Shape-Specific Gold Nanoparticles for Multiplex Biosensing Applications.

The biosensing field faces a significant challenge in efficiently detecting multiple analytes in a single diagnostic sample in order to compete with other established multiplex molecular diagnostic technologies such as PCR and ELISA. In response, we have developed a colorimetric nanobiosensor based on multiple morphological forms of functionalized gold nanoparticles (AuNPs) for the simultaneous detection of the influenza virus and SARS-CoV-2 virus. Gold nanospheres (GNSp) were modified with oligonucleotides specific for the influenza A virus, while gold nanoshells (GNSh) were modified with oligonucleotides specific for the SARS-CoV-2 virus. In the presence of their respective targets, AuNPs remain stable due to DNA-DNA interactions; conversely, in the absence of targets, AuNPs aggregate. Consequently, the hybrid system exhibits an indigo color with the SARS-CoV-2 target, a blue color with the Influenza A target, and a purple color with both targets, visible to the naked eye. Analytical sensitivity was 100 nM, and no cross-reactivity was observed with potentially confounding pathogens. This approach holds great promise for the simultaneous identification of multiple pathogens in a rapid manner without the need for equipment or trained personnel.

Predicting Survival of Metastatic Clear Cell Renal Cell Cancer Treated with VEGFR-TKI-Based Sequential Therapy.

(1) Objective: To develop a clinically useful nomogram that may provide a more individualized and accurate estimation of cancer-specific survival (CSS) for patients with clear-cell (CC) metastatic renal cell carcinoma (mRCC) treated with nephrectomy and vascular endothelial growth factor receptor-tyrosine kinase inhibitor (VEGFR-TKI)-based sequential therapy. (2) Methods: A prospectively maintained database of 145 patients with mRCC treated between 2008 and 2018 was analyzed to predict the CSS of patients receiving sunitinib and second- and third-line therapies according to current standards of practice. A nomogram based on four independent clinical predictors (Eastern Cooperative Oncology Group status, International Metastatic RCC Database Consortium score, the Morphology, Attenuation, Size and Structure criteria and Response Evaluation Criteria in Solid Tumors response criteria) was calculated. The corresponding 1- to 10-year CSS probabilities were then determined from the nomogram. (3) Results: The median age was 60 years (95% CI 57.9-61.4). The disease was metastatic at diagnosis in 59 (40.7%), and 86 (59.3%) developed metastasis during follow-up. Patients were followed for a median 48 (IQR 72; 95% CI 56-75.7) months after first-line VEGFR-TKI initiation. The concordance probability estimator value for the nomogram is 0.778 ± 0.02 (mean ± SE). (4) Conclusions: A nomogram to predict CSS in patients with CC mRCC that incorporates patient status, clinical risk classification and response criteria to first-line VEGFR-TKI at 3 months is presented. This new tool may be useful to clinicians assessing the risk and prognosis of patients with mRCC.

Efficient enrichment of free target sequences in an integrated microfluidic device for point-of-care detection systems.

Nucleic acid biomarker detection has great importance in the diagnosis of disease, the monitoring of disease progression and the classification of patients according to treatment decision making. Nucleic acid biomarkers found in the blood of patients have generated a lot of interest due to the possibility of being detected non-invasively which makes them ideal for monitoring and screening tests and particularly amenable to point-of-care (POC) or self-testing. A major challenge to POC molecular diagnostics is the need to enrich the target to optimise detection. In this work, we describe a microfabricated device for the enrichment of short dsDNA target sequences, which is especially valuable for potential detection methods, as it improves the probability of effectively detecting the target in downstream analyses. The device integrated a heating element and a temperature sensor with a microfluidic chamber to carry out the denaturation of the dsDNA combined with blocking-probes to enrich the target. This procedure was validated by fluorescence resonance energy transfer (FRET) technique, labelling DNA with a fluorophore and a quencher. As proof of concept, a 23-mer long dsDNA sequence corresponding to the L858R mutation of the EGFR gene was used. The qualitative results obtained determined that the most optimal blocking rate was obtained with the incorporation of 11/12-mer blocking-probes at a total concentration of 6 µM. This device is a powerful DNA preparation tool, which is an indispensable initial step for subsequent detection of sequences via nucleic acid hybridisation methods.

Identification of miRNAs and Their Target Genes Associated with Sunitinib Resistance in Clear Cell Renal Cell Carcinoma Patients.

Sunitinib has greatly improved the survival of clear cell renal cell carcinoma (ccRCC) patients in recent years. However, 20-30% of treated patients do not respond. To identify miRNAs and genes associated with a response, comparisons were made between biopsies from responder and non-responder ccRCC patients. Using integrated transcriptomic analyses, we identified 37 miRNAs and 60 respective target genes, which were significantly associated with the NF-kappa B, PI3K-Akt and MAPK pathways. We validated expression of the miRNAs (miR-223, miR-155, miR-200b, miR-130b) and target genes (FLT1, PRDM1 and SAV1) in 35 ccRCC patients. High levels of miR-223 and low levels of FLT1, SAV1 and PRDM1 were associated with worse overall survival (OS), and combined miR-223 + SAV1 levels distinguished responders from non-responders (AUC = 0.92). Using immunohistochemical staining of 170 ccRCC patients, VEGFR1 (FLT1) expression was associated with treatment response, histological grade and RECIST (Response Evaluation Criteria in Solid Tumors) score, whereas SAV1 and BLIMP1 (PRDM1) were associated with metachronous metastatic disease. Using in situ hybridisation (ISH) to detect miR-155 we observed higher tumoural cell expression in non-responders, and non-tumoural cell expression with increased histological grade. In summary, our preliminary analysis using integrated miRNA-target gene analyses identified several novel biomarkers in ccRCC patients that surely warrant further investigation.

Power of Light: Raman Spectroscopy and Machine Learning for the Detection of Lung Cancer.

Lung cancer is the leading cause of cancer-related deaths worldwide, emphasizing the urgent need for reliable and efficient diagnostic methods. Conventional approaches often involve invasive procedures and can be time-consuming and costly, thereby delaying the effective treatment. The current study explores the potential of Raman spectroscopy, as a promising noninvasive technique, by analyzing human blood plasma samples from lung cancer patients and healthy controls. In a benchmark study, 16 machine learning models were evaluated by employing four strategies: the combination of dimensionality reduction with classifiers; application of feature selection prior to classification; stand-alone classifiers; and a unified predictive model. The models showed different performances due to the inherent complexity of the data, achieving accuracies from 0.77 to 0.85 and areas under the curve for receiver operating characteristics from 0.85 to 0.94. Hybrid methods incorporating dimensionality reduction and feature selection algorithms present the highest figures of merit. Nevertheless, all machine learning models deliver creditable scores and demonstrate that Raman spectroscopy represents a powerful method for future in vitro diagnostics of lung cancer.

Non-coding RNA and gene expression analyses of papillary renal neoplasm with reverse polarity (PRNRP) reveal distinct pathological mechanisms from other renal neoplasms.

Papillary renal neoplasm with reversed polarity (PRNRP) is a recently described rare renal neoplasm. Traditionally, it was considered a variant of papillary renal cell carcinoma (PRCC). However, several studies reported significant differences between PRNRP and PRCC in terms of clinical, morphological, immunohistochemical and molecular features. Nonetheless, PRNRP remains a poorly understood entity. We used microarray analysis to elucidate the non-coding RNA (ncRNA) and gene expression profiles of 10 PRNRP cases and compared them with other renal neoplasms. Unsupervised cluster analysis showed that PRNRP had distinct expression profiles from either clear cell renal cell carcinoma (ccRCC) or PRCC cases at the level of ncRNA but were less distinct at the level of gene expression. An integrated omic approach determined miRNA:gene interactions that distinguished PRNRP from PRCC and we validated 10 differentially expressed miRNAs and six genes by quantitative RT-PCR. We found that levels of the miRNAs, miR-148a, miR-375 and miR-429, were up-regulated in PRNRP cases compared to ccRCC and PRCC. miRNA target genes, including KRAS and VEGFA oncogenes, and CXCL8, which regulates VEGFA, were also differentially expressed between renal neoplasms. Gene set enrichment analysis (GSEA) determined different activation of metabolic pathways between PRNRP and PRCC cases. Overall, this study is by far the largest molecular study of PRNRP cases and the first to investigate either ncRNA expression or their gene expression by microarray assays.

Robotic-assisted total hip arthroplasty utilizing a fluoroscopy-guided system resulted in improved intra-operative efficiency relative to a computerized tomography-based platform.

Robotic-assisted total hip arthroplasty (THA) using a computerized-tomography (CT) based workflow increases surgical time relative to traditional manual technique. The purpose of this investigation was to compare the intra-operative efficiencies of two robotic THA systems: a fluoroscopy-based platform (FL-RTHA) and a contemporary, CT-based (CT-RTHA) platform. A review of 107 consecutive FL-RTHA and 159 CT-RTHA primary, direct anterior approach (DAA) THA procedures was conducted. All cases were performed by one of two surgeons operating at the same institution, for a pre-operative diagnosis of osteoarthritis, avascular necrosis, or rheumatoid arthritis. Primary outcome variables included averages and consistencies (variances) for surgical times and operating room (OR) times. A secondary outcome was to quantify the duration of robot-active phases in the FL-RTHA workflow. The FL-RTHA cohort experienced shorter surgical times (38.71 min ± 7.00 vs. 75.33 min ± 11.38; p < 0.001) and OR times (101.35 min ± 12.22 vs. 156.74 min ± 17.79; p < 0.001) compared to the CT-RTHA cohort. Surgical times and OR times were both more consistent in the FL-RTHA cohort compared to the CT-RTHA cohort (p < 0.001). Patients who underwent DAA THA with the assistance of a fluoroscopy-based robotic system experienced shorter and more consistent surgical times and OR times compared to patients who underwent similar DAA THA procedures with a contemporary, CT-based robotic platform.

The role of microRNAs in the toxic mechanisms of furan and 3-MCPD and links to cancer risk - a review.

Contaminants formed during food processing are of increasing concern to public and food safety experts, as well as international risk assessment organizations. The emergence of 'omic' technologies (e.g. genomics and transcriptomics) have greatly increased the mechanistic knowledge of the toxicity associated with these compounds, and consequently have provided a better understanding of their potential adverse effects. Of note, microRNAs (miRNAs) have emerged as being of key importance during the development of cancer as well as being associated with food-processing contaminants. MiRNAs have been demonstrated to trigger toxic processes in hepatic and renal tissues due to exposure to toxic compounds such as furan and 3-monochloropropane-1,2-diol (3-MCPD), respectively. In this review, we consider the roles of miRNAs in the toxicity process and the challenges that lay ahead in order to translate this knowledge to the benefit of industrial food processing.

The learning curve for a novel, fluoroscopy-based robotic-assisted total hip arthroplasty system.

BACKGROUND: Adoption of robotic-assisted total hip arthroplasty (RA-THA) systems can improve the accuracy of acetabular cup placement, but no group has reported the learning curve of novel, fluoroscopy-based RA-THA systems. METHODS: A learning-curve cumulative summation (LC-CUSUM) analysis was performed on a consecutive series of the first 100 patients who received fluoroscopy-based RA-THA by the study surgeon. Operative times and specific robotic timepoints were compared between learning and proficiency phases. RESULTS: Implementation of fluoroscopy-based RA-THA was associated with a learning curve of 12 cases. A 6-min increase in operative time was seen during the learning phase compared to the proficiency phase (44.3 ± 4.4 vs. 38.0 ± 7.1 min; p < 0.001), with a 3-min longer robotic cup impaction sequence during the learning phase (7.8 ± 1.9 vs. 4.8 ± 1.3 min; p < 0.001). CONCLUSION: Adoption of fluoroscopy-based RA-THA is associated with a brief learning curve of 12 cases, with the most significant improvements in surgical efficiency realised during acetabular cup placement.

Chitranjan S. Ranawat Award: Tourniquet Use Does Not Impact Trajectory of Total Knee Arthroplasty Early Recovery: A Prospective, Randomized Controlled Trial.

BACKGROUND: The impact of tourniquet use on recovery after total knee arthroplasty (TKA) remains controversial. The purpose of this prospective, single blinded, randomized controlled trial was to investigate the effect of tourniquet use on early recovery after TKA using a smartphone app-based patient engagement platform (PEP) with a wrist-based activity monitor to obtain more robust data on early recovery. METHODS: There were 107 patients undergoing primary TKA for osteoarthritis who were enrolled (54 tourniquet [TQ+]; 53 no tourniquet [TQ-]). All patients utilized a PEP and wrist-based activity sensor for 2 weeks preoperatively and 90 days postoperatively to collect Visual Analog Scale (VAS) pain scores and opioid consumption, as well as weekly Oxford Knee Score (OKS) and monthly Forgotten Joint Score (FJS). There was no difference in demographics between groups. Formal physical therapy assessments were performed preoperatively and 3 months postoperatively. Independent sample t-tests were used for continuous data and Chi-square and Fisher's exact tests were used for discrete data. RESULTS: Tourniquet use did not have a statistically significant impact on daily VAS pain or opioid consumption during the first 30 days postoperatively (P > .05). Tourniquet use did not have a significant impact on OKS or FJS at 30 or 90 days postoperatively (P > .05), or on performance of formal physical therapy testing at 3 months postoperatively (P > .05). CONCLUSION: Using a digital technology to collect daily patient data, we found that tourniquet use has no clinically significant negative impact on pain and function in the first 90 days after primary TKA.

Targeting oncogenic functions of miR-301a in head and neck squamous cell carcinoma by PI3K/PTEN and MEK/ERK pathways.

Treatment of head and neck squamous cell carcinomas (HNSCC), the sixth most frequent cancer worldwide, remains challenging. miRNA dysregulation is closely linked to tumorigenesis and tumor progression, thus emerging as suitable targets for cancer treatment. Transcriptomic analysis of TCGA HNSCC dataset revealed that miR-301a expression levels significantly increased in primary tumors, as compared to patient-matched normal tissue. This prompted us to investigate its pathobiological role and potential as new therapeutic target using different preclinical HNSCC models. miR-301a overexpression in HNSCC-derived cell lines led to enhanced proliferation and invasion, whereas miR-301 inhibition reduced these effects. In vivo validation was performed using an orthotopic mouse model. Results concordantly showed that the mitotic counts, the percentage of infiltration depth and Ki67 proliferative index were significantly augmented in the subgroup of mice harboring miR-301a-overexpressing tumors. Further mechanistic characterization revealed PI3K/PTEN/AKT and MEK/ERK pathways as central signaling nodes responsible for mediating the oncogenic activity of miR-301a observed in HNSCC cells. Notably, pharmacological disruption of PI3K and ERK signals with BYL-719 and PD98059, respectively, was effective to completely revert/abolish miR-301a-promoted tumor cell growth and invasion. Altogether, these findings demonstrate that miR-301a dysregulation plays an oncogenic role in HNSCC, thus emerging as a candidate therapeutic target for this disease. Importantly, available PI3K and ERK inhibitors emerge as promising anti-tumor agents to effectively target miR-301a-mediated signal circuit hampering growth-promoting and pro-invasive functions.

Validation of Rapid and Economic Colorimetric Nanoparticle Assay for SARS-CoV-2 RNA Detection in Saliva and Nasopharyngeal Swabs.

Even with the widespread uptake of vaccines, the SARS-CoV-2-induced COVID-19 pandemic continues to overwhelm many healthcare systems worldwide. Consequently, massive scale molecular diagnostic testing remains a key strategy to control the ongoing pandemic, and the need for instrument-free, economic and easy-to-use molecular diagnostic alternatives to PCR remains a goal of many healthcare providers, including WHO. We developed a test (Repvit) based on gold nanoparticles that can detect SARS-CoV-2 RNA directly from nasopharyngeal swab or saliva samples with a limit of detection (LOD) of 2.1 × 105 copies mL-1 by the naked eye (or 8 × 104 copies mL-1 by spectrophotometer) in less than 20 min, without the need for any instrumentation, and with a manufacturing price of <$1. We tested this technology on 1143 clinical samples from RNA extracted from nasopharyngeal swabs (n = 188), directly from saliva samples (n = 635; assayed by spectrophotometer) and nasopharyngeal swabs (n = 320) from multiple centers and obtained sensitivity values of 92.86%, 93.75% and 94.57% and specificities of 93.22%, 97.96% and 94.76%, respectively. To our knowledge, this is the first description of a colloidal nanoparticle assay that allows for rapid nucleic acid detection at clinically relevant sensitivity without the need for external instrumentation that could be used in resource-limited settings or for self-testing.

Circulating MicroRNAs as Cancer Biomarkers in Liquid Biopsies.

Biological fluids such as blood, saliva, and urine offer a rich source of biomarkers that have the potential to change the paradigm of cancer management. By allowing routine noninvasive sampling that can offer new insights into cancer progression and response to treatment so-called liquid biopsies can play an important role in personalized medicine. MicroRNAs (miRNAs) are a particularly attractive class of biomarkers as they are not only resistant to the high levels of RNases found in biological fluids, but also able to confer clinically useful information about the disease relating to diagnosis, prognosis, and the response to treatment. Circulating miRNAs are either associated with proteins or extracellular vesicles (EV) and although their origin and implied functions as intracellular messengers remain somewhat controversial, they are implicated in the progression and the establishment of metastatic niches. In this chapter, we review the rapidly emerging field of circulating miRNA cancer biomarkers, their origin and function, techniques to detect these molecules, and the use of bioinformatic tools to derive implied regulatory function, as well as the challenges that lie ahead for their clinical implementation.

Sleep Disturbances and Disorders in Patients with Knee Osteoarthritis and Total Knee Arthroplasty.

➤: There is increasing evidence that patient-reported outcomes following total knee arthroplasty (TKA) are associated with psychosocial factors and pain catastrophizing. Sleep disturbance, pain, and mental health have a complex interaction, which, if unrecognized, can be associated with impaired patient-reported outcomes and dissatisfaction following TKA. ➤: The gold standard of objective sleep assessment is polysomnography, which is not feasible to use routinely for TKA patients. Wearable devices are a validated and less costly alternative. ➤: Subjective sleep measures, such as the Pittsburgh Sleep Quality Index, Epworth Sleepiness Scale, or Patient-Reported Outcomes Measurement Information System (PROMIS) computerized adaptive test sleep domains, are simple to administer and provide additional insight into sleep disturbance. Although objective and subjective measures do not correlate precisely, they can be informative together. ➤: Sleep disturbances in the elderly population are common and multifactorial in etiology, stemming from the interplay of sleep disorders, medication side effects, and pain. Commonly prescribed medications following TKA as well as postoperative pain can exacerbate underlying sleep disturbances. ➤: Obstructive sleep apnea (OSA) is prevalent in patients seeking TKA. In the setting of OSA, postoperative opioids can cause respiratory depression, resulting in consequences as severe as death. A standardized multimodal pain protocol including anti-inflammatories and gamma-aminobutyric acid (GABA) analogues may allow for decreased reliance on opioids for pain control. ➤: Surgeons should reassure patients that postoperative sleep disturbance is common and transient, collaborate with the patient's primary care doctor to address sleep disturbance, and avoid prescription of pharmaceutical sleep aids.

High SOX9 Maintains Glioma Stem Cell Activity through a Regulatory Loop Involving STAT3 and PML.

Glioma stem cells (GSCs) are critical targets for glioma therapy. SOX9 is a transcription factor with critical roles during neurodevelopment, particularly within neural stem cells. Previous studies showed that high levels of SOX9 are associated with poor glioma patient survival. SOX9 knockdown impairs GSCs proliferation, confirming its potential as a target for glioma therapy. In this study, we characterized the function of SOX9 directly in patient-derived glioma stem cells. Notably, transcriptome analysis of GSCs with SOX9 knockdown revealed STAT3 and PML as downstream targets. Functional studies demonstrated that SOX9, STAT3, and PML form a regulatory loop that is key for GSC activity and self-renewal. Analysis of glioma clinical biopsies confirmed a positive correlation between SOX9/STAT3/PML and poor patient survival among the cases with the highest SOX9 expression levels. Importantly, direct STAT3 or PML inhibitors reduced the expression of SOX9, STAT3, and PML proteins, which significantly reduced GSCs tumorigenicity. In summary, our study reveals a novel role for SOX9 upstream of STAT3, as a GSC pathway regulator, and presents pharmacological inhibitors of the signaling cascade.

James A. Rand Young Investigator's Award: Questioning the "Nickel Free" Total Knee Arthroplasty.

BACKGROUND: A study was performed to measure metal ions present in the knee joint after performing a total knee arthroplasty (TKA) with standard cobalt chromium (CoCr) components as well as with "nickel-free" oxidized zirconium femoral and titanium tibial (OxZr/Ti) components. METHODS: Knee joint fluid was collected prior to arthrotomy, and on postoperative day one to determine the amount of metal debris generated when performing a TKA with standard instrumentation from consecutive cases with CoCr components (n = 24) and OxZr/Ti components (n = 16). RESULTS: CoCr implant patients had statistically higher levels of nickel (Ni) (29.7%, P = .033), cobalt (Co), (1,100.7%, P < .0001) and chromium (Cr) (118.9%, P < .0001) postoperatively. The cutting blocks and sawblades do not contain Co, which therefore must have come from the components. The metal ions generated from the sawblades and cutting blocks, therefore, could be discerned from the OxZr/Ti whose components don't contain Co, Cr, or Ni. The OxZr patients had significantly higher Cr (9.5×, P < .001) and Ni (5.1×, P < .001) post-TKA vs pre-TKA; Co levels were not significantly different as expected with the absence of Co in the components (P = .60). The Ni levels generated in performing an Oxinium TKA was 3.3 times higher than when performing a CoCr TKA (1.37 vs. 41 ppb, P < .001). CONCLUSIONS: The substantial degree of Ni generation resulting from performing a hypoallergenic "nickel-free" TKA calls into questions the rationale of utilizing more expensive lower Ni components on the basis of known or suspected Ni or Cr allergy.

Clear Cell Renal Cell Carcinomas with Aggressive Behavior Display Low Intratumor Heterogeneity at the Histological Level.

PURPOSE OF REVIEW: Intratumor heterogeneity (ITH) is an inherent characteristic of most tumors and its detection remains a key task for pathologists. However, the clinical significance of the degree of development of this feature is still poorly understood. RECENT FINDINGS: A series of 28 clear cell renal cell carcinomas (CCRCC) have been exhaustively analyzed with two different sampling protocols [multisite tumor sampling (MSTS) and total sampling] to evaluate to what point the level (low vs. high) of histological ITH detected in routine practice influences tumor behavior and patients' survival. All CCRCC (n = 14) pursuing an aggressive clinical course presented low levels of ITH. A significant worse survival was detected in CCRCC with low ITH (p < 0.001). The simple quantification of the level of ITH using extensive sampling protocol may be of help in predicting tumor evolution, since all CCRCC with aggressive behavior demonstrated low levels of histological ITH.

The Impact of Component Design and Fixation on Stress Shielding After Modern Total Knee Arthroplasty.

BACKGROUND: Total knee arthroplasty (TKA) tibial baseplate thickness, metallurgy, and implant fixation with cement may influence stress shielding. The purpose of the present study is to compare bone mineral density of 2 cemented and press-fit TKA designs with differing tibial baseplate thicknesses and metallurgy over a 2-year period to assess for changes in stress shielding. METHODS: One-hundred one TKAs were performed in this Institutional Review Board-approved, prospective study. There were 4 cohorts: DePuy Attune cemented and press-fit, and Stryker Triathlon cemented and press-fit. The Attune tibial baseplate was thicker; both cemented tibial and femoral components were cobalt-chromium. The DePuy Attune press-fit had a cobalt-chromium sintered bead porous coating while the Stryker Triathlon was 3-dimensional printed highly porous titanium alloy. All patients had quantitative dual-energy X-ray absorptiometry scans performed at baseline (4-6 weeks postoperatively) and at 1 and 2 years postoperatively. Stress shielding was evaluated by comparing percent change in bone mineral density in 11 radiographic zones over 2 years. RESULTS: Over a 2-year period, there were no differences in stress shielding on the tibial side in either cemented or press-fit between Stryker Triathlon and DePuy Attune; however, there were differences on the femoral side. The press-fit tibial components of the Stryker Triathlon and DePuy Attune had either similar or less stress shielding over a 2-year period compared to their cemented counterparts. CONCLUSION: This study comparing 2 TKA implants with differing tibial tray thickness did not find significant differences in tibial stress shielding between designs. There was a difference in stress shielding on the femoral side between designs, suggesting that longer term follow-up is warranted.