshopping of newlyweds, downloading the latest movies, teens flirting on social network sites and virtual gaming may seem like typical behavior in the West; yet in the context of a village in Mali or a slum in Mumbai, it is seen as unusual and perhaps an anomaly in their new media practice. In recent years, some studies (Ganesh, 2010; Mitra, 2005; Arora, 2010; 2012; Rangaswamy & Nair, 2012; Kavoori, Chadha & Arceneaux, 2006) have documented these leisure-oriented behaviors in the global south and argued for the need to emphasize and reposition these user practices within larger and contemporary discourses on new media consumption. Yet, for the most part, studies in the field of Information and Communication Technologies for Deve

Preparation and Characterization of Phytosomal-Phospholipid Complex of P. Amarus and its Tablet Formulation

Present investigation was aimed at formulation, characterization and evaluation of phytosomal complex tablets for sustained delivery of Phyllanthus amarus complex. Phyllanthin, one of the active lignin present in this plant species was isolated from the aerial parts, by silica gel column chromatography employing gradient elution with hexane −ethyl acetate solvent mixture. It was obtained in high yields (1.23%), compared to reported procedures and the purity was ascertained by HPTLC analysis. Phyllanthin was characterized for M.P, UV −Visible spectrophotometry, FT-IR, 1H NMR, 13C and NMR analysis. Release kinetics was evaluated by using United States Pharmacopeia (USP)-22 type I dissolution apparatus. Scanning electron microscopy was used to visualize the effect of dissolution medium on matrix tablet surface. HPTLC was carried out for quantitative and qualitative estimation of Phyllanthin in Phyllanthus amarus and Rf of phyllanthin was found to be about 0.25. The content was found to be maximum for phytosomal complex of phyllanthus formed by vaccum drying of 1:1 drug excipient ratio. The in-vitro drug release study revealed that optimized formulation sustained the drug release for 12 hr (88.1% ± 4.1% release). Fitting the in vitro drug release data to Korsmeyer equation indicated that diffusion along with erosion could be the mechanism of drug release

Formulation and In-Vitro evaluation of Nanocrystal formulation of poorly soluble drugs

Introduction:-Poor solubility of drug compounds which accounts for 40% of new molecules investigated at present is an issue of great concern in pharmaceutical industry and reducing particle size (i,e to reduce below 1000 nm )of drug candidate to be investigated is one of the simplest and efficient ways to overcome this challenge. Drug nanocrystals, solid nanosized drug particles are defined as formulation having 100% drug, which are covered by a stabilizer layer. In this study attempt was made to formulate and evaluate nanocrystals of poorly soluble drugs having low oral bioavailability. Material and method:- Nanocrystals were prepared successfully by varying concentration of different stabilizers by anti-solvent precipitation method. The formulated nanocrystals were evaluated by determining physicochemical characteristics such as physical appearance, Differential Scanning Calorimetry (DSC), scanning electron microscopy (SEM), X-ray powder diffractometry, solubility studies, particle size distribution, zeta potential, and in vitro drug release profile studies. Results:- An in-vitro study was performed on the successful formulation in comparison to drug powder using dissolution apparatus The particle size of RVT and PSNC-3 was found to be 1975.3 nm and 790.1 nm respectively. Conclusion: Precipitated Nanocrystals formulated with different stablizer’s method resultedin formation of small and uniform RVT nanocrystals with an improved saturation solubility, dissolution rate. Keywords: Nanocrystal, poorly soluble drug

Over one third of patients with symptomatic femoroacetabular impingement display femoral or acetabular version abnormalities.

PURPOSE: The aim of this study was investigate the relationship between version and torsional abnormalities of the acetabulum, femur and tibia in patients with symptomatic FAI. METHODS: A systematic review was performed according to PRISMA guidelines using the EMBASE, MEDLINE, PubMed and Cochrane databases. Original research articles evaluating the described version and torsional parameters in FAI were included. The MINORS criteria were used to appraise study quality and risk of bias. Mean version and torsion values were displayed using forest plots and the estimated proportion of hips displaying abnormalities in version/torsion were calculated. RESULTS: A total of 1206 articles were identified from the initial search, with 43 articles, involving 8861 hips, meeting the inclusion criteria. All studies evaluating femoral or acetabular version in FAI reported 'normal' mean version values (10-25 °). However, distribution analysis revealed that an estimated 31% and 51% of patients with FAI displayed abnormal central acetabular and femoral version, respectively. CONCLUSION: Up to 51% of patients presenting with symptomatic FAI show an abnormal femoral version, whilst up to 31% demonstrate abnormal acetabular version. This high percentage of version abnormalities highlights the importance of evaluating these parameters routinely during assessment of patients with FAI, to guide clinical decision-making. LEVEL OF EVIDENCE: IV

A case series of IPOM-AS – intraperitoneal mesh fixation technique for ventral hernia using straight needle suture nylon 2–0

Intraperitoneal onlay mesh (IPOM) technique of mesh fixation using tackers for ventral hernia is widely done. But using tackers is costly (266.29 USD) and causes early and late post-operative pain. The aim of the study was to choose the better method of surgery for ventral hernia using composite mesh and straight needle suture nylon 2–0 for intraperitoneal mesh fixation (IPOM-AS) and will compare it with conventional IPOM technique in which Fixation of mesh is done by Tacker. Ten patients who were diagnosed with ventral hernia between November 2022 and December 2022 in the Department of Surgery, MGM Medical College and MY Hospital, Indore were taken in our study and IPOM-AS technique was performed using three ports on the left side of the abdomen, after reducing the contents of hernia, the axis for internal attachment of composite mesh is identified and marked with pair of sutures (vicryl and rapid vicryl) and intraperitoneal transabdominal fixation of mesh is done using straight needle suture nylon 2–0 with the help of suture passer. This technique was taken in our study and was assessed for cost-effectiveness, early and late post-operative pain, learning curve, early mobilization, early discharge, early return to work, and long-term complications. All the patients had less early and late post-operative pain according to VAS score (mean VAS score 2 on post-operative 1 and 1 on post-operative 2, no pain during the follow-up period), patients were discharged earlier (mostly on post-operative 1) and early return to work was possible and this technique of intraperitoneal mesh fixation using straight needle suture (2.03 USD) is more cost-effective when we compared these patients with those in whom conventional IPOM was done with the help of a tacker (266.29 USD). Good patient compliance was seen. IPOM using composite mesh and straight needle suture (IPOM-AS) for intraperitoneal mesh fixation in ventral hernia is better accepted by patients than the conventional IPOM using tacker for mesh fixation

Are DNA repair factors promising biomarkers for personalized therapy in gastric cancer?

Chronic inflammation is a driving force for gastric carcinogenesis. Reactive oxygen species (ROS) generated during the inflammatory process generates DNA damage that is processed through the DNA repair pathways. In this study, we profiled key DNA repair proteins (single-strand-selective monofunctional uracil-DNA glycosylase 1 [SMUG1], Flap endonuclease 1 [FEN1], X-ray repair cross-complementing gene 1 [XRCC1], and Ataxia telangiectasia mutated [ATM]) involved in ROS-induced oxidative DNA damage repair in gastric cancer and correlated to clinicopathological outcomes. High expression of SMUG1, FEN1, and XRCC1 correlated to high T-stage (T3/T4) (p-values: 0.001, 0.005, and 0.02, respectively). High expression of XRCC1 and FEN1 also correlated to lymph node-positive disease (p-values: 0.009 and 0.02, respectively). High expression of XRCC1, FEN1, and SMUG1 correlated with poor disease-specific survival (DSS) (p-values: 0.001, 0.006, and 0.05, respectively) and poor disease-free survival (DFS) (p-values: 0.001, 0.001, and 0.02, respectively). Low expression of ATM correlated to lymph node positivity (p=0.03), vascular invasion (p=0.05), and perineural invasion (p=0.005) and poor DFS (p=0.001) and poor DSS (p=0.003). In the multivariate Cox model, high XRCC1 and low ATM were independently associated with poor survival (p=0.008 and 0.011, respectively). Our observation supports the hypothesis that DNA repair factors are promising biomarkers for personalized therapy in gastric cancer. Antioxid. Redox Signal. 18, 2392–2398

Validating the Cambridge Protocol: Reliability of Hip Muscle Strength Measurements Using a Motorized Dynamometer and Electromyography

Background: Muscle weakness is common after injury in athletes and in the presence of hip pathology. It will cause abnormal hip biomechanics and can predict future injury. However, objective measurement of hip muscle strength is difficult to perform accurately and reliably. Therefore, it is challenging to determine when an athlete has returned to preinjury levels of strength. In addition, there is currently no standardized method of obtaining measurements, which prevents the data being compared or shared between research centers. Purpose: The purpose of this study is to comprehensively assess the inter- and intraobserver reliability of our standardized muscle strength measurement protocol. Study Design: Descriptive laboratory study. Level of Evidence: Level 3, inception cohort study. Methods: A total of 16 healthy male volunteers (age = 28.3 ± 7.9 years) were recruited. Those with a previous history of hip injuries or disorders were excluded. These volunteers underwent strength testing according to the Cambridge Protocol on 4 separate occasions, performed by 2 independent assessors. Maximal voluntary contractions, fatigue torque fluctuations, and electromyography measurements were recorded. Intra- and interobserver reliability was assessed using intraclass correlation coefficient (ICC). Results: Good-to-excellent correlation was seen for both intra- and interobserver reliability across almost all hip movements for maximal contractions: ICC ranged 0.78 to 0.93 and 0.78 to 0.96, respectively. The standard error of the mean for all hip movements was also extremely low at 2% to 3%. Conclusion: The Cambridge Protocol is a highly reliable method for objective measurement of hip muscle strength. We recommend future studies use this protocol, or the principles underpinning it, to enable data sharing and comparison across different studies. Clinical Relevance: This is a description and analysis of hip muscle strength measurement. If widely used, it will allow for accurate and objective strength assessment and closer monitoring of hip injuries and pathology

Transcriptomic and Protein Expression Analysis Reveals Clinicopathological Significance of Bloom Syndrome Helicase (BLM) in Breast Cancer

BLM has key roles in homologous recombination repair, telomere maintenance and DNA replication. Germ-line mutation in the BLM gene causes Bloom’s syndrome, a rare disorder characterised by premature aging and predisposition to multiple cancers including breast cancer. The clinicopathological significance of BLM in sporadic breast cancers is unknown. We investigated BLM mRNA expression in the Molecular Taxonomy of Breast Cancer International Consortium cohort (n=1950) and validated in an external dataset of 2413 tumours. BLM protein level was evaluated in the Nottingham Tenovus series comprising 1650 breast tumours. High BLM mRNA expression was highly significantly associated with high histological grade, larger tumour size, ER negative, PgR negative and triple negative phenotypes (ps<0.0001). High BLM mRNA expression was also linked to aggressive molecular phenotypes including PAM50.Her2 (p<0.0001), PAM50.Bas al (p<0.0001) and PAM50.LumB (p<0.0001) and Genufu subtype (ER+/Her2-/High proliferation) (p<0.0001). PAM50.LumA tumours and Genufu subtype (ER+/Her2-/low proliferation) were more likely to express low levels of BLM mRNA (ps<0.0001). Integrative molecular clusters (intClust) intClust.1 (p<0.0001), intClust.5 (p<0.0001), intClust.9 (p<0.0 001) and intClust.10 (p<0.0001) were also more likely in tumours with high BLM mRNA expression. High BLM mRNA expression was associated with poor breast cancer specific survival (BCSS) (ps<0.000001). At the protein level, altered sub-cellular localisation with high cytoplasmic BLM and low nuclear BLM was linked to aggressive phenotypes. In multivariate analysis, BLM mRNA and BLM protein levels independently influenced BCSS ( p=0.03). This is the first and the largest study to provide evidence that BLM is a promising biomarker in breast cancer