An internal pilot study of a novel rectal mucocellular sampling device to allow next-generation sequencing for colorectal disease.

BACKGROUND: The ORI-EGI-02 study was designed to test the hypothesis that rectal mucus collected using a novel rectal sampling device (OriCol™), contains sufficient human deoxyribonucleic acid (DNA) of the required quality for Next Generation Sequencing (NGS), for colorectal disease genetic signature discovery. METHODS: Using National Institute for Health and Care Research methodology, an internal pilot study was performed in January 2020-May 2021, at four sites in the United Kingdom, to assess the process of recruitment, consent, specimen acquisition and viability for analysis. Following an OriCol™ test, the sample was stabilized with a buffer solution to preserve the material, which was posted to the laboratory. Samples were processed using QIAamp® DNA Blood Midi kit to extract DNA and Quant-iT™ PicoGreen® dsDNA Reagent to quantify the retrieved DNA. DNA integrity was measured by Agilent TapeStation system. 25 ng of human amplifiable DNA was prepared for Next Generation Sequencing (NGS), which was performed on an Illumina NextSeq550 sequencer using the 300-cycle high output kit v2.5. RESULTS: This study assessed the first 300 patients enrolled to the ORI-EGI-02 Study (n = 800). 290/300 (96.67%) were eligible to undergo OriCol™ sampling procedure and 285/290 (98.27%) had a successful OriCol™ sample taken. After transportation, extraction and quantification of DNA, 96.20% (279/290) of the samples had NGS successfully performed for bioinformatic analysis. CONCLUSIONS: Our internal pilot study demonstrated that the OriCol™ sampling device can capture rectal mucus from unprepared bowel in subjects who could undergo a digital rectal examination. The technique could be applied irrespective of age, frailty, or co-morbidity. Completion of the study to 800 patients and analysis of NGS data for colorectal cancer mutations will now proceed.

A Pilot Study for a Quantitative Evaluation of Acoustic Coupling in US-guided Focused Ultrasound Surgery.

In Ultrasound-guided High Intensity Focused Ultrasound (USgHIFU) surgery, the verification of the acoustic coupling correctness between the HIFU transducer and the patient's body is a fundamental step for an efficient and safe therapy. Nowadays, clinicians perform this check by qualitative inspecting Ultrasound images. The aim of this study is the introduction of an objective index to quantitively evaluate the coupling on the base of the radiofrequency echo signals acquired during a low-energy HIFU shot. The experimental session involved a tissue mimicking phantom and a robotic system composed by a HIFU therapeutic transducer and a 2D confocal Ultrasound probe. 15 different coupling conditions between the phantom and the transducer were tested: in each of them, the maximum absolute value of the Fourier Transform of the echo signals was computed and employed to determine an Acoustic Coupling (AC) coefficient.This metrics showed a sigmoidal trend between AC coefficient and coupling increase. This curve can be employed as a calibration tool to quantitatively assess the correctness of the therapeutic set-up before starting the HIFU treatment.

Motion compensation with skin contact control for high intensity focused ultrasound surgery in moving organs.

High intensity focused ultrasound (HIFU) is an emerging therapeutic solution that enables non-invasive treatment of several pathologies, mainly in oncology. On the other hand, accurate targeting of moving abdominal organs (e.g. liver, kidney, pancreas) is still an open challenge. This paper proposes a novel method to compensate the physiological respiratory motion of organs during HIFU procedures, by exploiting a robotic platform for ultrasound-guided HIFU surgery provided with a therapeutic annular phased array transducer. The proposed method enables us to keep the same contact point between the transducer and the patient's skin during the whole procedure, thus minimizing the modification of the acoustic window during the breathing phases. The motion of the target point is compensated through the rotation of the transducer around a virtual pivot point, while the focal depth is continuously adjusted thanks to the axial electronically steering capabilities of the HIFU transducer. The feasibility of the angular motion compensation strategy has been demonstrated in a simulated respiratory-induced organ motion environment. Based on the experimental results, the proposed method appears to be significantly accurate (i.e. the maximum compensation error is always under 1 mm), thus paving the way for the potential use of this technique for in vivo treatment of moving organs, and therefore enabling a wide use of HIFU in clinics.

A computer-assisted robotic platform for Focused Ultrasound Surgery: Assessment of high intensity focused ultrasound delivery.

In the last century, medicine showed considerable advancements in terms of new technologies, devices and diagnostic/therapeutic strategies. Those advantages led to a significant reduction of invasiveness and an improvement of surgical outcomes. In this framework, a computer-assisted surgical robotic platform able to perform non-invasive Focused Ultrasound Surgery (FUS) - the FUTURA platform - has the ambitious goal to improve accuracy, safety and flexibility of the treatment, with respect to current FUS procedures. Aim of this work is to present the current implementation of the robotic platform and the preliminary results about high intensity focused ultrasound (HIFU) delivery in in-vitro conditions, under 3D ultrasound identification and monitoring. Tests demonstrated that the average accuracy of the HIFU delivery is lower than 0.7 mm in both X and Y radial directions and 3.7 mm in the axial direction (Z) with respect to the HIFU transducer active surface.

Bovine herpesvirus type 1 (BHV-1) up-regulates telomerase activity in MDBK cells.

The proliferative capacity of mammalian cells is regulated by telomerase, an enzyme uniquely specialised for telomeric DNA synthesis. The critical role of telomerase activation in tumor progression and maintenance has been well established in studies of cancer and of oncogenic transformation in cell culture. Experimental data suggest that telomerase activation has an important role in normal somatic cells, and that failure to activate sufficient telomerase also promotes disease. Evidence regarding the role of telomerase in the pathogenesis of several viruses including human immunodeficiency virus has led to an increased interest in the role of telomerase activity in other virus infections. In this research we evaluated the telomerase modulating activity of Bovine herpesvirus 1 (BHV-1) in MDBK cells. MDBK cells were infected at different multiplicity of infection with BHV-1 Cooper strain and telomerase activity at different times post-infection was measured by the TRAP assay. Our data indicate that BHV-1 significantly up-regulates telomerase activity at 3 and 6h post-infection decreasing after the 24h post-infection. Our data, showed that the effect was mediated by an immediate-early or early viral gene, and use of the protein translation inhibitor cycloheximide confirmed that an immediate early gene is primarily responsible.

Endemic goiter in Calabria: etiopathogenesis and thyroid function.

An endemic goiter study was carried out in a vast territory of the Calabria region, including the provinces of Catanzaro and Cosenza. About 50% of 7231 school-children, aged between 6 and 12 years, examined in 34 villages, presented a thyroid enlargement. The high prevalence of goiter seems to be related to a moderate iodine deficiency intake indicated by a mild urinary iodine excretion (lower than 100 micrograms/g creatinine). In a few villages, in which a two year voluntary iodine prophylaxis was carried out, an increase in the urinary iodine excretion with a decreased goiter prevalence was observed. A slight increase in TT3, FT3 and TSH in one endemic area studied, compared to the control area, was also observed. These data suggest that an effective program of iodoprophylaxis is fundamental in this region as well.

Influence of environmental iodine deficiency on neonatal thyroid screening results.

During a multicentric pilot screening program for congenital hypothyroidism a comparison between the results obtained from Urbino, an area characterized by low iodine supply and endemic goiter, and Rome, a non-endemic area, has been made. The evaluation of neonatal urinary iodine excretion in the two areas showed significantly lower iodine urinary excretion levels in Urbino than in Rome. A shift of TSH at screening toward higher values as well as a higher percentage of recall in Urbino area than in Rome was observed. This finding, which well correlates with a low environmental iodine supply, emphasizes the importance of screening for congenital hypothyroidism as a suitable index of the presence and action of goitrogenic factors in the environment.

Relationship between maternal parity, basal prolactin levels and neonatal breast milk intake.

Basal serum levels of prolactin (PRL) in 21 nursing mothers were measured by radioimmunoassay on the 2nd, 3rd and 4th days of the puerperium. The quantity of breast milk suckled during the 4th day of life was also evaluated by calculating the difference in the baby's weight before and after each feeding. During the first postpartum days, mean basal levels of PRL did not change. However these levels were noted to be significantly lower in the multiparas (p less than 0.05) than in the primiparas. In addition, the milk intake in neonates of multiparous mothers was significantly greater (p less than 0.05) than that in neonates of primiparous mothers. The author's hypothesis, based on the results of animal experimentation described in the literature, is that initiation of breast-feeding is facilitated in multiparas by the increased number of occupied PRL receptors in the mammary glands reflected by the lowered serum levels of the hormone.

Plasma thyrocalcitonin and parathyroid hormone concentrations in early neonatal hypocalcaemia.

During the first week of life serum calcium, phosphorus, magnesium, immunoreactive thyrocalcitonin hormone, and parathyroid hormone concentrations were determined daily in 36 preterm and 29 small for gestational age, full term, healthy infants. Preterm babies with early neonatal hypocalcaemia had significantly higher concentrations of serum thyrocalcitonin hormone in the first four days of life than normocalcaemic preterm babies. Parathyroid hormone concentrations were similar in hypocalcaemic and normocalcaemic infants. In contrast, in the full term group no significant differences were detected in thyrocalcitonin hormone and parathyroid hormone patterns between hypocalcaemic and normocalcaemic subjects. This suggests two different pathogeneses for early hypocalcaemia in low birthweight infants. Hyperthyrocalcitoninaemia seems to be the main determining factor in preterm infants, while a non-hormonal pathogenesis should be considered in full term infants who are small for gestational age.

Longitudinal assessment of children with congenital hypothyroidism detected by neonatal screening.

Clinical and laboratory data from 42 children (31 females and 11 males) with primary congenital hypothyroidism (CH) diagnosed by neonatal screening over a six-year period are reported. The mean age at onset of thyroid hormone therapy was 33 days. The adequacy of replacement therapy was assessed by repeated TT4, FT4, T3 and TSH serum determinations. The high serum TT4 concentrations frequently observed were not accompanied by clinical evidence of hyperthyroidism. rT3 levels determined in 28 CH children with TT4 greater than 15 micrograms/dl were clearly higher than in the controls. The mean weight, length and head circumference remained always between the 50th and 75th centile. The radiological assessment of the knee, mainly the distal femoral surface, has been considered as an important clinical value in the initial diagnosis and in the evaluation of both severity and duration of disease. The psychomotor development was assessed using Brunet-Lezine's test until age 36 months, Stanford-Binet at 4 and 5 and WISC at 6 years of age. The mean global developmental quotients (GDQ) were always between 85 and 97 at 6 to 72 months of age, only eight children were below 85. A significant correlation was found between GDQ at 6 months and the bone age. The neurological examination showed an impairment of posture, coordination and subtle deficits in motor and perceptual abilities in a small percentage of children.

Immunochemical properties of trypsin fragments of human thyroglobulin.

Purified human thyroglobulin (Tg) was enzymatically digested with trypsin. After completion of digestion, the tryptic digest was fractionated by gel filtration on a Biogel A 1.5 m column. Further separation and isolation of the major peak C was carried out on a Sephadex G-75 column. Nine fractions were separated and antigenic properties evaluated by a specific and sensitive radioimmunoassay using a rabbit antiserum to 19S Tg and three different antisera from patients with autoimmune thyroid diseases. The Tg fragments react with both hetero- and auto-antisera. The highest antigenic activity was found on larger fragments, but a fairly good activity was also observed on fragments (C6, C7) with lower molecular weight. Antigenic determinants of Tg differ in individual sera suggesting that different sets of determinants elicit the autoimmune process.