Technical refinements of the scapular tip-free flap for mandibular reconstruction.

BACKGROUND: The use of scapular tip chimeric free flaps (STFFs) for reconstructing mandibular defects has recently become popular, but its utility relative to other bone-containing free flaps remains debatable. The aim of the report is to describe how technical modification of STFF impacted in its use for mandibular reconstruction also commenting results obtained in a unicentric series of patients. PATIENTS AND METHODS: Patients undergoing mandibular reconstruction using an STFF from January 1, 2014 to June 1, 2022 were retrospectively enrolled in this report. We collected data on chimeric flap type, bone management, vascular pedicles, and the final outcomes. In total, 31 patients (13 men and 18 women) with a mean age of 68 years were enrolled. According to the classification system of Urken, 15 patients had body defects, while 7 had ramus defects, another 7 had symphysis defects, and 2 had both ramus and bodily defects. STFF was always harvested working in two equips simultaneously, in supine position. Dissection included preparation of chimeric components of the flap as latissimus dorsi, serratus and scapular tip. After pedicle dissection scapular bone was cut basing on reconstructive needing with a rectangular (stick) shape including the border of the scapula. In cases of longer bone harvesting, circumflex pedicle was also included to perfuse the upper portion of the scapular border. In five cases, the STFF was harvested with only the scapular angle component, and was thus a composite osteomuscular flap; for the remaining 26 cases, a chimeric STFF was used. Circumflex pedicle was included for eight patients. Six of the seven patients with symphyseal defects underwent a single osteotomy. RESULTS: The average length of the harvested was 69.92 mm (maximum length = 104 mm). The average height of transplanted bone was 26.78 mm (maximum height = 44.2 mm). Mouth-opening was normal in 25 patients, limited in 6 patients, and severely impaired in no patients. The cosmetic results were rated as excellent by 20 patients, good by 8 patients, and poor by 3 patients. CONCLUSION: The STFF is an excellent option for mandibular reconstruction when other flaps are not available and for patients in poor general condition. Technical innovations here presented made possible to harvest long bone segments with accurate shape thanks to osteotomies if needed and with adequate soft tissues components of the chimeric flap, ensuring satisfactory functional and cosmetic results.

Optimizing the results of facial animation surgery: Botulinum toxin injection into free functional gracilis flap transfer.

Although neuromuscular gracilis transplantation is the best choice for facial reanimation in patients with congenital or inveterate palsy, the results are not completely satisfactory. Ancillary procedures developed to achieve better symmetry of the smile and reduce the hypercontractility of the transplanted muscle have been reported. However, the intramuscular injection of botulinum toxin has not been described for this purpose. Patients undergoing gracilis injections of botulinum toxin after facial reanimation surgery between September 1, 2020, and June 1, 2022, were retrospectively enrolled in this study. We collected photographs taken before and 20-30 days after injection and compared the symmetry of the face using software. Nine patients with a mean age of 23.56 years (range, 7-56 years) were enrolled. Reinnervation of the muscle was provided by the contralateral healthy facial nerve via a sural cross-graft (four patients), by the ipsilateral masseteric nerve (three cases), and by the contralateral masseteric and facial nerve (two). Using Emotrics software, we identified differences in the commissure excursion discrepancy of 3.82 mm, the smile angle discrepancy of 0.084°, and the dental show discrepancy of 1.49 mm; the average difference in the commissure height deviation was 2.26 mm (P = 0.02), and those in the upper- and lower-lip height deviation were 1.05 mm and 1.49 mm, respectively. Gracilis injection of botulinum toxin after gracilis transplantation is a safe and feasible procedure that could be applicable to all patients with asymmetric smiles related to excessive transplant contraction. It yields good esthetic results with little to no related morbidity.

Special Issue on "Insights on Ecotoxicological Effects of Anthropogenic Contaminants in Aquatic Organisms".

In human history, many key points have characterized technological progress, such as the use of metals, which began in prehistoric times and continues to the present day, with many industrial uses [...].

Analysis of the P. lividus sea urchin genome highlights contrasting trends of genomic and regulatory evolution in deuterostomes.

Sea urchins are emblematic models in developmental biology and display several characteristics that set them apart from other deuterostomes. To uncover the genomic cues that may underlie these specificities, we generated a chromosome-scale genome assembly for the sea urchin Paracentrotus lividus and an extensive gene expression and epigenetic profiles of its embryonic development. We found that, unlike vertebrates, sea urchins retained ancestral chromosomal linkages but underwent very fast intrachromosomal gene order mixing. We identified a burst of gene duplication in the echinoid lineage and showed that some of these expanded genes have been recruited in novel structures (water vascular system, Aristotle's lantern, and skeletogenic micromere lineage). Finally, we identified gene-regulatory modules conserved between sea urchins and chordates. Our results suggest that gene-regulatory networks controlling development can be conserved despite extensive gene order rearrangement.

Inflammation and the Potential Implication of Macrophage-Microglia Polarization in Human ASD: An Overview.

Autism spectrum disorder (ASD) is a heterogeneous collection of neurodevelopmental disorders, difficult to diagnose and currently lacking treatment options. The possibility of finding reliable biomarkers useful for early identification would offer the opportunity to intervene with treatment strategies to improve the life quality of ASD patients. To date, there are many recognized risk factors for the development of ASD, both genetic and non-genetic. Although genetic and epigenetic factors may play a critical role, the extent of their contribution to ASD risk is still under study. On the other hand, non-genetic risk factors include pollution, nutrition, infection, psychological states, and lifestyle, all together known as the exposome, which impacts the mother's and fetus's life, especially during pregnancy. Pathogenic and non-pathogenic maternal immune activation (MIA) and autoimmune diseases can cause various alterations in the fetal environment, also contributing to the etiology of ASD in offspring. Activation of monocytes, macrophages, mast cells and microglia and high production of pro-inflammatory cytokines are indeed the cause of neuroinflammation, and the latter is involved in ASD's onset and development. In this review, we focused on non-genetic risk factors, especially on the connection between inflammation, macrophage polarization and ASD syndrome, MIA, and the involvement of microglia.

Carbonic anhydrases in development: morphological observations and gene expression profiling in sea urchin embryos exposed to acetazolamide.

Carbonic anhydrases (CANs) are conserved metalloenzymes catalysing the reversible hydration of carbon dioxide into protons and bicarbonate, with important roles in cells physiology. Some CAN-coding genes were found in sea urchin genome, although only one involved in embryonic skeletogenesis was described in Paracentrotus lividus. Here, we investigated gene expression patterns of P. lividus embryos cultured in the presence of acetazolamide (AZ), a CAN inhibitor, to combine morphological defects with their molecular underpinning. CAN inhibition blocked skeletogenesis, affected the spatial/temporal expression of some biomineralization-related genes, inhibited embryos swimming. A comparative analysis on the expression of 127 genes in control and 3 h/24 h AZ-treated embryos, using NanoString technology, showed the differential expression of genes encoding for structural/regulatory proteins, with different embryonic roles: biomineralization, transcriptional regulation, signalling, development and defence response. The study of the differentially expressed genes and the signalling pathways affected, besides in silico analyses and a speculative 'interactomic model', leads to predicting the presence of various CAN isoforms, possibly involved in different physiological processes/activities in sea urchin embryo, and their potential target genes/proteins. Our findings provide new valuable molecular data for further studies in several biological fields: developmental biology (biomineralization, axes patterning), cell differentiation (neural development) and drug toxicology (AZ effects on embryos/tissues).

Acute abdomen in post- Covid 19 multisystem inflammatory syndrome in children. A case report.

AIM: As more data about coronavirus disease-2019 (COVID-19) has been gathered it has become evident that children who have had or have been exposed to COVID-19 can develop a rare complication; multisystem inflammatory syndrome in children (MIS-C). We report the case of a 9-year-old boy, who was brought to the emergency room with suspected acute abdomen and was diagnosed with MIS-C. METHODS: The patient had had a positive molecular test for COVID-19, 25 days earlier and fever that started 4 days prior to presentation, He tested negative for COVID on arrival at the emergency room. After physical examination, and diagnostic tests were performed the differential diagnosis included appendiceal inflammation and MIS-C. Surgical exploration was performed laparoscopically. RESULTS: The immune morphological picture was reactive lymphogranular hyperplasia. Postoperatively the abdominal symptoms improved rapidly but the patient developed diffuse erythema as well as some cardiovascular and neurological disturbances. The patient was discharged on postoperative day 14 in good general condition with a diagnosis of MIS-C. CONCLUSIONS: In patients with a recent positive COVID test and mainly gastroenterological manifestations surgical exploration is necessary in order to prevent delayed diagnosis and inadequate/inappropriate treatment. KEY WORDS: Acute abdomen, COVID-19, MIS-C, Gastrointestinal symptoms.

Gene Expression Analysis of the Stress Response to Lithium, Nickel, and Zinc in Paracentrotus lividus Embryos.

Many anthropogenic pollutants such as metals are discharged into the marine environment through modern sources. Among these, lithium (Li), nickel (Ni), and zinc (Zn) can interfere with biological processes in many organisms when their concentration rises. These metals are toxic to sea urchin embryos, affecting their development. Indeed, animal/vegetal and dorso/ventral embryonic axes are differently perturbed: Li is a vegetalizing agent, Ni can disrupt dorso-ventral axis, Zn can be animalizing. To address the molecular response adopted by embryos to cope with these metals or involved in the gene networks regulating embryogenesis, and to detect new biomarkers for evaluating hazards in polluted environments in a well-known in vivo model, we applied a high-throughput screening approach to sea urchin embryos. After fertilization, Paracentrotus lividus embryos were exposed to Li, Ni, and Zn for 24/48 h. At both endpoints, RNAs were analyzed by NanoString nCounter technology. By in silico analyses, we selected a panel of 127 transcripts encoding for regulatory and structural proteins, ranked in categories: Apoptosis, Defense, Immune, Nervous, Development, and Biomineralization. The data analysis highlighted the dysregulation of many genes in a metal-dependent manner. A functional annotation analysis was performed by the KEEG Orthology database. This study provides a platform for research on metals biomarkers in sea urchins.

Post-surgery Rehabilitative Intervention Based on Imitation Therapy and Mouth-Hand Motor Synergies Provides Better Outcomes in Smile Production in Children and Adults With Long Term Facial Paralysis.

Rehabilitation after free gracilis muscle transfer (smile surgery, SS) is crucial for a functional recovery of the smiling skill, mitigating social and psychological problems resulting from facial paralysis. We compared two post-SS rehabilitation treatments: the traditional based on teeth clenching exercises and the FIT-SAT (facial imitation and synergistic activity treatment). FIT-SAT, based on observation/imitation therapy and on hand-mouth motor synergies would facilitate neuronal activity in the facial motor cortex avoiding unwanted contractions of the jaw, implementing muscle control. We measured the smile symmetry on 30 patients, half of whom after SS underwent traditional treatment (control group, CG meanage = 20 ± 9) while the other half FIT-SAT (experimental group, EG meanage= 21 ± 14). We compared pictures of participants while holding two postures: maximum and gentle smile. The former corresponds to the maximal muscle contraction, whereas the latter is strongly linked to the control of muscle strength during voluntary movements. No differences were observed between the two groups in the maximum smile, whereas in the gentle smile the EG obtained a better symmetry than the CG. These results support the efficacy of FIT-SAT in modulating the smile allowing patients to adapt their smile to the various social contexts, aspect which is crucial during reciprocal interactions.

Membrane Protein Production in Escherichia coli: Protocols and Rules.

Despite recent progresses in the use of eukaryotic expression system, production of membrane proteins for structural studies still relies on microbial expression systems. In this review, we provide protocols to achieve high level expression of membrane proteins in Escherichia coli, especially using the T7 RNA polymerase based expression system. From the design of the construct, the choice of the appropriate vector-host combination, the assessment of the bacterial fitness, to the selection of bacterial mutant adapted to the production of the target membrane protein, the chapter covers all necessary methods for a rapid optimization of a specific target membrane protein. In addition, we provide a protocol for membrane protein solubilization based on our recent analysis of the Protein Data Bank.

MiRNAs Expression Profiling in Raw264.7 Macrophages after Nfatc1-Knockdown Elucidates Potential Pathways Involved in Osteoclasts Differentiation.

Differentiation of macrophages toward osteoclasts is crucial for bone homeostasis but can be detrimental in disease states, including osteoporosis and cancer. Therefore, understanding the osteoclast differentiation process and the underlying regulatory mechanisms may facilitate the identification of new therapeutic targets. Hereby, we tried to reveal new miRNAs potentially involved in the regulation of early steps of osteoclastogenesis, with a particular focus on those possibly correlated with NFATc1 expression, by studying miRNAs profiling. During the first 24 h of osteoclastogenesis, 38 miRNAs were differentially expressed between undifferentiated and RANKL-stimulated RAW264.7 cells, while 10 miRNAs were differentially expressed between RANKL-stimulated cells transfected with negative control or NFATc1-siRNAs. Among others, the expression levels of miR-411, miR-144 and members of miR-29, miR-30, and miR-23 families changed after RANKL stimulation. Moreover, the potential role of miR-124 during osteoclastogenesis was explored by transient cell transfection with anti-miR-124 or miR-124-mimic. Two relatively unknown miRNAs, miR-880-3p and miR-295-3p, were differentially expressed between RANKL-stimulated/wild-type and RANKL-stimulated/NFATc1-silenced cells, suggesting their possible correlation with NFATc1. KEGG enrichment analyses showed that kinase and phosphatase enzymes were among the predicted targets for many of the studied miRNAs. In conclusion, our study provides new data on the potential role and possible targets of new miRNAs during osteoclastogenesis.

Neurotoxicity in Marine Invertebrates: An Update.

Invertebrates represent about 95% of existing species, and most of them belong to aquatic ecosystems. Marine invertebrates are found at intermediate levels of the food chain and, therefore, they play a central role in the biodiversity of ecosystems. Furthermore, these organisms have a short life cycle, easy laboratory manipulation, and high sensitivity to marine pollution and, therefore, they are considered to be optimal bioindicators for assessing detrimental chemical agents that are related to the marine environment and with potential toxicity to human health, including neurotoxicity. In general, albeit simple, the nervous system of marine invertebrates is composed of neuronal and glial cells, and it exhibits biochemical and functional similarities with the vertebrate nervous system, including humans. In recent decades, new genetic and transcriptomic technologies have made the identification of many neural genes and transcription factors homologous to those in humans possible. Neuroinflammation, oxidative stress, and altered levels of neurotransmitters are some of the aspects of neurotoxic effects that can also occur in marine invertebrate organisms. The purpose of this review is to provide an overview of major marine pollutants, such as heavy metals, pesticides, and micro and nano-plastics, with a focus on their neurotoxic effects in marine invertebrate organisms. This review could be a stimulus to bio-research towards the use of invertebrate model systems other than traditional, ethically questionable, time-consuming, and highly expensive mammalian models.

A preliminary gene expression analysis on Paracentrotus lividus embryos exposed to UVB, Cadmium and their combination.

Paracentrotus lividus is a Mediterranean and Eastern Atlantic sea urchin species, very sensitive to chemical and physical environmental changes and widely used in eco-toxicological studies. Here, we applied a high throughput screening approach on P. lividus embryos exposed to UVB radiation (UV), Cadmium Chloride (Cd) and their combination (Cd/UV), to deeply characterize the molecular responses adopted by embryos to cope with these stressors. in vitro eco-toxicological assays were performed by exposing embryos to Cd (10-4 M) soon after fertilization, to UV (200 and 400J/m2) at early stage of development, while in co-exposure experiments, Cd-exposed embryos were irradiated with UV at 200 J/m2. By NanoString nCounter technology, custom-made probes were developed and hybridized on total RNA extracted from exposed embryos at 51h after fertilization. By in silico analyses, we selected and retrieved at the NCBI nucleotide database a panel of P. lividus transcripts encoding for many regulatory and structural proteins that we ranked in categories, i.e., Apoptosis, Biomineralization, Defense, Development, Immunity, Signaling and Transcription Factors. The analysis of 127 transcripts highlighted the dysregulation of many genes, some specifically activated to cope with stress agents, others involved in the complex molecular network of genes that regulate embryo development. We revealed the downregulation of Biomineralization and Development genes and the upregulation of Defensive genes in Cd and Cd/UV embryos. Our approach, using sea urchin embryo as an in vivomodel, contributes to advance our knowledge about cellular responses to UV, Cd and their combination.

Osteoclasts Differentiation from Murine RAW 264.7 Cells Stimulated by RANKL: Timing and Behavior.

The development of multi-nucleated cells is critical for osteoclasts (OCs) maturation and function. Our objective was to extend knowledge on osteoclastogenesis, focusing on pre-OC fusion timing and behavior. RAW 264.7 cells, which is a murine monocyte-macrophage cell line, provide a valuable and widely used tool for in vitro studies on osteoclastogenesis mechanisms. Cells were treated with the receptor activator of nuclear factor κ-B ligand (RANKL) for 1-4 days and effects on cell morphology, cytoskeletal organization, protein distribution, and OC-specific gene expression examined by TEM, immunofluorescence, and qPCR. Multinucleated cells began to appear at two days of Receptor Activator of Nuclear factor κ-B Ligand (RANKL) stimulation, increasing in number and size in the following days, associated with morphological and cytoskeletal organization changes. Interesting cellular extensions were observed in three days within cells labeled with wheat germ agglutinin (WGA)-Fluorescein isothiocyanate (FITC). The membrane, cytoplasmic, or nuclear distribution of RANK, TRAF6, p-p38, pERK1/2, and NFATc1, respectively, was related to OCs maturation timing. The gene expression for transcription factors regulating osteoclastogenesis (NFATc1, c-fos, RelA, MITF), molecules involved in RANKL-signaling transduction (TRAF6), cytoskeleton regulation (RhoA), fusion (DC-STAMP), migration (MMP9), and OC-specific enzymes (TRAP, CtsK), showed different trends related to OC differentiation timing. Our findings provide an integrated view on the morphological and molecular changes occurring during RANKL stimulation of RAW 264.7 cells, which are important to better understand the OCs' maturation processes.

Maxillary reconstruction with scapular tip chimeric free flap.

PURPOSE: Purpose of the article is to discuss the use of the scapular tip free flap (STFF) for the reconstruction of maxillary defects. METHODS: A retrospective evaluation of patients who underwent maxillary reconstruction with STFF is presented. Patients were evaluated with respect to complications, function, and cosmesis. RESULTS: Study population consisted of 53 patients. All flaps survived and partial bone resorption only occurred in a young patient. Minor complications included two instances of partial muscular necrosis. The donor site was primarily closed in all patients. Mouth opening was assessed as good (>3 cm) in 41 patients, partially limited (2-3 cm) in 9 patients, and limited (<2 cm) in 3 patients. Dental rehabilitation was achieved in 35 patients; esthetic results were assessed by patient as excellent in 19 patients, good in 28 patients, and poor in 6 patients. CONCLUSIONS: The scapular tip chimeric free flap represents an indispensable tool for reconstructive head and neck microsurgery. The main advantages of this technique are very low donor site morbidity and a long pedicle, as well as the potential for harvesting multiple flaps in a chimeric design; STFF represents the first choice for treatment of small postero-lateral defects of the maxilla, and of wide and complex through-and-through defects involving all components of the midface.

Development and validation of new analytical methods using sea urchin embryo bioassay to evaluate dredged marine sediments.

Management of dredged materials disposal is regulated by several environmental normative requirements, and it is often supported by the integration of chemical data with ecotoxicological characterization. The reliability of a bioassay to assess the potential toxicity of dredged sediments requires the selection of quality criteria that should be based on simple analytical methods and easily understandable hazard for politicians and environmental managers. The sea urchin embryo-toxicity bioassay is considered an essential component for evaluating the quality of sediments in harbour areas but its use, when based exclusively on the observation of normal vs. abnormal embryos, may alter the interpretation of the results, overestimating the risk assessment. To improve the reliability of this assay in establishing a causative relationship between quality of sediments and sea urchin embryonic development, here we developed and validated three Integrative Toxicity Indexes (ITI 2.0, ITI 3.0, ITI 4.0), modifying the already-known ITI (here ITI 1.0). Based on this aim, we used Taranto harbour as a model pilot-study to compare results to those obtained from standard criteria. Among the tested indexes, the ITI 4.0, discriminating strictly developmental delay and morphological defects from fertilized egg to gastrula stage, resulted in the most promising.

Infant mandibular distraction in absence of ascending ramus: case series.

BACKGROUND: Severe microretrognathia with the absence of ascending mandibular ramus is a challenging deformity and treatment must aim to avoid tracheostomy or remove it as soon as possible. Although it is not often reported, mandibular distraction osteogenesis represents a valid treatment option in infants affected by hypoplastic mandible Pruzansky-Kaban type IIb and III. CASE PRESENTATION: The authors describe 3 cases of infants affected by severe respiratory insufficiency due to congenital mandibular hypoplasia, with follow up ranging from 4 to 8 years. Clinical and technical considerations on treatment choices and outcomes are discussed starting from review of the literature and direct clinical experience. CONCLUSION: Early mandibular distraction, specifically bidirectional distraction, is an effective and repeatable technique that leads to mandible lengthening with counterclockwise rotation, pogonion projection increase, anteropositioning of the tongue base, and expansion of oropharyngeal volume with positive effect on the respiratory problems of the infant. Even in Treacher Collins patients, known to have a low decannulation rate, all of these elements are essential for effective speech and swallowing therapy and for a subsequent attempt of decannulation.

Long-Lasting Activity of ERK Kinase Depends on NFATc1 Induction and Is Involved in Cell Migration-Fusion in Murine Macrophages RAW264.7.

Macrophages are mononuclear cells that become osteoclasts (OCs) in the presence of two cytokines, macrophage colony-stimulating factor (M-CSF), and receptor activator of NF-κB ligand (RANKL). RANKL binding to its specific receptor RANK leads to OCs differentiation mainly by nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). In our previous study, the analysis of the protein network in NFATc1-knockdown cells, using the Ingenuity Pathway Analysis (IPA), showed a link between NFATc1 and Mitogen-activated protein kinase kinase (MEK)-extracellular receptor kinase (ERK) signaling pathway. Therefore, this study aimed to extend our knowledge of the relationship between NFATc1 and the ERK. Here, we demonstrate that delayed ERK1/2 phosphorylation in pre-OC RANKL-induced depends on NFATc1. Indeed, the knockdown of NFATc1 reduced the phosphorylation of ERK1/2 (60%) and the pharmacological inhibition of the ERK1/2 kinase activity impairs the expression of NFATc1 without preventing its translocation into the nucleus. Furthermore, silencing of NFATc1 significantly reduced RANKL-induced migration (p < 0.01), and most pre-OCs are still mononuclear after 48 h (80 ± 5%), despite the presence of actin rings. On the other hand, the inhibitors FR180204 and PD98059 significantly reduced RANKL-induced cell migration (p < 0.01), leading to a reduction in the number of multinucleated cells. Finally, we suggest that long-lasting ERK activity depends on NFATc1 induction and is likely linked to cell migration, fusion, and OC differentiation.