Radiation response of head and neck squamous cell carcinoma using early markers of apoptosis and proliferation, utilising a microfluidic approach

IntroductionThe purpose of this study was to assess the response to irradiation of HNSCC tissue samples maintained in a microfluidic device, utilising early markers of apoptosis and proliferation.Materials and MethodsRat liver had initially been used to optimise the conditions so HNSCC tissue biopsies could be maintained in a pseudo in vivo environment. Parameters assessed were different flow rates, oxygenated media and concentrations of sera in the medium, buffer components and infusion times with bromodeoxyuridine. HNSCC tissue samples from the oral cavity, oropharynx, larynx, metastatic lymph node and maxillary sinus were obtained. Tissue from each subsite was irradiated with doses of 5Gy, 10Gy, 15Gy and 20Gy. Cell death was measured using LDH and the morphology of the tissue was assessed using H&E staining. Apoptosis was calculated using immunohistochemical techniques to detect cytokeratin and the M30 antibody; and proliferation was assessed using the bromodeoxyuridine assay.ResultsHNSCC tissue samples could be maintained in the microfluidic device after an initial rise in LDH levels and assessment of the tissue architecture using H&E staining. Statistically, there was no significant difference observed in LDH post irradiation. Apoptotic indices calculated for all subsites revealed variation within the same tissue with the same dose, statistically no significant difference between irradiation with different doses of the head and neck subsites. Proliferation was noted with the bromodeoxyuridine assay in three out of the five subsites but no significant pattern was observed.ConclusionThe microfluidic device is capable of maintaining HNSCC tissue in a viable state and is able to withstand rigorous testing. This study did not demonstrate a dose dependent relationship and suggests HNSCC intratumour heterogeneity would account for the variation in responses with irradiation. The microfluidic technique is a very useful method in assessing a patient’s response to radiotherapy and allows a personalised treatment approach for the future

An Open Core System-on-chip Platform

The design cycle required to produce a System-on-Chip can be reduced by providing pre-designed built-in features and functions such as configurable I/O, power and ground grids, block RAMs, timing generators and other embedded intellectual property (IP) blocks. A basic combination of such built-in features is known as a platform. The major objective of this thesis was to design and implement one such System-on-Chip platform using open IP cores targeting the TSMC-0.18 CMOS process. The integrated System-on-Chip platform, which contains approximately four million transistors, was synthesized using Synopsys - Design Compiler and placed and routed using Cadence - First Encounter, Silicon Ensemble. Design verification was done at the pre-synthesis, post-synthesis and post-layout levels using Mentor Graphics - ModelSim. Final layout was imported into Cadence - Virtuoso to perform design rule check. A tutorial was written to enable others to create derivative designs of this platform quickly

The stochastic quasi-steady-state assumption: Reducing the model but not the noise

Highly reactive species at small copy numbers play an important role in many biological reaction networks. We have described previously how these species can be removed from reaction networks using stochastic quasi-steady-state singular perturbation analysis (sQSPA). In this paper we apply sQSPA to three published biological models: the pap operon regulation, a biochemical oscillator, and an intracellular viral infection. These examples demonstrate three different potential benefits of sQSPA. First, rare state probabilities can be accurately estimated from simulation. Second, the method typically results in fewer and better scaled parameters that can be more readily estimated from experiments. Finally, the simulation time can be significantly reduced without sacrificing the accuracy of the solution

Generalization Error without Independence: Denoising, Linear Regression, and Transfer Learning

Studying the generalization abilities of linear models with real data is a central question in statistical learning. While there exist a limited number of prior important works (Loureiro et al. (2021A, 2021B), Wei et al. 2022) that do validate theoretical work with real data, these works have limitations due to technical assumptions. These assumptions include having a well-conditioned covariance matrix and having independent and identically distributed data. These assumptions are not necessarily valid for real data. Additionally, prior works that do address distributional shifts usually make technical assumptions on the joint distribution of the train and test data (Tripuraneni et al. 2021, Wu and Xu 2020), and do not test on real data. In an attempt to address these issues and better model real data, we look at data that is not I.I.D. but has a low-rank structure. Further, we address distributional shift by decoupling assumptions on the training and test distribution. We provide analytical formulas for the generalization error of the denoising problem that are asymptotically exact. These are used to derive theoretical results for linear regression, data augmentation, principal component regression, and transfer learning. We validate all of our theoretical results on real data and have a low relative mean squared error of around 1% between the empirical risk and our estimated risk

Measuring the response of human head and neck squamous cell carcinoma to irradiation in a microfluidic model allowing customized therapy

Radiotherapy is the standard treatment for head and neck squamous cell carcinoma (HNSCC), however, radioresistance remains a major clinical problem despite significant improvements in treatment protocols. Therapeutic outcome could potentially be improved if a patient's tumour response to irradiation could be predicted ex vivo before clinical application. The present study employed a bespoke microfluidic device to maintain HNSCC tissue whilst subjecting it to external beam irradiation and measured the responses using a panel of cell death and proliferation markers. HNSCC biopsies from five newly-presenting patients [2 lymph node (LN); 3 primary tumour (PT)] were divided into parallel microfluidic devices and replicates of each tumour were subjected to single-dose irradiation (0, 5, 10, 15 and 20 Gy). Lactate dehydrogenase (LDH) release was measured and tissue sections were stained for cytokeratin (CK), cleaved-CK18 (cCK18), phosphorylated-H2AX (λH2AX) and Ki.67 by immunohistochemistry. In addition, fragmented DNA was detected using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Compared with non.irradiated controls, higher irradiation doses resulted in elevated CK18-labelling index in two lymph nodes [15 Gy; 34.8% on LN1 and 31.7% on LN2 (p=0.006)] and a single laryngeal primary tumour (20 Gy; 31.5%; p=0.014). Significantly higher levels of DNA fragmentation were also detected in both lymph node samples and one primary tumour but at varying doses of irradiation, i.e., LN1 (20 Gy; 27.6%; p=0.047), LN2 (15 Gy; 15.3%; p=0.038) and PT3 (10 Gy; 35.2%; p=0.01). The λH2AX expression was raised but not significantly in the majority of samples. The percentage of Ki.67 positive nuclei reduced dose-dependently following irradiation. In contrast no significant difference in LDH release was observed between irradiated groups and controls. There is clear interand intra-patient variability in response to irradiation when measuring a variety of parameters, which offers the potential for the approach to provide clinically valuable information

Sequence based polymorphic (SBP) marker technology for targeted genomic regions: its application in generating a molecular map of the Arabidopsis thaliana genome

<p>Abstract</p> <p>Background</p> <p>Molecular markers facilitate both genotype identification, essential for modern animal and plant breeding, and the isolation of genes based on their map positions. Advancements in sequencing technology have made possible the identification of single nucleotide polymorphisms (SNPs) for any genomic regions. Here a sequence based polymorphic (SBP) marker technology for generating molecular markers for targeted genomic regions in Arabidopsis is described.</p> <p>Results</p> <p>A ~3X genome coverage sequence of the <it>Arabidopsis thaliana </it>ecotype, Niederzenz (Nd-0) was obtained by applying Illumina's sequencing by synthesis (Solexa) technology. Comparison of the Nd-0 genome sequence with the assembled Columbia-0 (Col-0) genome sequence identified putative single nucleotide polymorphisms (SNPs) throughout the entire genome. Multiple 75 base pair Nd-0 sequence reads containing SNPs and originating from individual genomic DNA molecules were the basis for developing co-dominant SBP markers. SNPs containing Col-0 sequences, supported by transcript sequences or sequences from multiple BAC clones, were compared to the respective Nd-0 sequences to identify possible restriction endonuclease enzyme site variations. Small amplicons, PCR amplified from both ecotypes, were digested with suitable restriction enzymes and resolved on a gel to reveal the sequence based polymorphisms. By applying this technology, 21 SBP markers for the marker poor regions of the Arabidopsis map representing polymorphisms between Col-0 and Nd-0 ecotypes were generated.</p> <p>Conclusions</p> <p>The SBP marker technology described here allowed the development of molecular markers for targeted genomic regions of Arabidopsis. It should facilitate isolation of co-dominant molecular markers for targeted genomic regions of any animal or plant species, whose genomic sequences have been assembled. This technology will particularly facilitate the development of high density molecular marker maps, essential for cloning genes based on their genetic map positions and identifying tightly linked molecular markers for selecting desirable genotypes in animal and plant breeding experiments.</p

Hedgehog Signaling Antagonist GDC-0449 (Vismodegib) Inhibits Pancreatic Cancer Stem Cell Characteristics: Molecular Mechanisms

Recent evidence from in vitro and in vivo studies has demonstrated that aberrant reactivation of the Sonic Hedgehog (SHH) signaling pathway regulates genes that promote cellular proliferation in various human cancer stem cells (CSCs). Therefore, the chemotherapeutic agents that inhibit activation of Gli transcription factors have emerged as promising novel therapeutic drugs for pancreatic cancer. GDC-0449 (Vismodegib), orally administrable molecule belonging to the 2-arylpyridine class, inhibits SHH signaling pathway by blocking the activities of Smoothened. The objectives of this study were to examine the molecular mechanisms by which GDC-0449 regulates human pancreatic CSC characteristics in vitro.GDC-0499 inhibited cell viability and induced apoptosis in three pancreatic cancer cell lines and pancreatic CSCs. This inhibitor also suppressed cell viability, Gli-DNA binding and transcriptional activities, and induced apoptosis through caspase-3 activation and PARP cleavage in pancreatic CSCs. GDC-0449-induced apoptosis in CSCs showed increased Fas expression and decreased expression of PDGFRα. Furthermore, Bcl-2 was down-regulated whereas TRAIL-R1/DR4 and TRAIL-R2/DR5 expression was increased following the treatment of CSCs with GDC-0449. Suppression of both Gli1 plus Gli2 by shRNA mimicked the changes in cell viability, spheroid formation, apoptosis and gene expression observed in GDC-0449-treated pancreatic CSCs. Thus, activated Gli genes repress DRs and Fas expressions, up-regulate the expressions of Bcl-2 and PDGFRα and facilitate cell survival.These data suggest that GDC-0499 can be used for the management of pancreatic cancer by targeting pancreatic CSCs

In-vitro antibacterial activity of Zingiber officinale (ginger) against bacteria isolated from reproductive tract of clinical endometritic Murrah buffaloes

Endometritis, an inflammation of the innermost uterine layer, is caused by a variety of bacteria, including Gram- positive and Gram-negative aerobes and anaerobes. This study aimed to identify the active compounds in Zingiber officinale (ginger) and assess its effectiveness against common bacterial pathogens found in Murrah buffaloes suffering from endometritis. The infrared spectrum of ginger powder displayed a range of wavelengths from 3361.89/cm to 664.26/cm, indicating the presence of eight functional groups. In the hydro-ethanolic ginger extract, the spectrum ranged from 3402.8/cm to 765.35/cm, revealing 12 functional groups. A total of 42 vaginal mucus samples were collected, with E. coli being the most prevalent in 23 samples, followed by S. aureus in 21 samples, Pseudomonas spp. in 12 samples, and Campylobacter spp. in 11 samples. To evaluate the in vitro antibacterial activity of ginger extracts, antimicrobial susceptibility tests were conducted at concentrations of 2.5, 5, 10, 20, 30, 40, and 50 mg/ml. Staphylococcus aureus exhibited the highest sensitivity to the ginger extract, while E. coli showed substantial resistance. In conclusion, ginger demonstrates potent antibacterial activity against multidrug-resistant uterine pathogens and could serve as an alternative therapy to mitigate the risk of drug resistance in treating uterine infections

Congenital Absence of Posterior Elements of C2 Vertebra with Atlanto-Axial Dislocation and Basilar Invagination: A Case Report and Review of Literature

Developmental anomalies of the axis are commonly encountered, especially anomalies involving the odontoid process. Anomalies of the posterior elements are uncommon. We describe a unique case of agenesis of posterior elements of C2 with basilar invagination and atlanto-axial dislocation. An obese 8-year-old boy presented with symptoms of cervical myelopathy. Radiological workup revealed a craniovertebral junction anomaly with occipitalised atlas, absent posterior elements of axis, and hypertrophied C3 spinous process. Atlanto-axial instability and basilar invagination was present. Magnetic resonance angiography revealed hypoplastic left vertebral artery. Traction with cervical tongs failed to improve the alignment and symptoms. Anterior trans-oral release, followed by posterior decompression and custom-made instrumentation, was done. The patient recovered completely and was asymptomatic at the end of two years. X-ray and computed tomography scan demonstrated reduction of basilar invagination and maintenance of alignment. This is the first case to be reported of agenesis of posterior elements of axis associated with basilar invagination. One should look for this condition in patients with hypertrophied spinous process of C3. Utilization of hypoplastic pedicle of axis serves as an additional fixation point to increase the stability of the construct

The Versatile Approach: A Novel Single Incision Combined with Anterior and Posterior Approaches for Decompression and Instrumented Fusion to Treat Tuberculosis of the Thoracic Spine

Study DesignRetrospective case series.PurposeTo describe a novel single incision that combines anterior and posterior approaches for decompression and instrumented fusion to treat tuberculosis of the thoracic spine and study the neurological and radiological outcomes.Overview of LiteratureTuberculosis of the spine remains a major health issue in many developing countries. The options for treating tuberculosis of the thoracic spine include the anterior, posterior, and combined approaches, each with its advantages and disadvantages.MethodsTotally, 143 patients with tuberculosis of the thoracic spine were surgically treated using the “Versatile approach”. Posterior fixation was performed using sublaminar wires and a Hartshill rectangle in all patients. Anterior reconstruction was accomplished using bone graft harvested from autologous rib, iliac crest, or fibula.ResultsThe study included 45 males and 98 females, with a mean age of 33.18±18.65 years (range, 3–82 years) and a mean follow-up of 60.23±24.56 months (range, 18–156 months). Kyphosis improved from a mean value of 24.02 preoperatively to 10.25 postoperatively. A preoperative neurological deficit was observed in 131 patients, with 130 patients regaining ambulatory power. No patient had deterioration of neurological status following surgery. Fusion was achieved in all cases. The visual analogscale score improved from an average score of 7.02 preoperatively to 1.51 at final follow-up. Eight patients had superficial macerations, which healed spontaneously. One patient had buckling of the anterior graft, and one patient had implant breakage following road traffic accident.ConclusionsThe “Versatile approach” is an effective, single-stage, single-incision method that combines anterior and posterior approaches for the surgically treating tuberculosis of the thoracic spine. It offers the advantage of direct visualization for decompression and reconstruction of the anterior and posterior vertebral columns, thus providing an excellent, long-lasting clinical outcome