Impact of COVID-19 lockdown on glycemic levels during pregnancy: A retrospective analysis.

Studies on the COVID-19 pandemic effects on gestational diabetes mellitus (GDM) remain limited and controversial. This study aimed to investigate the impact of the COVID-19 lockdown on the glycemic balance of pregnant women and GDM risk. To this aim, a single-center retrospective cohort analysis assessing glucose homeostasis using the oral glucose tolerance test in 862 pregnant women before (from March 9, 2019 to March 8, 2020 - Group 1), during (from March 9, 2020 to March 8, 2021 - Group 2), and after (from March 9, 2021 to March 8, 2022 - Group 3) the COVID-19 lockdown in Molise, a region of central Italy, was conducted. We observed that the blood glucose concentration of pregnant women was significantly lower during the COVID-19 lockdown than during the previous and following years at all time points evaluated (time 0, 60', and 120'). Specifically, at time 0, it was 82.14 mg/dl for group 2 vs 85.94 for group 1 (p = 0.0001) and 85.87 for group 3 (p = 0.001). Similarly, at 60', it was 121.38 mg/dl for group 2 vs 129.30 mg/dl for group 1 (p = 0.0029) and 131.68 mg/dl for group 3 (p = 0.0006). Moreover, at 120', it was 104.20 mg/dl for group 2 vs 111.51 mg/dl (p = 0.0004) for group 1, and 116.06 mg/dl for group 3 (p = 0.0001). In contrast with previous findings, the COVID-19 lockdown was associated with an improved glycemic balance. Further studies are needed to better clarify the influence of lockdown restrictions on glucose metabolism and, consequently, on GDM risk.

LimpiAD foam and the potential control of the pressure ulcers onset.

Pressure ulcers development is an undesirable event that often worsens the clinical condition of patients already affected by severe pathologies. Since the aetiology of this clinical complication is unclear yet, at current the primary approach to treat the problem is the adoption of suitable patients' assistance procedures. At the same time, the research focuses on finding medicaments or treatment strategies that could prevent the lesions and/or accelerate their healing. The international market's wide range of cosmetic/pharmaceuticals products is mainly topical preparations based on emollient agents to preserve or restore skin homeostasis. On the other hand, the skin microbiome's implication in the pressure ulcers occurrence is mainly unknown. Based on these assumptions, here we tested an innovative preparation, the LimpiAD foam, as a potential preventive strategy of pressure ulcers onset. The active component of this product is composed of hyaluronic acid conjugated with a bacterial cell wall fragment of C. acnes DSM 28251. For LimpiAD foam, we hypothesised a combined action of the two components on the skin tissue, an emollient effect due to the hyaluronic acid properties together with a modulatory effect on the skin microbiota carried out by the component of bacterial derivation. Our results supported the hypothesis and suggested a potential role of LimpiAD foam in pressure ulcers prevention.

Developmental Coordination Disorder in a Patient with Mental Disability and a Mild Phenotype Carrying Terminal 6q26-qter Deletion.

Terminal deletion of chromosome 6q is a rare chromosomal abnormality associated with variable phenotype spectrum. Although intellectual disability, facial dysmorphism, seizures and brain abnormalities are typical features of this syndrome, genotype-phenotype correlation needs to be better understood. We report the case of a 6-year-old Caucasian boy with a clinical diagnosis of intellectual disability, delayed language development and dyspraxia who carries an approximately 8 Mb de novo heterozygous microdeletion in the 6q26-q27 locus identified by karyotype and defined by high-resolution SNP-array analysis. This patient has no significant structural brain or other organ malformation, and he shows a very mild phenotype compared to similar 6q26-qter deletion. The patient phenotype also suggests that a dyspraxia susceptibility gene is located among the deleted genes.

Inhibition of type 5 phosphodiesterase counteracts ß2-adrenergic signalling in beating cardiomyocytes.

AIMS: Compartmentalization of cAMP and PKA activity in cardiac muscle cells plays a key role in maintaining basal and enhanced contractility stimulated by sympathetic nerve activity. In cardiomyocytes, activation of adrenergic receptor increases cAMP production, which is countered by the hydrolytic activity of selective phosphodiesterases (PDEs). The intracellular regional dynamics of cAMP production and hydrolysis modulate downstream signals resulting in different biological responses. The interplay between beta receptors (ßARs) signalling and phosphodiesterase 5 (PDE5) activity remains to be addressed. METHODS AND RESULTS: Using combined strategies with pharmacological inhibitors and genetic deletion of PDEs and ßAR isoforms, we revealed a specific pool of cAMP that is under dual regulation by PDE2 and, indirectly, PDE5 activity. Inhibition of PDE5 with sildenafil produces a cGMP-dependent activation of PDE2 that attenuates cAMP generation induced by ßAR agonists, with concomitant modulation of stimulated contraction rate and calcium transients. PDE2 haploinsufficiency abolished the effects of sildenafil. The negative chronotropic effect of PDE5 inhibition through PDE2 activation was also observed in sinoatrial node tissue from adult mice. PDE5 inhibition selectively lowered contraction rate stimulated by ß2AR, but not ß1AR activation, supporting a compartmentalization of the cGMP-modulated pool of cAMP. CONCLUSION: These data identify a new effect of PDE5 inhibitors on the modulation of cardiomyocyte response to adrenergic stimulation via PDE5-PDE2-mediated cross-talk.

From genetics to genomics of epilepsy.

The introduction of DNA microarrays and DNA sequencing technologies in medical genetics and diagnostics has been a challenge that has significantly transformed medical practice and patient management. Because of the great advancements in molecular genetics and the development of simple laboratory technology to identify the mutations in the causative genes, also the diagnostic approach to epilepsy has significantly changed. However, the clinical use of molecular cytogenetics and high-throughput DNA sequencing technologies, which are able to test an entire genome for genetic variants that are associated with the disease, is preparing a further revolution in the near future. Molecular Karyotype and Next-Generation Sequencing have the potential to identify causative genes or loci also in sporadic or non-familial epilepsy cases and may well represent the transition from a genetic to a genomic approach to epilepsy.

Cell adaptation to activated FGFR3 includes Sprouty4 up regulation to inhibit the receptor-mediated ERKs activation from the endoplasmic reticulum.

The kinase activity of the thanatophoric dysplasia type II-fibroblast growth factor receptor 3 mutant (TDII-FGFR3) hampers its maturation. As a consequence, the immature receptor activates extracellular regulated kinases (ERKs) from the endoplasmic reticulum (ER), which leads to apoptosis. On the other hand, in stable TDII-FGFR3 cells receptor biosynthesis is restored and ERKs are activated from the cell surface. To identify potential mediators of cell adaptation to the activated receptor we investigated gene products that are differently regulated in TDII and wild-type FGFR3 cells. cDNA representational difference analysis reveals Sprouty4 up regulation in the TDII-FGFR3 cells. Interestingly, Sprouty4 inhibits the TDII-FGFR3-mediated ERKs activation from the ER, but fails to suppress ERKs activation from cell surface. We conclude that cell adaptation to activated FGFR3 include Sprouty4 activity, which silences the premature receptor signaling and suppress apoptosis.

Transient dimerization and interaction with ERGIC-53 occur in the fibroblast growth factor receptor 3 early secretory pathway.

The fibroblast growth factor receptor 3 (FGFR3) secretory pathway includes N-linked glycosylation in the endoplasmic reticulum where a stringent quality control system ensures that only correctly folded receptor reaches the cell surface from where mature-functional FGFR3 signals upon ligand-mediated dimerization. We have previously shown that the increased kinase activity associated with FGFR3 bearing the thanatophoric dysplasia type II (TDII) mutation hampers its maturation, enabling the receptor to signal from the endoplasmic reticulum. Here we investigate if this biosynthetic disturbance could be explained by premature dimerization of the receptor. Our observations show that a limited fraction of the immature high-mannose, mutant receptor dimerizes in the early secretory pathway, as does the immature wild type FGFR3. In contrast, the mature fully glycosylated wild type receptor reaches the cell surface as monomer suggesting that dimerization is a transient event. The kinase activity of mutant FGFR3 is not required for dimerization to occur, although it increases dimerization efficiency. Furthermore, mutant FGFR3 trans-phosphorylates the immature wild type receptor indicating that dimerization occurs in the endoplasmic reticulum. Visualization of protein interaction inside the secretory pathway confirms receptor dimerization. In addition, it shows that both wild type and TDII FGFR3 interact with the mannose-specific lectin ERGIC-53. We conclude that transient dimerization is an obligatory step in FGFR3 biosynthesis acting as a pre-assembly quality control mechanism. Furthermore, the TDII/ERGIC-53 complex formation may function as a checkpoint for FGFR3 sorting downstream the endoplasmic reticulum. These findings have implications for understanding the pathogenesis of FGFR3-related disorders.

Overexpression of the C-type natriuretic peptide (CNP) is associated with overgrowth and bone anomalies in an individual with balanced t(2;7) translocation.

Longitudinal bone growth is determined by the process of endochondral ossification in the cartilaginous growth plate, which is located at both ends of vertebrae and long bones and involves many systemic hormones and local regulators. We report the molecular characterization of a de novo balanced t(2;7)(q37.1;q21.3) translocation in a young female with Marfanoid habitus and skeletal anomalies. The translocation was characterized by fluorescence in situ hybridization (FISH), checked for other abnormalities by array-comparative genomic hybridization (CGH), and finally, the breakpoints were cloned, sequenced, and compared. Biochemical dosage was applied to study the possible mechanisms that may cause the proposita's phenotype. The breakpoint on chromosome 2 disrupts the hypothetical gene MGC42174 (HUGO-approved symbol DIS3L2) and is located in the proximity of the NPPC gene coding for C-type natriuretic peptide (CNP), a molecule that regulates endochondral bone growth. CNP plasma concentration was doubled in the proband compared to five normal controls, while NPPC was substantially overexpressed in her fibroblasts. A transgenic mouse generated to target NPPC overexpression in bone showed a phenotype highly reminiscent of the patient's phenotype. The breakpoint on chromosome 7 is localized proximally at about 75 kb from the COL1A2 gene. The COL1A2 allele on the derivative chromosome was strongly underexpressed in fibroblasts, but total collagen was not significantly different from controls. Several evidences support the conclusion that the proband's abnormal phenotype is associated with C-type natriuretic peptide overexpression.

The immediate upstream sequence of the mouse Ret gene controls tissue-specific expression in transgenic mice.

The RET gene is tightly regulated at the transcriptional level during embryo development, however in vitro experiments in cultured cells have failed to clarify the molecular mechanism of cell-type specificity of RET promoter activity. Therefore, we have generated transgenic mice in which the LacZ reporter gene is controlled by murine Ret promoter sequences to clarify in an in vivo model how this transcriptional regulation is achieved. We describe here the results of reporter gene expression in mice in which the transgene contained 380- and 1962-bp sequence upstream of the ATG start codon, derived from the mouse Ret promoter region, fused to the beta-galactosidase coding sequence. Transgenic mice showed well-defined patterns of beta-galactosidase staining obtained with both transgenes, suggesting that they were able per se to direct the reporter gene expression in specific districts, such as cranial ganglia, dorsal root ganglia, the heart and the kidney, partially recapitulating the profile of the endogenous Ret gene. In particular, proper expression in the developing excretory system seemed quite significant when considering that the appropriate regulation was obtained with a very short, 380 bp, fragment of Ret 5' flanking sequence.

Targeted expression of SHH affects chondrocyte differentiation, growth plate organization, and Sox9 expression.

UNLABELLED: The role of Hedgehogs (Hh) in murine skeletal development was studied by overexpressing human Sonic Hedgehog (SHH) in chondrocytes of transgenic mice using the collagen II promoter/enhancer. Overexpression caused a lethal craniorachischisis with major alterations in long bones because of defects in chondrocyte differentiation. INTRODUCTION: Hedgehogs (Hhs) are a family of secreted polypeptides that play important roles in vertebrate development, controlling many critical steps of cell differentiation and patterning. Skeletal development is affected in many different ways by Hhs. Genetic defects and anomalies of Hhs signaling pathways cause severe abnormalities in the appendicular, axial, and cranial skeleton in man and other vertebrates. MATERIALS AND METHODS: Genetic manipulation of mouse embryos was used to study in vivo the function of SHH in skeletal development. By DNA microinjection into pronuclei of fertilized oocytes, we have generated transgenic mice that express SHH specifically in chondrocytes using the cartilage-specific collagen II promoter/enhancer. Transgenic skeletal development was studied at different embryonic stages by histology. The expression pattern of specific chondrocyte molecules was studied by immunohistochemistry and in situ hybridization. RESULTS: Transgenic mice died at birth with severe craniorachischisis and other skeletal defects in ribs, sternum, and long bones. Detailed analysis of long bones showed that chondrocyte differentiation was blocked at prehypertrophic stages, hindering endochondral ossification and trabecular bone formation, with specific defects in different limb segments. The growth plate was highly disorganized in the tibia and was completely absent in the femur and humerus, leading to skeletal elements entirely made of cartilage surrounded by a thin layer of bone. In this cartilage, chondrocytes maintained a columnar organization that was perpendicular to the bone longitudinal axis and directed toward its outer surface. The expression of SHH receptor, Patched-1 (Ptc1), was greatly increased in all cartilage, as well as the expression of parathyroid hormone-related protein (PTHrP) at the articular surface; while the expression of Indian Hedgehog (Ihh), another member of Hh family that controls the rate of chondrocyte maturation, was greatly reduced and restricted to the displaced chondrocyte columns. Transgenic mice also revealed the ability of SHH to upregulate the expression of Sox9, a major transcription factor implicated in chondrocyte-specific gene expression, in vivo and in vitro, acting through the proximal 6.8-kb-long Sox9 promoter. CONCLUSION: Transgenic mice show that continuous expression of SHH in chondrocytes interferes with cell differentiation and growth plate organization and induces high levels and diffuse expression of Sox9 in cartilaginous bones.

Depletion of cartilage collagen fibrils in mice carrying a dominant negative Col2a1 transgene affects chondrocyte differentiation.

We have generated transgenic mice harboring the deletion of exon 48 in the mouse alpha1(II) procollagen gene (Col2a1). This was the first dominant negative mutation identified in the human alpha1(II) procollagen gene (COL2A1). Patients carrying a single allele with this mutation suffer from a severe skeletal disorder called spondyloepiphyseal dysplasia congenita (SED). Transgenic mice phenotype was neonatally lethal with severe respiratory failure, short bones, and cleft palate. Transgene mRNA was expressed at high levels. Growth plate cartilage of transgenic mice presented morphological abnormalities and reduced number of collagen type II fibrils. Chondrocytes carrying the mutation showed altered expression of several differentiation markers, like fibroblast growth factor receptor 3 (Fgfr3), Indian hedgehog (Ihh), runx2, cyclin-dependent kinase inhibitor P21CIP/WAF (Cdkn1a), and collagen type X (Col10a1), suggesting that a defective extracellular matrix (ECM) depleted of collagen fibrils affects chondrocytes differentiation and that this defect participates in the reduced endochondral bone growth observed in chondrodysplasias caused by mutations in COL2A1.

Y-position collagen II mutation disrupts cartilage formation and skeletal development in a transgenic mouse model of spondyloepiphyseal dysplasia.

Mice were generated by pronuclear injection of a type II collagen transgene harboring an Arg789Cys (R789C) mutation that has been found in patients with spondyloepiphyseal dysplasia (SED). Expression was directed to cartilage by the murine Col2a1 promoter to examine the consequences of mutations involving the Y-position of the collagen helix Gly-X-Y triplet on skeletogenesis. The transgenic mice had very short limbs, short trunk, short snout, and cleft palate; they died at birth. Their growth plates were disorganized and collagen fibrils were sparse in cartilage matrix. When the transgene was expressed in RCS cells, there was no evidence that R789C-bearing collagen chains were incorporated into stable collagen molecules. Molecular modeling of the mutation raised the possibility that it destabilizes the collagen triple helix. Together our results suggest that Y-position mutations, such as R789C, can act in a dominant negative manner to destabilize collagen molecules during assembly, reducing their availability to form fibrils, the deficiency of which profoundly disturbs the template functions of cartilage during skeletogenesis.