Search

The perinatal histories of 27 newborn infants with NEC were compared to those of 54 infants of equivalent birth weight who did not have NEC during an 8-year study period to see if possible predisposing factors were independent of the confounding effect of birth weight. No differences were observed in gestational age, degree of intrauterine growth retardation, premature rupture of membranes, perinatal asphyxia, skin temperature at admission, haematocrit, presence or absence of respiratory distress syndrome, umbilical catheter placement, start and type of feeding or presence of positive blood cultures. Prematurity is the greatest risk factor predisposing to the development of NEC and the perinatal problems which precede the onset of NEC are common among all premature infants. [ABSTRACT FROM AUTHOR]

Integrins are fundamental for cell adhesion and the formation of focal adhesions (FA). Accordingly, these receptors guide embryonic development, tissue maintenance, and haemostasis but are also involved in cancer invasion and metastasis. A detailed understanding of the molecular interactions that drive integrin activation, FA assembly, and downstream signalling cascades is critical. Here, we reveal a direct association of paxillin, a marker protein of FA sites, with the cytoplasmic tails of the integrin β1 and β3 subunits. The binding interface resides in paxillin's LIM3 domain, where based on the NMR structure and functional analyses, a flexible, 7-amino acid loop engages the unstructured part of the integrin cytoplasmic tail. Genetic manipulation of the involved residues in either paxillin or integrin β3 compromises cell adhesion and motility of murine fibroblasts. This direct interaction between paxillin and the integrin cytoplasmic domain identifies an alternative, kindlin-independent mode of integrin outside-in signalling particularly important for integrin β3 function., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Baade et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Olmesartan is an angiotensin II receptor blocker, approved in 2002 by the Food and Drug Administration for the treatment of hypertension. During chronic therapy with olmesartan, sprue-like enteropathy can occur, being mainly characterised by non-bloody diarrhoea, weight loss and variable degrees of duodenal mucosal damage, which resolved after withdrawal of olmesartan. We hereby report the case of a 77-year-old, poli-treated male patient with a 3-month history of diarrhoea, vomiting and weight loss, associated with severe intestinal villous atrophy and lymphocytic infiltration of gastric and colonic mucosa. After extensive investigations aimed at excluding other possible causes of chronic diarrhoea, a diagnosis of olmesartan-associated enteropathy was made, which was later confirmed by clinical improvement after the discontinuation of the drug. Repeated endoscopy 8 months later showed complete healing of duodenal mucosa with normal villous architecture. Villous atrophy and lymphocytic infiltration of duodenal mucosa are the most described pathologic finding, but several cases of gastric and colonic involvement have also been reported. We, therefore, reviewed the available literature, focussing on the extent of mucosal damage throughout the whole intestine and on its possible causative factors., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Control of integrin activity is vital during development and tissue homeostasis, while derailment of integrin function contributes to pathophysiological processes. Phosphorylation of a conserved threonine motif (T788/T789) in the integrin β cytoplasmic domain increases integrin activity. Here, we report that T788/T789 functions as a phospho-switch, which determines the association with either talin and kindlin-2, the major integrin activators, or filaminA, an integrin activity suppressor. A genetic screen identifies the phosphatase PPM1F as the critical enzyme, which selectively and directly dephosphorylates the T788/T789 motif. PPM1F-deficient cell lines show constitutive integrin phosphorylation, exaggerated talin binding, increased integrin activity, and enhanced cell adhesion. These gain-of-function phenotypes are reverted by reexpression of active PPM1F, but not a phosphatase-dead mutant. Disruption of the ppm1f gene in mice results in early embryonic death at day E10.5. Together, PPM1F controls the T788/T789 phospho-switch in the integrin β1 cytoplasmic tail and constitutes a novel target to modulate integrin activity., (© 2020 Grimm et al.)

Magnesium (Mg 2+ ) is an enzyme co-factor that plays a key role in many biochemical reactions, as well as in glucose metabolism. Clinical evidences have demonstrated that depletion of serum Mg 2+ increases exponentially with the duration of type 2 diabetes mellitus (T2DM). Diabetes is associated with low Mg 2+ , and hypomagnesemia is associated with insulin resistance, inflammation, and increased risk for cardiovascular disease. In subjects at high risk of inflammation and insulin resistance, supplementation of Mg 2+ alone ameliorates both phenotypes, slowing the development and progression of hepatic steatosis. We analyze the relationship between serum Mg 2+ levels and the onset of T2DM in a large cohort of well-characterized adult white individuals participating in the CATAMERI study, who were reexamined after a mean follow-up of 5.6 ± 0.9 years. In our analysis we acquired a significant negative correlation between Mg 2+ levels, fasting glucose, and 2h-post load glucose in subjects who underwent an OGTT. Moreover, Mg 2+ levels correlated negatively with fasting insulin levels, and positively with the lipid profile. As for the detrimental effect of lower circulating Mg 2+ levels, our data revealed a significant reduction of T2DM risk of about 20% for each 1 mg/dL increase of circulating Mg 2+ . The present results are consistent with the theory that Mg 2+ supplementation could ameliorate insulin sensitivity reducing the risk to develop T2DM., Competing Interests: The authors declare no conflict of interest.

Sinus node dysfunction (SND) and atrial fibrillation (AF) often coexist; however, the molecular mechanisms linking both conditions remain elusive. Mutations in the homeobox-containing SHOX2 gene have been recently associated with early-onset and familial AF. Shox2 is a key regulator of sinus node development, and its deficiency leads to bradycardia, as demonstrated in animal models. To provide an extended SHOX2 gene analysis in patients with distinct arrhythmias, we investigated SHOX2 as a susceptibility gene for SND and AF by screening 98 SND patients and 450 individuals with AF. The functional relevance of the novel mutations was investigated in vivo and in vitro , together with the previously reported p.H283Q variant. A heterozygous missense mutation (p.P33R) was identified in the SND cohort and four heterozygous variants (p.G77D, p.L129=, p.L130F, p.A293=) in the AF cohort. Overexpression of the pathogenic predicted mutations in zebrafish revealed pericardial edema for p.G77D and the positive control p.H283Q, whereas the p.P33R and p.A293= variants showed no effect. In addition, a dominant-negative effect with reduced heart rates was detected for p.G77D and p.H283Q. In vitro reporter assays demonstrated for both missense variants p.P33R and p.G77D significantly impaired transactivation activity, similar to the described p.H283Q variant. Also, a reduced Bmp4 target gene expression was revealed in zebrafish hearts upon overexpression of the p.P33R mutant. This study associates additional rare variants in the SHOX2 gene implicated in the susceptibility to distinct arrhythmias and allows frequency estimations in the AF cohort (3/990). We also demonstrate for the first time a genetic link between SND and AF involving SHOX2 . Moreover, our data highlight the importance of functional investigations of rare variants.

Integrins and integrin-dependent cell-matrix adhesions are essential for a number of physiological processes. Integrin function is tightly regulated via binding of cytoplasmic proteins to integrin intracellular domains. Yet, the complexity of cell-matrix adhesions in mammals, with more than 150 core adhesome proteins, complicates the analysis of integrin-associated protein complexes. Interestingly, the evolutionary origin of integrins dates back before the transition from unicellular life to complex multicellular animals. Though unicellular relatives of metazoa have a less complex adhesome, nothing is known about the initial steps of integrin activation and adhesion complex assembly in protozoa. Therefore, we developed a minimal, microscope-based system using chimeric integrins to investigate receptor-proximal events during focal adhesion assembly. Clustering of the human integrin β1 tail led to recruitment of talin, kindlin, and paxillin and mutation of the known talin binding site abolished recruitment of this protein. Proteins indirectly linked to integrins, such as vinculin, migfilin, p130 CAS , or zyxin were not enriched around the integrin β1 tail. With the exception of integrin β4 and integrin β8, the cytoplasmic domains of all human integrin β subunits supported talin binding. Likewise, the cytoplasmic domains of integrin β subunits expressed by the protozoan Capsaspora owczarzaki readily recruited talin and this interaction was based on an evolutionary conserved NPXY/F amino acid motif. The results we present here validate the use of our novel microscopic assay to uncover details of integrin-based protein-protein interactions in a cellular context and suggest that talin binding to integrin β cytoplasmic tails is an ancient feature of integrin regulation.

Cardiovascular disease (CVD) is still the leading cause of death in all western world countries and genetic predisposition in combination with traditional risk factors frequently mediates their manifestation. Genome-wide association (GWA) studies revealed numerous potentially disease modifying genetic loci often including several SNPs and associated genes. However, pure genetic association does not prove direct or indirect relevance of the modifier region on pathogenesis, nor does it define within the associated region the exact genetic driver of the disease. Therefore, the relevance of the identified genetic disease associations needs to be confirmed either in monogenic traits or in experimental in vivo model system by functional genomic studies. In this review, we focus on the use of functional genomic approaches such as gene knock-down or CRISPR/Cas9-mediated genome editing in the zebrafish model to validate disease-associated genomic loci and to identify novel cardiovascular disease genes. We summarize the benefits of the zebrafish for cardiovascular research and highlight examples demonstrating the successful combination of GWA studies and functional genomics in zebrafish to broaden our knowledge on the genetic and molecular underpinnings of cardiovascular diseases.

Mutations in the mitochondrially located protein CHCHD10 cause motoneuron disease by an unknown mechanism. In this study, we investigate the mutations p.R15L and p.G66V in comparison to wild-type CHCHD10 and the non-pathogenic variant p.P34S in vitro, in patient cells as well as in the vertebrate in vivo model zebrafish. We demonstrate a reduction of CHCHD10 protein levels in p.R15L and p.G66V mutant patient cells to approximately 50%. Quantitative real-time PCR revealed that expression of CHCHD10 p.R15L, but not of CHCHD10 p.G66V, is already abrogated at the mRNA level. Altered secondary structure and rapid protein degradation are observed with regard to the CHCHD10 p.G66V mutant. In contrast, no significant differences in expression, degradation rate or secondary structure of non-pathogenic CHCHD10 p.P34S are detected when compared with wild-type protein. Knockdown of CHCHD10 expression in zebrafish to about 50% causes motoneuron pathology, abnormal myofibrillar structure and motility deficits in vivo. Thus, our data show that the CHCHD10 mutations p.R15L and p.G66V cause motoneuron disease primarily based on haploinsufficiency of CHCHD10., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Sarcomeric protein turnover needs to be tightly balanced to assure proper assembly and renewal of sarcomeric units within muscle tissues. The mechanisms regulating these fundamental processes are only poorly understood, but of great clinical importance since many cardiac and skeletal muscle diseases are associated with defective sarcomeric organization. The SET- and MYND domain containing protein 1b (Smyd1b) is known to play a crucial role in myofibrillogenesis by functionally interacting with the myosin chaperones Unc45b and Hsp90α1. In zebrafish, Smyd1b, Unc45b and Hsp90α1 are part of the misfolded myosin response (MMR), a regulatory transcriptional response that is activated by disturbed myosin homeostasis. Genome duplication in zebrafish led to a second smyd1 gene, termed smyd1a. Morpholino- and CRISPR/Cas9-mediated knockdown of smyd1a led to significant perturbations in sarcomere structure resulting in decreased cardiac as well as skeletal muscle function. Similar to Smyd1b, we found Smyd1a to localize to the sarcomeric M-band in skeletal and cardiac muscles. Overexpression of smyd1a efficiently compensated for the loss of Smyd1b in flatline (fla) mutant zebrafish embryos, rescued the myopathic phenotype and suppressed the MMR in Smyd1b-deficient embryos, suggesting overlapping functions of both Smyd1 paralogs. Interestingly, Smyd1a is not transcriptionally activated in Smyd1b-deficient fla mutants, demonstrating lack of genetic compensation despite the functional redundancy of both zebrafish Smyd1 paralogs., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

VCP/p97 (valosin containing protein) is a key regulator of cellular proteostasis. It orchestrates protein turnover and quality control in vivo, processes fundamental for proper cell function. In humans, mutations in VCP lead to severe myo- and neuro-degenerative disorders such as inclusion body myopathy with Paget disease of the bone and frontotemporal dementia (IBMPFD), amyotrophic lateral sclerosis (ALS) or and hereditary spastic paraplegia (HSP). We analyzed here the in vivo role of Vcp and its novel interactor Washc4/Swip (WASH complex subunit 4) in the vertebrate model zebrafish (Danio rerio). We found that targeted inactivation of either Vcp or Washc4, led to progressive impairment of cardiac and skeletal muscle function, structure and cytoarchitecture without interfering with the differentiation of both organ systems. Notably, loss of Vcp resulted in compromised protein degradation via the proteasome and the macroautophagy/autophagy machinery, whereas Washc4 deficiency did not affect the function of the ubiquitin-proteasome system (UPS) but caused ER stress and interfered with autophagy function in vivo. In summary, our findings provide novel insights into the in vivo functions of Vcp and its novel interactor Washc4 and their particular and distinct roles during proteostasis in striated muscle cells.

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase (FAK) family and is mainly expressed in neuronal and hematopoietic cells. As FAK family members are involved in signaling connections downstream of integrins, we studied the role of Pyk2 in complement-receptor 3 (CR3, also known as Mac-1, integrin αMβ2, CD11b/CD18)-mediated phagocytosis, a key process in innate immunity. Using 3 independent approaches, we observed that Pyk2 contributes to CR3-dependent phagocytosis by RAW 264.7 macrophages, but is dispensable for Fcγ receptor (FcγR)-mediated uptake. Reduction of Pyk2 expression levels via siRNA, the pharmacological inhibition of Pyk2 kinase activity as well as macrophage treatment with a cell permeable TAT fusion protein containing the C-terminus of Pyk2 (TAT-PRNK) significantly impaired CR3-mediated phagocytosis without affecting FcγR-mediated uptake. In addition, Pyk2 was strongly recruited to complement opsonized Escherichia coli and the pharmacological inhibition of Pyk2 significantly decreased uptake of the bacteria. Finally, CRISPR/Cas-mediated disruption of the pyk2 gene in RAW 264.7 macrophages confirmed the role of this protein tyrosine kinase in CR3-mediated phagocytosis. Together, our data demonstrate that Pyk2 selectively contributes to the coordination of phagocytosis-promoting signals downstream of CR3, but is dispensable for FcγR-mediated phagocytosis., (© 2016 S. Karger AG, Basel.)

Objective: To investigate the role of azathioprine in maintaining improvement after 1-year low-dose IV pulse CYC therapy in patients with early diffuse Systemic Sclerosis (dcSSc)., Methods: Thirteen patients with early dcSSc who had completed a year of treatment with low-dose IV pulse CYC underwent AZA treatment (100 mg/day) in a prospective 1-year study. Modified Rodnan skin score (mRss), Health Assessment Questionnaire-Disability Index (HAQ-DI), forced vital capacity (FVC), and diffusing lung capacity for CO (DLCO) were assessed as outcome measures. In addition, the nine organ/system Medsger et al. severity scores and the European Scleroderma Study Group (ESSG) activity index were evaluated., Results: The improvement from a year of CYC therapy was maintained by AZA treatment. No outcome measures deteriorated (mRss 8.23 +/- 2.9 vs. 6.38 +/- 3.4; HAQ-DI 0.38 +/- 0.4 vs. 0.32 +/- 0.3; FVC 89.5 +/- 13.2 vs. 89.4 +/- 15.9; DLCO 73.6 +/- 14.4 vs. 75.0 +/- 19.5), nor were there any increases in any organ/system severity scores or ESSG activity index detected., Conclusion: This study suggests a role of AZA in maintaining the improvement induced by low dose pulse CYC in early dcSSc, making it possible a short duration of treatment at a low cumulative dose of the drug. These results, however, await confirmation in controlled studies.

The majority of cutaneous squamous cell carcinoma (SCC) are diagnosed early and cured using local treatment, although a minority of cases metastasize to regional structures. In this report the authors describe an unknown feature of skin SCC, namely, distant brain metastasis. This 54-year-old man, who had undergone surgery for moderately differentiated SCC of the dorsum (T2NOM0 stage), was admitted to our institution 11 months later with headache, vomiting, and ataxia. A magnetic resonance image documented a cerebellar lesion, which was totally removed. Results of histological studies revealed SCC. The patient received whole-brain radiotherapy (30 Gy over 2 weeks using a linear accelerator). A metastatic work-up showed enlarged inguinal and para-aortic lymph nodes that were histologically examined using excisional biopsy. Inguinal lymph nodes were tumor-positive and were dissected. The patient was subjected to two cycles of chemotherapy with cisplatin (75 mg/m2). After 3 months, a significant reduction in the size of the para-aortic lymph nodes was documented on control computerized tomography studies. Although the described case is unique, knowledge of the potential for this uncommon behavior in cutaneous SCC may be useful, especially because of its increasing incidence.

Congenital presentation of neonatal lupus erythematous is very rare. We describe an infant who had congenital neonatal lupus erythematosus with atrophic lesions on the face and scarring lesions on the trunk. All radiologic, virologic, and hematologic assays were normal. Skin biopsy specimen and immunofluorescence findings led us to suspect neonatal lupus, a diagnosis confirmed by positivity for anti-Sjörgen syndrome and antinuclear antibodies both in the child and her mother. In this infant, skin manifestations represented the stable and irreversible outcome of an inflammatory stage that occurred during pregnancy.

Both oral and intravenous high-dose cyclophosphamide (CYC) regimens are associated with serious side effects when used for the treatment of systemic sclerosis (SSc). The aim of the present trial was to test the safety of low-dose intravenous CYC in patients with SSc. Eight SSc patients, in whom CYC treatment was warranted, were studied at baseline and after 6 months' intravenous CYC treatment (500 mg pulses at weeks 0, 1, 2, 6, 10, 14, 18 and 22). Side effects probably related to CYC treatment were carefully investigated. The development of amenorrhea was assessed during the period of treatment and over the following 12 months. The therapy was well tolerated overall. No patient discontinued treatment because of side effects. Leukopenia, premature ovarian failure, hemorrhagic cystitis, microscopic hematuria and liver toxicity were never detected. The most common adverse events were mild and self-limiting nausea and weakness. Our data suggest that low-dose intravenous CYC is relatively safe, at least in the short term. Further studies are needed to assess both the efficacy and the long-term safety.