Hydrochlorination of ruthenaphosphaalkenyls: unexpectedly facile access to alkylchlorohydrophosphane complexes

The novel ruthenaphosphaalkenyls [Ru{P═C(H)SiMe2R}Cl- (CO)(PPh3)2] (R = p-C6H4CF3, nBu) have been prepared for the first time, and studied alongside precedent analogues (R = Me, Ph, p-tol) for their reactions with HCl. In contrast to chemistry defined for the tert-butyl congener [Ru{P═C(H)tBu}Cl(CO)(PPh3)2], which initially adds a single equivalent of HCl across the Ru−P linkage, all five silyl derivatives undergo spontaneous addition of a second equivalent to afford [Ru{η1-PHCl−CH2SiMe2R}Cl(CO)(PPh3)2], extremely rare examples of coordinated “PHXR” type ligands. Where R = SiMe3, a distorted octahedral geometry with a conformationally restricted “PHXR” ligand is observed crystallographically; this structure is appreciably retained in solution, as determined from multinuclear NMR spectroscopic features, which include a Karplus-like PPh3−Ru−P−H spin−spin coupling dependence. Computational data suggest a silyl-induced increase in negative charge density at the phosphaalkenic carbon, rather than an intrinsic thermodynamic driver, as the likely origin of the disparate reactivity

Natriuretic Peptide Expression and Function in GH3 Somatolactotropes and Feline Somatotrope Pituitary Tumours.

Patients harbouring mutations in genes encoding C-type natriuretic peptide (CNP; NPPC) or its receptor guanylyl cyclase B (GC-B, NPR2) suffer from severe growth phenotypes; loss-of-function mutations cause achondroplasia, whereas gain-of-function mutations cause skeletal overgrowth. Although most of the effects of CNP/GC-B on growth are mediated directly on bone, evidence suggests the natriuretic peptides may also affect anterior pituitary control of growth. Our previous studies described the expression of NPPC and NPR2 in a range of human pituitary tumours, normal human pituitary, and normal fetal human pituitary. However, the natriuretic peptide system in somatotropes has not been extensively explored. Here, we examine the expression and function of the CNP/GC-B system in rat GH3 somatolactotrope cell line and pituitary tumours from a cohort of feline hypersomatotropism (HST; acromegaly) patients. Using multiplex RT-qPCR, all three natriuretic peptides and their receptors were detected in GH3 cells. The expression of Nppc was significantly enhanced following treatment with either 100 nM TRH or 10 µM forskolin, yet only Npr1 expression was sensitive to forskolin stimulation; the effects of forskolin and TRH on Nppc expression were PKA- and MAPK-dependent, respectively. CNP stimulation of GH3 somatolactotropes significantly inhibited Esr1, Insr and Lepr expression, but dramatically enhanced cFos expression at the same time point. Oestrogen treatment significantly enhanced expression of Nppa, Nppc, Npr1, and Npr2 in GH3 somatolactotropes, but inhibited CNP-stimulated cGMP accumulation. Finally, transcripts for all three natriuretic peptides and receptors were expressed in feline pituitary tumours from patients with HST. NPPC expression was negatively correlated with pituitary tumour volume and SSTR5 expression, but positively correlated with D2R and GHR expression. Collectively, these data provide mechanisms that control expression and function of CNP in somatolactotrope cells, and identify putative transcriptional targets for CNP action in somatotropes

Natriuretic Peptide Expression and Function in GH3 Somatolactotropes and Feline Somatotrope Pituitary Tumours.

Patients harbouring mutations in genes encoding C-type natriuretic peptide (CNP; NPPC) or its receptor guanylyl cyclase B (GC-B, NPR2) suffer from severe growth phenotypes; loss-of-function mutations cause achondroplasia, whereas gain-of-function mutations cause skeletal overgrowth. Although most of the effects of CNP/GC-B on growth are mediated directly on bone, evidence suggests the natriuretic peptides may also affect anterior pituitary control of growth. Our previous studies described the expression of NPPC and NPR2 in a range of human pituitary tumours, normal human pituitary, and normal fetal human pituitary. However, the natriuretic peptide system in somatotropes has not been extensively explored. Here, we examine the expression and function of the CNP/GC-B system in rat GH3 somatolactotrope cell line and pituitary tumours from a cohort of feline hypersomatotropism (HST; acromegaly) patients. Using multiplex RT-qPCR, all three natriuretic peptides and their receptors were detected in GH3 cells. The expression of Nppc was significantly enhanced following treatment with either 100 nM TRH or 10 µM forskolin, yet only Npr1 expression was sensitive to forskolin stimulation; the effects of forskolin and TRH on Nppc expression were PKA- and MAPK-dependent, respectively. CNP stimulation of GH3 somatolactotropes significantly inhibited Esr1, Insr and Lepr expression, but dramatically enhanced cFos expression at the same time point. Oestrogen treatment significantly enhanced expression of Nppa, Nppc, Npr1, and Npr2 in GH3 somatolactotropes, but inhibited CNP-stimulated cGMP accumulation. Finally, transcripts for all three natriuretic peptides and receptors were expressed in feline pituitary tumours from patients with HST. NPPC expression was negatively correlated with pituitary tumour volume and SSTR5 expression, but positively correlated with D2R and GHR expression. Collectively, these data provide mechanisms that control expression and function of CNP in somatolactotrope cells, and identify putative transcriptional targets for CNP action in somatotropes

View-dependent accuracy in body mass judgements of female bodies

A fundamental issue in testing body image perception is how to present the test stimuli. Previous studies have almost exclusively used images of bodies viewed in front-view, but this potentially obscures key visual cues used to judge adiposity reducing the ability to make accurate judgements. A potential solution is to use a three-quarter view, which combines visual cues to body fat that can be observed in front and profile. To test this hypothesis, 20 female observers completed a 2-alternative forced choice paradigm to determine the smallest difference in body fat detectable in female bodies in front, three-quarter, and profile view. There was a significant advantage for three-quarter and profile relative to front-view. Discrimination accuracy is predicted by the saliency of stomach depth, suggesting that this is a key visual cue used to judge body mass. In future, bodies should ideally be presented in three-quarter to accurately assess body size discrimination

Can Animal Models of Disease Reliably Inform Human Studies?

H. Bart van der Worp and colleagues discuss the controversies and possibilities of translating the results of animal experiments into human clinical trials

Investigation of SARS-CoV-2 faecal shedding in the community: a prospective household cohort study (COVID-LIV) in the UK

Background SARS-CoV-2 is frequently shed in the stool of patients hospitalised with COVID-19. The extent of faecal shedding of SARS-CoV-2 among individuals in the community, and its potential to contribute to spread of disease, is unknown. Methods In this prospective, observational cohort study among households in Liverpool, UK, participants underwent weekly nasal/throat swabbing to detect SARS-CoV-2 virus, over a 12-week period from enrolment starting July 2020. Participants that tested positive for SARS-CoV-2 were asked to provide a stool sample three and 14 days later. In addition, in October and November 2020, during a period of high community transmission, stool sampling was undertaken to determine the prevalence of SARS-CoV-2 faecal shedding among all study participants. SARS-CoV-2 RNA was detected using Real-Time PCR. Results A total of 434 participants from 176 households were enrolled. Eighteen participants (4.2%: 95% confidence interval [CI] 2.5–6.5%) tested positive for SARS-CoV-2 virus on nasal/throat swabs and of these, 3/17 (18%: 95% CI 4–43%) had SARS-CoV-2 detected in stool. Two of three participants demonstrated ongoing faecal shedding of SARS-CoV-2, without gastrointestinal symptoms, after testing negative for SARS-CoV-2 in respiratory samples. Among 165/434 participants without SARS-CoV-2 infection and who took part in the prevalence study, none had SARS-CoV-2 in stool. There was no demonstrable household transmission of SARS-CoV-2 among households containing a participant with faecal shedding. Conclusions Faecal shedding of SARS-CoV-2 occurred among community participants with confirmed SARS-CoV-2 infection. However, during a period of high community transmission, faecal shedding of SARS-CoV-2 was not detected among participants without SARS-CoV-2 infection. It is unlikely that the faecal-oral route plays a significant role in household and community transmission of SARS-CoV-2

Improving lung aeration in ventilated newborn preterm rabbits with a partially aerated lung

Preterm newborns commonly receive intermittent positive pressure ventilation (iPPV) at birth, but the optimal approach that facilitates uniform lung aeration is unknown, particularly in a partially aerated lung. As both inflation time and exogenous surfactant facilitate uniform lung aeration, we investigated whether they can improve lung aeration and lung mechanics in a partially aerated lung immediately after birth. Preterm rabbit kittens (29 days of gestation, term similar to 32 days) were delivered by caesarean section and partial lung aeration was created by intubating and mechanically ventilating the right lung. The tube was then withdrawn to ventilate both lungs using inflation times of 0.2 s or 1.0 s, with or without exogenous surfactant (200 mg/kg; Curosurf) and a tidal volume (Vt) of 8 mL/kg. Simultaneous phase contrast X-ray imaging and plethysmography were used to measure lung aeration and mechanics. Kittens ventilated with longer inflation times (1.0 s) reached their target Vt with fewer inflations, required lower inflation pressures (28.5 +/- 1.1 vs. 33.5 +/- 1.3 cmH(2)O, P = 0.01) and had higher dynamic lung compliances (0.54 +/- 0.3 vs. 0.40 +/- 0.3 cmH(2)O.mL(-1).kg(-1), P = 0.003). Surfactant increased functional residual capacity (FRC; 31.9 +/- 3.2 vs. 18.0 +/- 3.9 mL/kg, P = 0.02) and the proportion of the Vt entering the previously unaerated lung but had no effect on dynamic lung compliance. Combining early surfactant treatment with longer inflation times increases FRC levels, improves dynamic lung compliance, reduces inflation pressures and markedly increases the proportion of the lungs being ventilated during iPPV in preterm kittens with a partially aerated lung.NEW & NOTEWORTHY Preterm newborns commonly receive intermittent positive pressure ventilation (iPPV) at birth, but the optimal approach that facilitates uniform lung aeration is unknown, particularly in a partially aerated lung. Using phase contrast X-ray imaging, we showed that combining a long inflation time (1.0 s) with surfactant improved lung mechanics and aeration in the immediate newborn period. The current clinical practice of using short inflation times during iPPV might be suboptimal and a different approach is needed

Development of a Web-Based Query Tool for Quality Assurance of Clinical Molecular Genetic Test Results

The College of American Pathologists molecular pathology checklist item (MOL.20550) calls for periodic review of molecular genetic statistics, including percentages of normal and abnormal findings and allele frequencies. A web-based query tool application for clinical molecular genetic test results was developed to plot dynamically and display genotype and/or allele frequencies for any time period. This tool is used to produce plots of all high-volume molecular genetic assays (>50 samples per month). A single web page contains pull-down menus, enabling the user to select the type of chart to be generated (genotype or allele frequency), the molecular genetic assays to chart (from one to all), the ending date for data in the chart (month and year), and the duration of the time period to plot (1 to 12 months). The rendered graphical and textual frequency data can then be viewed or printed. This tool can be used by any laboratory and interfaced with a standard laboratory information system. Monthly quality control charts and tables are now generated in minutes compared with the hours it took using manual charting applications. This simplified process enables timely compliance with a College of American Pathologists checklist item

Figures for Pituitary pathology and gene expression in acromegalic cats

<div> <p>Figure 1</p> <p>Representative photomicrographs of growth hormone (A - C) and prolactin (D – F) immunostaining. A and D are x40 photomicrographs demonstrating specific immunostaining for somatrophs and lactotrophs, respectively. B and E are photomicrographs from a control cat C and F are from an acromegalic cat. </p><p><br></p> <p> </p> <p>Figure 2</p> <p>Representative images of SSTR2 immunoreactivity using feline pituitary tissue. A - D represent pituitary tissue exhibiting SSTR2 immunohistochemistry scores 0, 1, 2 and 3, respectively using the following criteria:.0 = absent; 1 = cytoplasmic staining; 2 = membranous staining in less than 50% cells or incomplete membranous staining; and 3 = circumferential membranous staining in >50% cells. All presented photomicrographs collected at x100 magnification using Leica DM400 B, Leica Microsystems Cambridge, UK.</p><p><br></p> <p> </p> <p>Figure 3</p> <p>An electropherogram results from PCR products using multiplex 1 primer sets The blue peaks represent PCR products from gene specific primers and red peaks represent product size standards.</p><p><br></p> <p> </p> <p>Figure 4</p> <p>All images stained using Silver stain for reticulin fibres and counter stained using Nuclear Fast Red solution. A and C; reconstructed stitched pituitary x100 magnification photomicrographs from two control pituitaries. B and D; x400 magnification photomicrographs from A and C, respectively. The acinar pattern of reticulin staining is identified in B and D. This pattern of reticulin staining was demonstrated in all reticulin staining control pituitaries.</p><p><br></p> <p> </p> <p>Figure 5</p> <p>All images stained using Silver stain for reticulin fibres and counter stained using Nuclear Fast Red solution. A – D; selected images taken from reconstructed stitched pituitary x100 magnification photomicrographs from four HST pituitaries. A; disrupted reticulin staining and loss of acinar structure. B; areas of enlarged acini (blue stars) and areas of loss of acinar structure (blue cross). C; enlarged acini (blue stars) adjacent to normal sized and small acini. D; loss of acinar structure in the bottom right of the image (blue stars), and adenomatous tissue has compressed the normal pituitary tissue resulting in compression of the acini and a ring of cords of acini giving the impression of a pseudocapsule.</p><p><br></p> <p> </p> <p>Figure 6</p> <p>A: Bar charts comparing the relative gene expression of <i>SSTR1</i>, <i>SSTR2 and</i> <i>SSTR5</i> in pituitary tissue from control (CTRL) and acromegalic (Acro) cats determine using GeXP multiplex technique. <i>RPL18</i> is the reference gene. Bar height represents mean and error bars are 95% confidence intervals ** represents <i>P</i> < 0.01 and *** represents <i>P</i> < 0.001. Dot plot of the individual somatostatin profiles from each of the 19 acromegalic cats.</p> </div> <br> <p>Table 1</p> <p>Clinical data of cats in the control and acromegalic groups</p><p><br></p> <p> </p> <p>Table 2</p> <p>Gene expression data and GH, PRL and SSTR2 immunohistochemistry scoring of cats in the control and acromegalic groups.</p><p><br></p> <p> </p> <p>Table 3</p> <p>Summary of Spearman rank correlation gene expression data in the control group and acromegalic groups</p><p><br></p> <p> </p><p>Supplementary material 1</p><p>Nucleotide sequences of primers used in multiplexes 1, 2 and 3.</p><p><br></p><p>Supplementary material 2</p><p>Amino acid multiple sequence alignment for human and feline SSTR2</p><div><br></div><p>Supplementary material 3</p><p>Amino acid multiple sequence alignment for porcine and feline growth hormone </p><div><br></div><p>Supplementary material 4</p><p>Amino acid multiple sequence alignment for porcine and feline prolactin </p><div><br></div><div><br></div