Mechanistic studies on the uptake and intracellular trafficking of novel cyclodextrin transfection complexes by intestinal epithelial cells

Oral delivery of gene therapeutics would facilitate treatment of local intestinal disease, including colon cancer and inflammatory bowel disease, thus avoiding invasive surgery. The aims of this study were to investigate; if the orientation of the lipid tail on the cyclodextrin (CD) influenced the efficacy of a novel poly-6-cationic amphiphilic CD to transfect intestinal enterocytes; the endocytotic uptake pathway(s), and, the intracellular trafficking of the CD.DNA complexes. Inhibitors of clathrin- and caveolae- mediated endocytosis and macropinocytosis were used to determine the mechanism(s) of CD.DNA uptake by both undifferentiated and differentiated Caco-2 cells. Cell surface heparan sulphate proteoglycans were involved in the association of CD.DNA complexes with undifferentiated Caco-2 cells. Complexation of pDNA with CD facilitated significant levels of pDNA uptake and gene expression (comparable to PEI) in both undifferentiated and differentiated Caco-2 cells. Disruption of intracellular vesicular trafficking reduced transfection activity. CD was also capable of transfecting the more physiologically relevant differentiated Caco-2 model. Macropinocytosis was responsible for the uptake of CD.DNA transfection complexes by both undifferentiated and differentiated Caco-2 cells. The ability of this novel CD to transfect differentiated intestinal cells indicates the potential of this vector for oral gene delivery

ATR promotes cilia signalling: links to developmental impacts

Mutations in ATR (ataxia telangiectasia and RAD3-related) cause Seckel syndrome (ATR-SS), a microcephalic primordial dwarfism disorder. Hitherto, the clinical manifestation of ATR deficiency has been attributed to its canonical role in DNA damage response signalling following replication fork stalling/collapse. Here, we show that ATR regulates cilia-dependent signalling in a manner that can be uncoupled from its function during replication. ATR-depleted or patient-derived ATR-SS cells form cilia of slightly reduced length but are dramatically impaired in cilia-dependent signalling functions, including growth factor and Sonic hedgehog signalling. To better understand the developmental impact of ATR loss of function, we also used zebrafish as a model. Zebrafish embryos depleted of Atr resembled ATR-SS morphology, showed a modest but statistically significant reduction in cilia length and other morphological features indicative of cilia dysfunction. Additionally, they displayed defects in left-right asymmetry including ambiguous expression of southpaw, incorrectly looped hearts and randomized localization of internal organs including the pancreas, features typically conferred by cilia dysfunction. Our findings reveal a novel role for ATR in cilia signalling distinct from its canonical function during replication and strengthen emerging links between cilia function and development

Proteomic screening of glucose-responsive and glucose non-responsive MIN-6 beta cells reveals differential expression of proteins involved in protein folding, secretion and oxidative stress

The glucose-sensitive insulin-secretion (GSIS) phenotype is relatively unstable in long-term culture of beta cells. The purpose of this study was to investigate relative changes in the proteome between glucose-responsive (low passage) and glucose non-responsive (high passage) murine MIN-6 pancreatic beta cells. The 2D-DIGE and subsequent DeCyder analysis detected 3351 protein spots in the pH range of 4–7. Comparing MIN-6(H) to MIN-6(L) and using a threshold of 1.2-fold, the number of proteins with a decrease in expression level was 152 (4.5%), similar was 3140 (93.7%) and increased 59 (1.8%). From the differentially expressed proteins identified in this study, groups of proteins associated with the endoplasmic reticulum (ER) and proteins involved in oxidative stress were found to be significantly decreased in the high-passage (H passage) cells. These proteins included endoplasmic reticulum protein 29 (ERp29); 78-kDa glucoserelated protein, (GRP78); 94-kDa glucose-related protein (GRP94); protein disulphide isomerase; carbonyl reductase 3; peroxidoxin 4 and superoxide dismutase 1. These results suggest that non- GSIS MIN-6 cells do not have the same ability/capacity of glucose-responsive MIN-6 cells to successfully fold, modify or secrete proteins and counteract the problems associated with oxidative stress

Mutations in pericentrin cause Seckel syndrome with defective ATR-dependent DNA damage signaling

Large brain size is one of the defining characteristics of modern humans. Seckel syndrome (MIM 210600), a disorder of markedly reduced brain and body size, is associated with defective ATR-dependent DNA damage signaling. Only a single hypomorphic mutation of ATR has been identified in this genetically heterogeneous condition. We now report that mutations in the gene encoding pericentrin (PCNT)--resulting in the loss of pericentrin from the centrosome, where it has key functions anchoring both structural and regulatory proteins--also cause Seckel syndrome. Furthermore, we find that cells of individuals with Seckel syndrome due to mutations in PCNT (PCNT-Seckel) have defects in ATR-dependent checkpoint signaling, providing the first evidence linking a structural centrosomal protein with DNA damage signaling. These findings also suggest that other known microcephaly genes implicated in either DNA repair responses or centrosomal function may act in common developmental pathways determining human brain and body size

Investigation of the molecular profile of basal cell carcinoma using whole genome microarrays

BACKGROUND: Skin cancer accounts for 1/3 of all newly diagnosed cancer. Although seldom fatal, basal cell carcinoma (BCC) is associated with severe disfigurement and morbidity. BCC has a unique interest for researchers, as although it is often locally invasive, it rarely metastasises. This paper, reporting the first whole genome expression microarray analysis of skin cancer, aimed to investigate the molecular profile of BCC in comparison to non-cancerous skin biopsies. RNA from BCC and normal skin specimens was analysed using Affymetrix whole genome microarrays. A Welch t-test was applied to data normalised using dCHIP to identify significant differentially-expressed genes between BCC and normal specimens. Principal component analysis and support vector machine analysis were performed on resulting genelists, Genmapp was used to identify pathways affected, and GOstat aided identification of areas of gene ontology more highly represented on these lists than would be expected by chance. RESULTS: Following normalisation, specimens clustered into groups of BCC specimens and of normal skin specimens. Of the 54,675 gene transcripts/variants analysed, 3,921 were differentially expressed between BCC and normal skin specimens. Of these, 2,108 were significantly up-regulated and 1,813 were statistically significantly down-regulated in BCCs. CONCLUSION: Functional gene sets differentially expressed include those involved in transcription, proliferation, cell motility, apoptosis and metabolism. As expected, members of the Wnt and hedgehog pathways were found to be significantly different between BCC and normal specimens, as were many previously undescribed changes in gene expression between normal and BCC specimens, including basonuclin2 and mrp9. Quantitative-PCR analysis confirmed our microarray results, identifying novel potential biomarkers for BCC

Evidence that autosomal recessive spastic cerebral palsy-1 (CPSQ1) is caused by a missense variant in HPDL.

A subset of individuals diagnosed with cerebral palsy will have an underlying genetic diagnosis. Previously, a missense variant in GAD1 was described as a candidate mutation in a single family diagnosed with autosomal recessive spastic cerebral palsy-1 (CPSQ1; OMIM 603513). Following the ascertainment of a further branch of the CPSQ1 kindred, we found that the previously reported GAD1 variant did not segregate with the neurological disease phenotype in the recently ascertained branch of the kindred. Following genetic linkage studies to map autozygous regions and whole-exome sequencing, a missense variant (c.527 T > C; p. Leu176Pro, rs773333490) in the HPDL gene was detected and found to segregate with disease status in both branches of the kindred. HPDL encodes a 371-amino acid protein (4-Hydroxyphenylpyruvate Dioxygenase Like) that localizes to mitochondria but whose function is uncertain. Recently, biallelic loss of function variants and missense substitution-causing variants in HPDL were reported to cause a childhood onset progressive spastic movement disorder with a variable presentation. These findings suggest that HPDL-related neurological disease may mimic spastic cerebral palsy and that GAD1 should not be included in diagnostic gene panels for inherited cerebral palsy.NIH

Enacting person-centredness in integrated care: A qualitative study of practice and perspectives within Multidisciplinary Groups in the care of older people

BACKGROUND: Person-centredness is important in delivering care for long-term conditions. New models of care aim to co-ordinate care through integration of health and social care which require new ways of working, often remotely from the patient. OBJECTIVE: To describe how person-centred care is enacted within multidisciplinary groups (MDGs) created as part of a new service, integrating health and social care for older people. METHODS: We followed the implementation of eight neighbourhood MDGs, observing and interviewing staff from three MDGs at different phases of programme implementation using semi-structured topic guides. RESULTS: Thirty-four MDG meetings were observed and 32 staff interviewed. Three core themes were identified which impacted on enactment of person-centred care: the structural context of MDGs enabling person-centred care; interaction of staff and knowledge sharing during the MDG meetings; and direct staff involvement of the person outside the MDG discussion. CONCLUSIONS: This study provides new insights into attempts to enact person-centred care within a new model of service delivery. Teams did what they could to enact person-centred care in the absence of the "real" patient within MDG meetings. They were successful in delivering and co-ordinating some aspects of care (eg prompting medication reviews, referring to social worker, health improvement and arranging further multidisciplinary team meetings for complex cases). This "absence of patients" and time pressures within the MDGs led to reliance on the "virtual" record, enhanced by additional "soft" knowledge provided by staff, rather than ensuring the patient's voice was included

Translabyrinthine resection of NF2 associated vestibular schwannoma with cochlear implant insertion

The authors present the case of a 24-year-old female with neurofibromatosis type 2. Growth of the left vestibular schwannoma and progressive hearing loss prompted the decision to proceed to translabyrinthine resection with cochlear nerve preservation and cochlear implant insertion. Complete resection with preservation of the facial and cochlear nerves was achieved. The patient had grade 1 facial function and was discharged on postoperative day 4 following suturing of a minor CSF leak. This case highlights the feasibility of cochlear nerve preservation and cochlear implant insertion in appropriately selected patients, offering a combination of effective tumor control and hearing rehabilitation. The video can be found here: https://stream.cadmore.media/r10.3171/2021.7.FOCVID2112

Oxygen therapy for acute myocardial infarction

BACKGROUND: Oxygen (O2) is widely used in people with acute myocardial infarction (AMI). Previous systematic reviews concluded that there was insufficient evidence to know whether oxygen reduced, increased or had no effect on heart ischaemia or infarct size. Our first Cochrane review in 2010 also concluded there was insufficient evidence to know whether oxygen should be used. Since 2010, the lack of evidence to support this widely used intervention has attracted considerable attention, prompting further trials of oxygen therapy in myocardial infarction patients. It is thus important to update this Cochrane review. OBJECTIVES: To assess the effects of routine use of inhaled oxygen for acute myocardial infarction (AMI). SEARCH METHODS: We searched the following bibliographic databases on 6 June 2015: the Cochrane Central Register of Controlled Trials (CENTRAL) in the Cochrane Library, MEDLINE (OVID), Embase (OVID), CINAHL (EBSCO) and Web of Science (Thomson Reuters). LILACS (Latin American and Caribbean Health Sciences Literature) was last searched in September 2016. We also contacted experts to identify eligible studies. We applied no language restrictions. SELECTION CRITERIA: Randomised controlled trials in people with suspected or proven AMI (ST-segment elevation myocardial infarction (STEMI) or non-STEMI) within 24 hours after onset, in which the intervention was inhaled oxygen (at normal pressure) compared to air, regardless of co-therapies provided to participants in both arms of the trial. DATA COLLECTION AND ANALYSIS: Two authors independently reviewed the titles and abstracts of identified studies to see if they met the inclusion criteria and independently undertook the data extraction. We assessed the quality of studies and the risk of bias according to guidance in the Cochrane Handbook for Systematic Reviews of Interventions. The primary outcome was death. The measure of effect used was the risk ratio (RR) with a 95% confidence interval (CI). We used the GRADE approach to evaluate the quality of the evidence and the GRADE profiler (GRADEpro) to import data from Review Manager 5 and create 'Summary of findings' tables. MAIN RESULTS: The updated search yielded one new trial, for a total of five included studies involving 1173 participants, 32 of whom died. The pooled risk ratio (RR) of all-cause mortality in the intention-to-treat analysis was 0.99 (95% CI 0.50 to 1.95; 4 studies, N = 1123; I(2) = 46%; quality of evidence: very low) and 1.02 (95% CI 0.52 to 1.98; 4 studies, N = 871; I(2) = 49%; quality of evidence: very low) when only analysing participants with confirmed AMI. One trial measured pain directly, and two others measured it by opiate usage. The trial showed no effect, with a pooled RR of 0.97 for the use of opiates (95% CI 0.78 to 1.20; 2 studies, N = 250). The result on mortality and pain are inconclusive. There is no clear effect for oxygen on infarct size (the evidence is inconsistent and low quality). AUTHORS' CONCLUSIONS: There is no evidence from randomised controlled trials to support the routine use of inhaled oxygen in people with AMI, and we cannot rule out a harmful effect. Given the uncertainty surrounding the effect of oxygen therapy on all-cause mortality and on other outcomes critical for clinical decision, well-conducted, high quality randomised controlled trials are urgently required to inform guidelines in order to give definitive recommendations about the routine use of oxygen in AMI