Finite Element Analysis of the Influence of Implant Tilting and the Direction of Loading on the Displacement and Micromotion of Immediately Loaded Implants.

Objectives: To investigate the outcome of the loading direction and implant tilting on the micromotion and displacement of immediately placed implants with finite element analysis (FEA). Materials and Method: Eight blocks of synthetic bone were created. Eight screw-type implants were inserted, four axially and four slanted, each measuring 11 mm in length and 4.5 mm in diameter. The axial implants and the tilted implants were distally inclined by 30°. The top of the abutment was subjected to 180 N vertical and mesiodistal oblique (45° angle) loads, and the displacement of the abutment was measured. The abutment displacement and micromotion were estimated, and nonlinear finite element models simulating the in vitro experiment were built. In vitro studies and FEA data on abutment displacement were compared, and the reliability of the finite element model was assessed. Result: Under oblique stress, abutment displacement was larger than under axial loading, and it was also greater for tilted implants than for axial implants. The consistency of the in vitro and FEA data was satisfactory. Under vertical stress, the highest micromotion values in the axial and tilted implants were extremely near. Conclusion: Under mesiodistal oblique stress, tilted implants may have a smaller maximum amount of micromotion than axial implants. The loading direction had a significant impact on the highest micromotion values. The abutment displacement values were not reflected in the maximum micromotion measurements.

Assessment of the impact of a personalised nutrition intervention in impaired glucose regulation over 26 weeks: a randomised controlled trial.

Dietary interventions can reduce progression to type 2 diabetes mellitus (T2DM) in people with non-diabetic hyperglycaemia. In this study we aimed to determine the impact of a DNA-personalised nutrition intervention in people with non-diabetic hyperglycaemia over 26 weeks. ASPIRE-DNA was a pilot study. Participants were randomised into three arms to receive either (i) Control arm: standard care (NICE guidelines) (n = 51), (ii) Intervention arm: DNA-personalised dietary advice (n = 50), or (iii) Exploratory arm: DNA-personalised dietary advice via a self-guided app and wearable device (n = 46). The primary outcome was the difference in fasting plasma glucose (FPG) between the Control and Intervention arms after 6 weeks. 180 people were recruited, of whom 148 people were randomised, mean age of 59 years (SD = 11), 69% of whom were female. There was no significant difference in the FPG change between the Control and Intervention arms at 6 weeks (- 0.13 mmol/L (95% CI [- 0.37, 0.11]), p = 0.29), however, we found that a DNA-personalised dietary intervention led to a significant reduction of FPG at 26 weeks in the Intervention arm when compared to standard care (- 0.019 (SD = 0.008), p = 0.01), as did the Exploratory arm (- 0.021 (SD = 0.008), p = 0.006). HbA1c at 26 weeks was significantly reduced in the Intervention arm when compared to standard care (- 0.038 (SD = 0.018), p = 0.04). There was some evidence suggesting prevention of progression to T2DM across the groups that received a DNA-based intervention (p = 0.06). Personalisation of dietary advice based on DNA did not result in glucose changes within the first 6 weeks but was associated with significant reduction of FPG and HbA1c at 26 weeks when compared to standard care. The DNA-based diet was effective regardless of intervention type, though results should be interpreted with caution due to the low sample size. These findings suggest that DNA-based dietary guidance is an effective intervention compared to standard care, but there is still a minimum timeframe of adherence to the intervention before changes in clinical outcomes become apparent.Trial Registration: www.clinicaltrials.gov.uk Ref: NCT03702465.

Pooled sputum to optimise the efficiency and utility of rapid, point-of-care molecular SARS-CoV-2 testing.

BACKGROUND: As SARS-CoV-2 testing expands, particularly to widespread asymptomatic testing, high sensitivity point-of-care PCR platforms may optimise potential benefits from pooling multiple patients' samples. METHOD: We tested patients and asymptomatic citizens for SARS-CoV-2, exploring the efficiency and utility of CovidNudge (i) for detection in individuals' sputum (compared to nasopharyngeal swabs), (ii) for detection in pooled sputum samples, and (iii) by modelling roll out scenarios for pooled sputum testing. RESULTS: Across 295 paired samples, we find no difference (p = 0.1236) in signal strength for sputum (mean amplified replicates (MAR) 25.2, standard deviation (SD) 14.2, range 0-60) compared to nasopharyngeal swabs (MAR 27.8, SD 12.4, range 6-56). At 10-sample pool size we find some drop in absolute strength of signal (individual sputum MAR 42.1, SD 11.8, range 13-60 vs. pooled sputum MAR 25.3, SD 14.6, range 1-54; p < 0.0001), but only marginal drop in sensitivity (51/53,96%). We determine a limit of detection of 250 copies/ml for an individual test, rising only four-fold to 1000copies/ml for a 10-sample pool. We find optimal pooled testing efficiency to be a 12-3-1-sample model, yet as prevalence increases, pool size should decrease; at 5% prevalence to maintain a 75% probability of negative first test, 5-sample pools are optimal. CONCLUSION: We describe for the first time the use of sequentially dipped sputum samples for rapid pooled point of care SARS-CoV-2 PCR testing. The potential to screen asymptomatic cohorts rapidly, at the point-of-care, with PCR, offers the potential to quickly identify and isolate positive individuals within a population "bubble".

Assessing a novel, lab-free, point-of-care test for SARS-CoV-2 (CovidNudge): a diagnostic accuracy study.

BACKGROUND: Access to rapid diagnosis is key to the control and management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Laboratory RT-PCR testing is the current standard of care but usually requires a centralised laboratory and significant infrastructure. We describe our diagnostic accuracy assessment of a novel, rapid point-of-care real time RT-PCR CovidNudge test, which requires no laboratory handling or sample pre-processing. METHODS: Between April and May, 2020, we obtained two nasopharyngeal swab samples from individuals in three hospitals in London and Oxford (UK). Samples were collected from three groups: self-referred health-care workers with suspected COVID-19; patients attending emergency departments with suspected COVID-19; and hospital inpatient admissions with or without suspected COVID-19. For the CovidNudge test, nasopharyngeal swabs were inserted directly into a cartridge which contains all reagents and components required for RT-PCR reactions, including multiple technical replicates of seven SARS-CoV-2 gene targets (rdrp1, rdrp2, e-gene, n-gene, n1, n2 and n3) and human ribonuclease P (RNaseP) as sample adequacy control. Swab samples were tested in parallel using the CovidNudge platform, and with standard laboratory RT-PCR using swabs in viral transport medium for processing in a central laboratory. The primary analysis was to compare the sensitivity and specificity of the point-of-care CovidNudge test with laboratory-based testing. FINDINGS: We obtained 386 paired samples: 280 (73%) from self-referred health-care workers, 15 (4%) from patients in the emergency department, and 91 (23%) hospital inpatient admissions. Of the 386 paired samples, 67 tested positive on the CovidNudge point-of-care platform and 71 with standard laboratory RT-PCR. The overall sensitivity of the point-of-care test compared with laboratory-based testing was 94% (95% CI 86-98) with an overall specificity of 100% (99-100). The sensitivity of the test varied by group (self-referred healthcare workers 94% [95% CI 85-98]; patients in the emergency department 100% [48-100]; and hospital inpatient admissions 100% [29-100]). Specificity was consistent between groups (self-referred health-care workers 100% [95% CI 98-100]; patients in the emergency department 100% [69-100]; and hospital inpatient admissions 100% [96-100]). Point of care testing performance was similar during a period of high background prevalence of laboratory positive tests (25% [95% 20-31] in April, 2020) and low prevalence (3% [95% 1-9] in inpatient screening). Amplification of viral nucleocapsid (n1, n2, and n3) and envelope protein gene (e-gene) were most sensitive for detection of spiked SARS-CoV-2 RNA. INTERPRETATION: The CovidNudge platform was a sensitive, specific, and rapid point of care test for the presence of SARS-CoV-2 without laboratory handling or sample pre-processing. The device, which has been implemented in UK hospitals since May, 2020, could enable rapid decisions for clinical care and testing programmes. FUNDING: National Institute of Health Research (NIHR) Imperial Biomedical Research Centre, NIHR Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance at Oxford University in partnership with Public Health England, NIHR Biomedical Research Centre Oxford, and DnaNudge.

HER2/neu Testing In 432 Consecutive Breast Cancer Cases using FISH and IHC - A Comparative Study.

INTRODUCTION: The development of trastuzumab, a HER-2/neu targeted monoclonal antibody resulted in significant improvements in clinical response and survival in HER-2/neu gene amplified group of patients. Thus, accurate assessment of HER-2/neu status becomes critical. Fluorescence In Situ Hybridization (FISH) and Immunohistochemistry (IHC) are the most commonly used methods for this purpose and specific recommendations exist with regard to the concordance to be observed between the two tests. AIM: Here, we report and evaluate the concordance rate between FISH and IHC for HER-2/neu status in breast cancer specimens. MATERIALS AND METHODS: Archival paraffin blocks of tumour tissue from 450 patients of breast cancer were analyzed for Her-2/neu status using FISH and IHC. RESULTS: There was a highly significant concordance between the results of FISH and IHC assays in HER-2/neu status assessment in invasive breast cancer cases. There were inverse associations between the expression of Oestrogen Receptors/Progesterone Receptors (ER/PR) and HER-2/neu amplification. CONCLUSION: Although, IHC gave significant concordant results with FISH in determining HER-2/neu status, its subjective grading system precludes its use as a gold standard. FISH should always be used to determine true gene amplification when the IHC results are equivocal.

Antiproliferative Effect of Lapatinib in HER2-Positive and HER2-Negative/HER3-High Breast Cancer: Results of the Presurgical Randomized MAPLE Trial (CRUK E/06/039).

PURPOSE: Not all breast cancers respond to lapatinib. A change in Ki67 after short-term exposure may elucidate a biomarker profile for responsive versus nonresponsive tumors. EXPERIMENTAL DESIGN: Women with primary breast cancer were randomized (3:1) to 10 to 14 days of preoperative lapatinib or placebo in a multicenter phase II trial (ISRCTN68509377). Biopsies pre-/posttreatment were analyzed for Ki67, apoptosis, HER2, EGFR, ER, PgR, pAKT, pERK, and stathmin by IHC. Further markers were measured by RT-PCR. Primary endpoint was change in Ki67. HER2(+) was defined as 2+/3+ by IHC and FISH(+). RESULTS: One hundred twenty-one patients (lapatinib, 94; placebo, 27) were randomized; of these, 21% were HER2(+), 78% were HER2(-) nonamplified, 26% were EGFR(+). Paired samples containing tumor were obtained for 98% (118 of 121). Ki67 fell significantly with lapatinib (-31%; P < 0.001), but not with placebo (-3%). Whereas Ki67 reduction with lapatinib was greatest in HER2(+) breast cancer (-46%; P = 0.003), there was a significant Ki67 decrease in HER2(-) breast cancer (-27%; P = 0.017) with 14% of HER2(-) breast cancer demonstrating ≥50% Ki67 reduction with lapatinib. Among HER2(+) patients, the only biomarker predictive of Ki67 response was the EGFR/HER4 ligand epiregulin (EREG) (rho = -0.7; P = 0.002). Among HER2(-) tumors, only HER3 mRNA levels were significantly associated with Ki67 response on multivariate analysis (P = 0.01). In HER2(-) breast cancer, HER2 and HER3 mRNA levels were highly correlated (rho = 0.67, P < 0.001), with all Ki67 responders having elevated HER3 and HER2 expression. CONCLUSIONS: Lapatinib has antiproliferative effects in a subgroup of HER2(-) nonamplified tumors characterized by high HER3 expression. The possible role of high HER2:HER3 heterodimers in predicting response to lapatinib merits investigation in HER2(-) tumors.

A novel alternative to cryopreservation for the short-term storage of stem cells for use in cell therapy using alginate encapsulation.

Efficient transport of stem/progenitor cells without affecting their survival and function is a key factor in any practical cell-based therapy. However, the current approach using liquid nitrogen for the transfer of stem cells requires a short delivery time window is technically challenging and financially expensive. The present study aims to use semipermeable alginate hydrogels (crosslinked by strontium) to encapsulate, store, and release stem cells, to replace the conventional cryopreservation method for the transport of therapeutic cells within world-wide distribution time frame. Human mesenchymal stem cell (hMSC) and mouse embryonic stem cells (mESCs) were successfully stored inside alginate hydrogels for 5 days under ambient conditions in an air-tight environment (sealed cryovial). Cell viability, of the cells extracted from alginate gel, gave 74% (mESC) and 80% (hMSC) survival rates, which compared favorably to cryopreservation. More importantly, the subsequent proliferation rate and detection of common stem cell markers (both in mRNA and protein level) from hMSCs and mESCs retrieved from alginate hydrogels were also comparable to (if not better than) results gained following cryopreservation. In conclusion, this new and simple application of alginate hydrogel encapsulation may offer a cheap and robust alternative to cryopreservation for the transport and storage of stem cells for both clinical and research purposes.

Relationship between p53 overexpression, human papillomavirus infection, and lifestyle in Indian patients with head and neck cancers.

Exposure to pollutants in the environment, tobacco and alcohol consumption, poor oral hygiene and opportunistic viral infections are important aetiological factors in head and neck cancers. In this study, we evaluate the complex interrelationships between these factors and molecular events such as p53 overexpression in causation of head and neck cancers. Tissue samples from 110 patients with histopathologically confirmed carcinoma of head and neck were analyzed from our tissue biorepository with patient consent. Data pertaining to their dietary habits, tobacco and alcohol consumption were abstracted. P53 overexpression was analysed by immunohistochemistry and HPV (high-risk genotype) were studied by Chromogenic in situ Hybridization using an ultra sensitive DNA probe. Chi-square analysis was done to determine relationships between proportions of dependent and independent variables. Bivariate relationships were determined between these variables using Spearman's rank correlation. Linear regression analysis was used to determine the best predictor variable influencing p53 expression. Tobacco consumption especially smoking cigarettes and all forms of tobacco consumption put together and HPV infection significantly influenced p53 overexpression. Forty-five percent of the studied cohort was positive for HPV. Regression analysis showed interaction between tobacco and HPV infection to be a primary predictor (ß = 0.31, p = 0.02) for p53 expression. Tobacco in any form: chewing, smoking and snuffing, along with HPV infection is significantly associated with p53 overexpression. There is a high prevalence of HPV infection (45%) in Indian patients suggesting its possible role in the aetiology of head and neck cancer.

An uncommon case of an adult with del(5)(q) in acute lymphoblastic leukemia.

Del(5)(q) is a common chromosomal abnormality with favourable prognosis in Myelodysplastic Syndrome (MDS) and Acute myeloid leukemia (AML). However, del(5)(q) is also seen rarely in Acute lymphoblastic leukemia (ALL) and its significance remains poorly understood. We present here, a case report of diagnosis of an adult 75 year old patient of ALL with a cytogenetic abnormality of del(5)(q32). His clinical features, morphology and immunophenotyping findings were suggestive of T-ALL. Relevant literature has been reviewed and discussed.

Her-2/neu overexpression due to polysomy 17 in breast cancer: molecular testing to guide therapeutic options.

BACKGROUND: Fluorescent in situ hybridization (FISH) equivocal results for Her-2/neu still pose a diagnostic dilemma in oncology practice. In this study, we evaluate if Her-2/neu mRNA expression is an alternative to FISH for detecting Her-2/neu positivity. PATIENTS AND METHODS: Archival paraffin blocks of 54 breast cancer patients were analyzed for Her-2/neu status using immunohistochemistry (IHC), FISH, and Her-2/neu gene expression using mRNA. RESULTS: There was a 100% positive agreement and 64.7% negative agreement of Her-2/neu mRNA expression with respect to the reference standard (FISH), with the kappa value for agreement being 0.36. mRNA levels correlated positively and strongly with FISH ratio and IHC positivity. For Her-2/neu mRNA expression, Her-2/neu copy number was a significant predictor indicating that mRNA expression is independent of polysomy status. CONCLUSIONS: Her-2/neu mRNA expression may help tide over ambiguity posed by polysomy and FISH equivocal samples.

Screening for EGFR mutations in lung cancer, a report from India.

Epidermal growth factor receptor (EGFR) is one of the targeted molecular markers in many cancers including the lung malignancy. Genetic modifications such as deletions, insertions and Single Nucleotide Polymorphisms in the tyrosine kinase (TK) domain of EGFR is a common feature observed in most lung cancers. Gefitinib and erlotinib are commonly available therapeutic drugs which act as specific inhibitors for the tyrosine kinase domain of EGFR and associated with EGFR mutations in exons 18-21. However the prevalence of mutation varies among ethnicity, grade, age and gender. This is the first report on the prevalence of EGFR mutation in non-small cell lung cancer patients using DNA obtained from samples such as biopsy/cytology/pleural fluid and Fine Needle Aspiration (FNA), across India. We have screened for 29 somatic mutations which span exons 18, 19, 20 and 21 of EGFR gene using Scorpion probe based ARMS-PCR technique. DNA from 220 NSCLC tissue samples were analyzed for EGFR mutations and mutations were detected in 51.8% of the study population. Among the mutant positive cases, the deletions in exon 19 (52%) and a missense mutation L858R in exon 21 (26%) were most predominant. There was a significant increase in overall mutations (p=0.01) as a function of age, mutation in exons 19 and 21 together (p=0.003), mutations in exons 18, 19 and 21 (p=0.04) and mutations in exons 18 and 19 (p=0.03) in females. Mutations did not seem to significantly correlate metastases or disease progression. Mutations in exons [19] and 21 together were significant in non-smokers compared to smokers (p=0.01) using Mann-Whitney tests. The study suggests high prevalence of EGFR positivity in NSCLC in Indian sub-population and provides opportunities for targeted therapies for this group.

Gene-specific methylation: potential markers for colorectal cancer.

PURPOSE: Aberrant methylation of the promoter region is associated with silencing of many genes in neoplasia. CpG island methylation is an epigenetic mechanism for transcriptional silencing that occurs at various stages of colon tumorigenesis. In this study, we tested the promoter methylation and expression of seven genes from various pathways of DNA repair, apoptosis and inflammation, i.e., sFRP1, MLH1, RASSF1A, CDA, v-fgr, LYN-B, and TNFR10d. METHOD: The genes were analyzed by quantitative polymerase chain reaction for the level of gene expression. The promoter methylation status of the genes was studied by methylation-specific polymerase chain reaction. RESULT: The correlation of promoter methylation status with suppressed gene expression patterns suggested a potential role for the silencing these genes in colon cancer progression. CONCLUSION: Promoter methylations of the studied genes could be explored as promising biomarkers for new diagnostic, prognostic and therapeutic targets of colorectal cancer.

Ethanol inhibits methionine adenosyltransferase II activity and S-adenosylmethionine biosynthesis and enhances caspase-3-dependent cell death in T lymphocytes: relevance to alcohol-induced immunosuppression.

An important aspect in alcohol abuse-associated immune suppression is the loss of T helper CD4(+) lymphocytes, leading to impairment of multiple immune functions. Our work has shown that ethanol can sensitize CD4(+) T lymphocytes to caspase-3-dependent activation-induced cell death (AICD). It has been demonstrated that the formation of S-adenosylmethionine (SAMe) catalyzed by methionine adenosyltransferase (MAT) II is essential for CD4(+) T-cell activation and proliferation. Since ethanol is known to affect SAMe metabolism in hepatocytes, we investigated the effect of ethanol on MAT II activity/expression, SAMe biosynthesis and cell survival in CD4(+) T lymphocytes. We demonstrate for the first time that ethanol at a physiologically relevant concentration (25 mM) substantially decreased the enzymatic activity of MAT II in T lymphocytes. Ethanol was observed to decrease the transcription of MAT2A, which encodes the catalytic subunit of MAT II and is vital for MAT II activity and SAMe biosynthesis. Furthermore, correspondent to its effect on MAT II, ethanol decreased intracellular SAMe levels and enhanced caspase-3-dependent AICD. Importantly, restoration of intracellular SAMe levels by exogenous SAMe supplementation considerably decreased both caspase-3 activity and apoptotic death in T lymphocytes. In conclusion, our data show that MAT II and SAMe are critical molecular components essential for CD4(+) T-cell survival that are affected by ethanol, leading to enhanced AICD. Furthermore, these studies provide a clinical paradigm for the development of much needed therapy using SAMe supplementation in the treatment of immune dysfunction induced by alcohol abuse.

Porphyromonas gingivalis enhances FasL expression via up-regulation of NFkappaB-mediated gene transcription and induces apoptotic cell death in human gingival epithelial cells.

The interaction between epithelial cells and micro-organisms is often a crucial initiating event in infectious diseases. Infection with Porphyromonas gingivalis, a Gram-negative anaerobe, is strongly associated with severe periodontal disease. This bacterium possesses an array of virulence factors, some of which can induce apoptosis. The tumour necrosis factor (TNF) receptor family is involved in the regulation of cellular homeostasis, cell surface molecules involved in phagocytosis, Fas ligand (L) expression and activation of the caspase cascade resulting in DNA fragmentation and cell blebbing. The current study examined the role of nuclear factor-kappaB (NFkappaB) in FasL-mediated apoptotic cell death in primary human gingival epithelial cells (HGEC) induced by heat-killed P. gingivalis, probably through TLR signalling pathways. A marked up-regulation of TLR2 and Fas-FasL was detected in HGEC stimulated with P. gingivalis. Activation of NFkappaB by P. gingivalis in HGEC was demonstrated by an NFkappaB promoter luciferase assay as well as by phosphorylation of p65 as detected by Western blotting. Activation of cleaved caspase-3 and caspase-8 resulted in apoptotic cell death of HGEC. The survival proteins c-IAP-1/c-IAP-2 were decreased in HGEC exposed to P. gingivalis. HGEC apoptosis induced by P. gingivalis was inhibited by an anti-human FasL monoclonal antibody. Blockade of NFkappaB by helenalin resulted in down-regulation of FasL whereas a caspase-8 inhibitor did not decrease FasL. Taken together, these studies show that P. gingivalis can induce epithelial cell apoptosis through Fas-FasL up-regulation and activation of caspase-3 and caspase-8.

S-adenosylmethionine (SAMe) modulates interleukin-10 and interleukin-6, but not TNF, production via the adenosine (A2) receptor.

S-adenosylmethionine (SAMe) is the first product in methionine metabolism and serves as a precursor for glutathione (GSH) as well as a methyl donor in most transmethylation reactions. The administration of exogenous SAMe has beneficial effects in many types of liver diseases. One mechanism for the hepatoprotective action is its ability to regulate the immune system by modulating cytokine production from LPS stimulated monocytes. In the present study, we investigated possible mechanism(s) by which exogenous SAMe supplementation modulated production of TNF, IL-10 and IL-6 in LPS stimulated RAW 264.7 cells, a murine monocyte cell line. Our results demonstrated that exogenous SAMe supplementation inhibited TNF production but enhanced both IL-10 and IL-6 production. SAMe increased intracellular GSH level, however, N-acetylcysteine (NAC), the GSH pro-drug, decreased the production of all three cytokines. Importantly, SAMe increased intracellular adenosine levels, and exogenous adenosine supplementation had effects similar to SAMe on TNF, IL-10 and IL-6 production. 3-Deaza-adenosine (DZA), a specific inhibitor of S-adenosylhomocysteine (SAH) hydrolase, blocked the elevation of IL-10 and IL-6 production induced by SAMe, which was rescued by the addition of exogenous adenosine. Furthermore, the enhancement of LPS-stimulated IL-10 and IL-6 production by both SAMe and adenosine was inhibited by ZM241385, a specific antagonist of the adenosine (A(2)) receptor. Our results suggest that increased adenosine levels with subsequent binding to the A(2) receptor account, at least in part, for SAMe modulation of IL-10 and IL-6, but not TNF production, from LPS stimulated monocytes.