Vitamin D treatment induces in vitro and ex vivo transcriptomic changes indicating anti-tumor effects.

Vitamin D deficiency is associated with risk of several common cancers, including colorectal cancer (CRC). Here we have utilized patient derived epithelial organoids (ex vivo) and CRC cell lines (in vitro) to show that calcitriol (1,25OHD) increased the expression of the CRC tumor suppressor gene, CDH1, at both the transcript and protein level. Whole genome expression analysis demonstrated significant differential expression of a further six genes after 1,25OHD treatment, including genes with established links to carcinogenesis GADD45, EFTUD1 and KIAA1199. Furthermore, gene ontologies relevant to carcinogenesis were enriched by 1,25OHD treatment (e.g., 'regulation of Wnt signaling pathway', 'regulation of cell death'), with common enriched processes across in vitro and ex vivo cultures including 'negative regulation of cell proliferation', 'regulation of cell migration' and 'regulation of cell differentiation'. Our results identify genes and pathways that are modifiable by calcitriol that have links to CRC tumorigenesis. Hence the findings provide potential mechanism to the epidemiological and clinical trial data indicating a causal association between vitamin D and CRC. We suggest there is strong rationale for further well-designed trials of vitamin D supplementation as a novel CRC chemopreventive and chemotherapeutic agent.

Dual Josephson Impedance Bridge: Towards a Universal Bridge for Impedance Metrology.

This paper presents a full characterization of a Dual Josephson Impedance Bridge (DJIB) at frequencies up to 80 kHz by using the DJIB to compare the best available impedance standards that are (a) directly traceable to the quantum Hall effect, (b) used as part of international impedance comparisons, or (c) believed to have calculable frequency dependence. The heart of the system is a dual Josephson Arbitrary Waveform Synthesizer (JAWS) source that offers unprecedented flexibility in high-precision impedance measurements. The JAWS sources allow a single bridge to compare impedances with arbitrary ratios and phase angles in the complex plane. The uncertainty budget shows that both the traditional METAS bridges and the DJIB have comparable uncertainties in the kilohertz range. This shows that the advantages of the DJIB, including the flexibility which allows the comparison of arbitrary impedances, the wide frequency range, and the automated balancing procedure, are obtained without compromising the measurement uncertainties. These results demonstrate that this type of instrument can considerably simplify the realization and maintenance of the various impedance scales. In addition, the DJIB is a very sensitive tool for investigating the frequency-dependent systematic-errors that can occur in impedance construction and in the voltage provided by the JAWS source at frequencies greater than 10 kHz.

Characterization of Uniformity in Nb/NbxSi1-x/Nb Josephson Junctions.

The uniformity of the barriers in Josephson junctions (JJs) is a critical parameter in determining performance and operating margins for a wide variety of superconducting electronic circuits. We present an automated measurement system capable of measuring individual JJs across a 1 × 1 cm die at both ambient temperature and 4 K. This technique allows visualization of the spatial variation over a large area of the critical electrical properties of the junctions and allows for the direct correlation between room-temperature (RT) resistance and low temperature properties. The critical current variation of NbxSi1-x (x = 15%) barriers is found to be about 2.6% (one standard deviation) for 1024 junctions across an individual die and only weakly correlates with RT resistance measurements.

Quantized Pulse Propagation in Josephson Junction Arrays.

We present time-domain electrical measurements and simulations of the quantized voltage pulses that are generated from series-connected Josephson junction (JJ) arrays. The transmission delay of the JJ array can lead to a broadening of the net output pulse, depending on the direction of the output pulse propagation relative to the input bias pulse. To demonstrate this, we compare time-domain measurements of output pulses from radio-frequency Josephson Arbitrary Waveform Synthesizer (RF-JAWS) circuits fabricated with two different output measurement configurations, so that the backward-propagating and forward-propagating pulses can be measured. Measurements were made on arrays with 1200 and 3600 JJs and show that the net backward-propagating output pulse is broadened by timing delays in the JJ array while the net forward-propagating output pulse is insensitive to delay effects and can theoretically be further scaled to longer JJ array lengths without significant output pulse broadening. These measurements match well with simulations and confirm the expectation that the net output pulses arise from the time-delayed superposition of individual JJ output pulses from the series array of JJs. The measurements and analysis shown here have important implications for the realization of RF-JAWS circuits to be used as quantum-based reference sources for communications metrology.

The NIST Johnson Noise Thermometry System for the Determination of the Boltzmann Constant.

In preparation for the redefinition of the International System of Units (SI), five different electronic measurements of the Boltzmann constant have been performed using different Johnson noise thermometry (JNT) systems over the past seven years. In this paper, we describe in detail the JNT system and uncertainty components associated with the most recent National Institute of Standards and Technology (NIST) determination of the Boltzmann constant: k = 1.380642 9(69) × 10-23 J/K, with a relative standard uncertainty of 5.0 × 10-6 and relative offset of -4.05 × 10-6 from the Committee on Data for Science and Technology (CODATA) 2014 recommended value. We discuss the input circuits and the approach we used to match the frequency response of two noise sources. We present new measurements of the correlated noise of the 4 K on-chip resistors in the quantum-accurate, pseudorandom, voltage-noise source, which we used to estimate the correlated, frequency-dependent, nonthermal noise in our system. Finally, we contrast our system with those used in other measurements and speculate on future improvements.

Josephson Arbitrary Waveform Synthesis With Multilevel Pulse Biasing.

We describe the implementation of new commercial pulse-bias electronics that have enabled an improvement in the generation of quantum-accurate waveforms both with and without low-frequency compensation biases. We have used these electronics to apply a multilevel pulse bias to the Josephson arbitrary waveform synthesizer and have generated, for the first time, a quantum-accurate bipolar sinusoidal waveform without the use of a low-frequency compensation bias current. This uncompensated 1 kHz waveform was synthesized with an rms amplitude of 325 mV and maintained its quantum accuracy over a1.5 mA operating current range. The same technique and equipment was also used to synthesize a quantum-accurate 1 MHz sinusoid with a 1.2 mA operating margin. In addition, we have synthesized a compensated 1 kHz sinusoid with an rms amplitude of 1 V and a 2.7 mA operating margin.

Two-Volt Josephson Arbitrary Waveform Synthesizer Using Wilkinson Dividers.

The root-mean-square (rms) output voltage of the NIST Josephson arbitrary waveform synthesizer (JAWS) has been doubled from 1 V to a record 2 V by combining two new 1 V chips on a cryocooler. This higher voltage will improve calibrations of ac thermal voltage converters and precision voltage measurements that require state-of-the-art quantum accuracy, stability, and signal-to-noise ratio. We achieved this increase in output voltage by using four on-chip Wilkinson dividers and eight inner-outer dc blocks, which enable biasing of eight Josephson junction (JJ) arrays with high-speed inputs from only four high-speed pulse generator channels. This approach halves the number of pulse generator channels required in future JAWS systems. We also implemented on-chip superconducting interconnects between JJ arrays, which reduces systematic errors and enables a new modular chip package. Finally, we demonstrate a new technique for measuring and visualizing the operating current range that reduces the measurement time by almost two orders of magnitude and reveals the relationship between distortion in the output spectrum and output pulse sequence errors.

A specific inhibitor of PfCDPK4 blocks malaria transmission: chemical-genetic validation.

Malaria parasites are transmitted by mosquitoes, and blocking parasite transmission is critical in reducing or eliminating malaria in endemic regions. Here, we report the pharmacological characterization of a new class of malaria transmission-blocking compounds that acts via the inhibition of Plasmodia CDPK4 enzyme. We demonstrate that these compounds achieved selectivity over mammalian kinases by capitalizing on a small serine gatekeeper residue in the active site of the Plasmodium CDPK4 enzyme. To directly confirm the mechanism of action of these compounds, we generated P. falciparum parasites that express a drug-resistant methionine gatekeeper (S147 M) CDPK4 mutant. Mutant parasites showed a shift in exflagellation EC50 relative to the wild-type strains in the presence of compound 1294, providing chemical-genetic evidence that CDPK4 is the target of the compound. Pharmacokinetic analyses suggest that coformulation of this transmission-blocking agent with asexual stage antimalarials such as artemisinin combination therapy (ACT) is a promising option for drug delivery that may reduce transmission of malaria including drug-resistant strains. Ongoing studies include refining the compounds to improve efficacy and toxicological properties for efficient blocking of malaria transmission.

Multicentre study of the clinical relevance of screening IVF patients for carrier status of the annexin A5 M2 haplotype.

Thrombophilia and impaired placental vasculature are a major cause of adverse pregnancy outcome. In 2007, a new hereditary factor for obstetric complications and recurrent pregnancy loss (RPL) was identified as a sequence variation in the core promoter of the annexin A5 gene, ANXA5, called the M2 haplotype. M2 carriership has been demonstrated in couples with recurrent miscarriage and its origin is embryonic rather than specifically maternal, confirmed by subsequent papers. The M2 haplotype is the first report of a hereditary factor related to pregnancy pathology caused by embryonic-induced anticoagulation. It has been demonstrated that couples with RPL had equal and significantly increased M2 carriership and that maternal and paternal carriership confers equal risk. Given its importance for patients with RPL, and potentially implantation failure, this study assessed the incidence of carrier status for the M2 ANXA5 haplotype in both the male and female of couples attending five CARE IVF centres. In 314 patients (157 couples), 44% of couples (one or both partners), 24% of females, 26% of males and 37% of couples with unexplained infertility were M2 carriers. This high incidence has provoked further urgent studies on specific patient populations and on the value of post embryo-transfer therapy.

The gatekeeper residue and beyond: homologous calcium-dependent protein kinases as drug development targets for veterinarian Apicomplexa parasites.

Specific roles of individual CDPKs vary, but in general they mediate essential biological functions necessary for parasite survival. A comparative analysis of the structure-activity relationships (SAR) of Neospora caninum, Eimeria tenella and Babesia bovis calcium-dependent protein kinases (CDPKs) together with those of Plasmodium falciparum, Cryptosporidium parvum and Toxoplasma gondii was performed by screening against 333 bumped kinase inhibitors (BKIs). Structural modelling and experimental data revealed that residues other than the gatekeeper influence compound-protein interactions resulting in distinct sensitivity profiles. We subsequently defined potential amino-acid structural influences within the ATP-binding cavity for each orthologue necessary for consideration in the development of broad-spectrum apicomplexan CDPK inhibitors. Although the BKI library was developed for specific inhibition of glycine gatekeeper CDPKs combined with low inhibition of threonine gatekeeper human SRC kinase, some library compounds exhibit activity against serine- or threonine-containing CDPKs. Divergent BKI sensitivity of CDPK homologues could be explained on the basis of differences in the size and orientation of the hydrophobic pocket and specific variation at other amino-acid positions within the ATP-binding cavity. In particular, BbCDPK4 and PfCDPK1 are sensitive to a larger fraction of compounds than EtCDPK1 despite the presence of a threonine gatekeeper in all three CDPKs.

A novel calcium-dependent protein kinase inhibitor as a lead compound for treating cryptosporidiosis.

Cryptosporidium parasites infect intestinal cells, causing cryptosporidiosis. Despite its high morbidity and association with stunting in the developing world, current therapies for cryptosporidiosis have limited efficacy. Calcium-dependent protein kinases (CDPKs) are essential enzymes in the biology of protozoan parasites. CDPK1 was cloned from the genome of Cryptosporidium parvum, and potent and specific inhibitors have been developed based on structural studies. In this study, we evaluated the anti-Cryptosporidium activity of a novel CDPK1 inhibitor, 1294, and demonstrated that 1294 significantly reduces parasite infection in vitro, with a half maximal effective concentration of 100 nM. Pharmacokinetic studies revealed that 1294 is well absorbed, with a half-life supporting daily administration. Oral therapy with 1294 eliminated Cryptosporidium parasites from 6 of 7 infected severe combined immunodeficiency-beige mice, and the parasites did not recur in these immunosuppressed mice. Mice treated with 1294 had less epithelial damage, corresponding to less apoptosis. Thus, 1294 is an important lead for the development of drugs for treatment of cryptosporidiosis.

An Exploration of Yoga in Occupational Therapy Practice for Multiple Sclerosis.

The purpose of this exploratory qualitative study was to provide insight on the use of yoga in occupational therapy (OT) for people with multiple sclerosis (PwMS). This study aimed to answer how and why OT practitioners (OTPs) integrate yoga into clinical practice for PwMS. Eight OTPs, half of whom have also completed yoga teacher training, participated in a semi-structured telephone interview. Interviews were transcribed verbatim, inductively open-coded, and analyzed using thematic data analysis. Themes that emerged were: (a) OT and yoga are a natural fit; (b) improved performance and participation; (c) leveraging personal ties to yoga; and (d) influenced by client factors and clinical environment. The qualitative data provide valuable information about OTPs' justification for, and unique application of, yoga in clinical practice for PwMS. Future researchers should further explore the use of yoga for OT-related outcomes and the experience of PwMS.

Development of Preclinical Ultrasound Imaging Techniques to Identify and Image Sentinel Lymph Nodes in a Cancerous Animal Model.

Lymph nodes (LNs) are believed to be the first organs targeted by colorectal cancer cells detached from a primary solid tumor because of their role in draining interstitial fluids. Better detection and assessment of these organs have the potential to help clinicians in stratification and designing optimal design of oncological treatments for each patient. Whilst highly valuable for the detection of primary tumors, CT and MRI remain limited for the characterization of LNs. B-mode ultrasound (US) and contrast-enhanced ultrasound (CEUS) can improve the detection of LNs and could provide critical complementary information to MRI and CT scans; however, the European Federation of Societies for Ultrasound in Medicine and Biology (EFSUMB) guidelines advise that further evidence is required before US or CEUS can be recommended for clinical use. Moreover, knowledge of the lymphatic system and LNs is relatively limited, especially in preclinical models. In this pilot study, we have created a mouse model of metastatic cancer and utilized 3D high-frequency ultrasound to assess the volume, shape, and absence of hilum, along with CEUS to assess the flow dynamics of tumor-free and tumor-bearing LNs in vivo. The aforementioned parameters were used to create a scoring system to predict the likelihood of a disease-involved LN before establishing post-mortem diagnosis with histopathology. Preliminary results suggest that a sum score of parameters may provide a more accurate diagnosis than the LN size, the single parameter currently used to predict the involvement of an LN in disease.

Gene Co-Expression Network Analysis Identifies Vitamin D-Associated Gene Modules in Adult Normal Rectal Epithelium Following Supplementation.

Colorectal cancer (CRC) is a common, multifactorial disease. While observational studies have identified an association between lower vitamin D and higher CRC risk, supplementation trials have been inconclusive and the mechanisms by which vitamin D may modulate CRC risk are not well understood. We sought to perform a weighted gene co-expression network analysis (WGCNA) to identify modules present after vitamin D supplementation (when plasma vitamin D level was sufficient) which were absent before supplementation, and then to identify influential genes in those modules. The transcriptome from normal rectal mucosa biopsies of 49 individuals free from CRC were assessed before and after 12 weeks of 3200IU/day vitamin D (Fultium-D3) supplementation using paired-end total RNAseq. While the effects on expression patterns following vitamin D supplementation were subtle, WGCNA identified highly correlated genes forming gene modules. Four of the 17 modules identified in the post-vitamin D network were not preserved in the pre-vitamin D network, shedding new light on the biochemical impact of supplementation. These modules were enriched for GO terms related to the immune system, hormone metabolism, cell growth and RNA metabolism. Across the four treatment-associated modules, 51 hub genes were identified, with enrichment of 40 different transcription factor motifs in promoter regions of those genes, including VDR:RXR. Six of the hub genes were nominally differentially expressed in studies of vitamin D effects on adult normal mucosa organoids: LCN2, HLA-C, AIF1L, PTPRU, PDE4B and IFI6. By taking a gene-correlation network approach, we have described vitamin D induced changes to gene modules in normal human rectal epithelium in vivo, the target tissue from which CRC develops.

Aspirin Rescues Wnt-Driven Stem-like Phenotype in Human Intestinal Organoids and Increases the Wnt Antagonist Dickkopf-1.

BACKGROUND & AIMS: Aspirin reduces colorectal cancer (CRC) incidence and mortality. Understanding the biology responsible for this protective effect is key to developing biomarker-led approaches for rational clinical use. Wnt signaling drives CRC development from initiation to progression through regulation of epithelial-mesenchymal transition (EMT) and cancer stem cell populations. Here, we investigated whether aspirin can rescue these proinvasive phenotypes associated with CRC progression in Wnt-driven human and mouse intestinal organoids. METHODS: We evaluated aspirin-mediated effects on phenotype and stem cell markers in intestinal organoids derived from mouse (ApcMin/+ and Apcflox/flox) and human familial adenomatous polyposis patients. CRC cell lines (HCT116 and Colo205) were used to study effects on motility, invasion, Wnt signaling, and EMT. RESULTS: Aspirin rescues the Wnt-driven cystic organoid phenotype by promoting budding in mouse and human Apc deficient organoids, which is paralleled by decreased stem cell marker expression. Aspirin-mediated Wnt inhibition in ApcMin/+ mice is associated with EMT inhibition and decreased cell migration, invasion, and motility in CRC cell lines. Chemical Wnt activation induces EMT and stem-like alterations in CRC cells, which are rescued by aspirin. Aspirin increases expression of the Wnt antagonist Dickkopf-1 in CRC cells and organoids derived from familial adenomatous polyposis patients, which contributes to EMT and cancer stem cell inhibition. CONCLUSIONS: We provide evidence of phenotypic biomarkers of response to aspirin with an increased epithelial and reduced stem-like state mediated by an increase in Dickkopf-1. This highlights a novel mechanism of aspirin-mediated Wnt inhibition and potential phenotypic and molecular biomarkers for trials.

Incidence and aetiology of talipes equino-varus with recent population changes.

This study looks at the changing incidence and aetiology of congenital talipes equinovarus due to the recent population changes within the area. Between 1st June 1992 and the 31st May 2006, 83 consecutive children (121 feet) born with fixed talipes equinovarus (TEV) were assessed and treated (an incidence of 1.6 per 1000 live births) in an observational longitudinal cohort study assessing associated factors. There were 17 syndromal cases in the fixed group (20.8%), 6 cases of non-syndromal distal arthrogryposis (7.2%), and a strong family history in 12 cases (14.5%). This study would suggest that genetic and primary causes of fixed TEV are more common than previously considered. Many of the primary aetiologies were diagnosed months or years after birth.

Joint sparing management of hallux rigidus: Cartiva SCI vs cheilectomy a comparative review.

BACKGROUND: Hallux rigidus, or first metatarsophalangeal joing arthritis, is a very common condition which causes pain and loss of motion in the joing which can be debilitating. METHODS: Patient reported outcome measures were utilised to study the difference in day to day functionality between two common surgical management options for hallux rigidus. RESULTS: 55 Cartiva and 23 Cheilectomy patients were included in this study. The Manchester Oxford Foot and Ankle Questionnaire revealed statistically significant improvements in the cheilectomy patients when compared to Cartiva SCI.Foot and Ankle Ability Measure found no statistical difference was found between the two surgical treatments. CONCLUSION: Cheilectomy offers good post-operative functional and sporting outcomes after surgery for the management of hallux rigidus at 5 years.

Cartiva case series: The efficacy of the cartiva synthetic cartilage implant interpositional arthroplasty at one year.

BACKGROUND: The Cartiva synthetic cartilage implant (SCI) is licenced for use in management of symptomatic hallux rigidus in several countries including the UK. As of now, there are no independent series for treatment of hallux rigidus utilising polyvinyl alcohol implants. METHODS: Patients at a single centre with symptomatic hallux rigidus who underwent Cartiva implant interpositional arthroplasty were identified. First metatarsophalangeal joint arthritis was radiographically graded according to the Hattrup and Johnson (HJ) classification. Pre-operative and post-operative patient-reported outcomes (PROMs) were evaluated using the Foot and Ankle Ability Measure (FAAM) activities of daily living subscale and the Manchester-Oxford Foot Questionnaire (MOXFQ). RESULTS: 55 patients (14M, 41F) (37R and 18L) were followed up for an average of 21 months (min = 12, max = 38). 14 patients suffered from HJ2/moderate arthritis and 41 patients with grade HJ3/severe arthritis.Post-operative mean FAAM scores showed statistically significant improvement (p < 0.0001). Patients reported a 40% increase in functionality during activities of daily living.All 3 MOXFQ Domain scores improved significantly (p < 0.02). The Index score improved by 34 points (p < 0.0001).There was no correlation between length of follow up or age and PROMs (r = 0.129). No statistical difference was demonstrated between sexes. Clinically, however, males and older patients exhibited better outcomes. CONCLUSIONS: Our study shows excellent results with statistically significant improvements in functional outcomes, and promising short-term follow-up with low early revision rates. Durability and survivability of the implant will continue to be studied in this cohort.

Comparing the efficacy of Cartiva synthetic cartilage implant hemiarthroplasty vs osteotomy for the treatment of conditions affecting the second metatarsal head.

BACKGROUND: Cartiva synthetic cartilage implants (SCI) have been designed for treatment of conditions affecting the second metatarsal head. Osteotomies are regularly performed for the treatment of conditions affecting the second metatarsal head such as Freiburg's disease. A comparative study between these two procedures has not yet been performed. METHODS: Patients at a single centre with symptomatic conditions affecting the second metatarsal head who received Cartiva SCI or a primary Osteotomy were identified, and patient-reported outcomes were evaluated using MOXFQ and the FAAM questionnaires. RESULTS: Six Cartiva SCI and 7 Osteotomy patients were identified and included in this study. All patients were female and were followed up for an average of 19 months (SD +/- 5.6) and 27 months (SD +/- 10.9) respectively. Cartiva SCI MOXFQ Index scores improved by 13 points from 57 to 44 (33%). Walking/and Pain domain scores improved, however, Social interaction deteriorated. Mean FAAM scores improved by 10% from 65% to 75%, and subjective FAAM scores improved by 7% from 48% to 55%. Four of the six Cartiva patients (3 Freiberg's disease and 1 Osteochondral defect) had revisions to Weil osteotomies at a mean of 15 months post-operatively. The Osteotomy group had improved MOXFQ Index and 3 Domain scores. FAAM scores improved by 26% from 66% to 92% and subjective FAAM scores improved by 28% from 60% to 88%. CONCLUSIONS: Second metatarsal head osteotomies result in high functional outcomes and should be the mainstay of treatment, especially when there is avascular necrosis of the metatarsal head.

Observation of non-exponential absorption of ultra-fast pulses in water.

We observe non-exponential absorption as a function of path length for pulses propagating in pure water. Two types of pulses with differing time duration, bandwidth, and repetition rate were compared with simulated absorption predictions. Deviations from exponential behaviour occurred when the launched pulse had a temporal width of 60fs and a repetition rate of 1 kHz. Under these conditions we observe more than 2 orders of magnitude less absorption after propagation through 6 m of water compared to Beer's law prediction. No significant deviation was observed for launched pulses of varying bandwidth with temporal widths of 900fs and repetition rates of 80MHz.