How future surgery will benefit from SARS-COV-2-related measures: a SPIGC survey conveying the perspective of Italian surgeons

COVID-19 negatively affected surgical activity, but the potential benefits resulting from adopted measures remain unclear. The aim of this study was to evaluate the change in surgical activity and potential benefit from COVID-19 measures in perspective of Italian surgeons on behalf of SPIGC. A nationwide online survey on surgical practice before, during, and after COVID-19 pandemic was conducted in March-April 2022 (NCT:05323851). Effects of COVID-19 hospital-related measures on surgical patients' management and personal professional development across surgical specialties were explored. Data on demographics, pre-operative/peri-operative/post-operative management, and professional development were collected. Outcomes were matched with the corresponding volume. Four hundred and seventy-three respondents were included in final analysis across 14 surgical specialties. Since SARS-CoV-2 pandemic, application of telematic consultations (4.1% vs. 21.6%; p < 0.0001) and diagnostic evaluations (16.4% vs. 42.2%; p < 0.0001) increased. Elective surgical activities significantly reduced and surgeons opted more frequently for conservative management with a possible indication for elective (26.3% vs. 35.7%; p < 0.0001) or urgent (20.4% vs. 38.5%; p < 0.0001) surgery. All new COVID-related measures are perceived to be maintained in the future. Surgeons' personal education online increased from 12.6% (pre-COVID) to 86.6% (post-COVID; p < 0.0001). Online educational activities are considered a beneficial effect from COVID pandemic (56.4%). COVID-19 had a great impact on surgical specialties, with significant reduction of operation volume. However, some forced changes turned out to be benefits. Isolation measures pushed the use of telemedicine and telemetric devices for outpatient practice and favored communication for educational purposes and surgeon-patient/family communication. From the Italian surgeons' perspective, COVID-related measures will continue to influence future surgical clinical practice

Development of a novel genetic marker for nematode transgenesis

La construction d’animaux transgéniques est une technique clef qui a permis l’étude de nombreux aspects de la biologie du nématode Caenorhabditis elegans. Les animaux transgéniques peuvent être construits soit en injectant l’ADN exogène dans les gonades syncitiales de l’hermaphrodite adulte, soit en bombardant une population de vers avec des microbilles enrobées d’ADN. Dans les deux cas, l’utilisation de marqueurs génétiques est indispensable pour l’identification des individus transgéniques et la maintenance des lignées. Nous avons développé un vecteur d’expression pour les nématodes contenant le gène de résistance à la néomycine (neo), qui fonctionne comme marqueur génétique. Le gène neo confère la résistance au G-418, un antibiotique qui inhibe la synthèse de protéines chez les eucaryotes et qui est létal pour les nématodes sauvages. Nous avons montré que le marqueur neo est un marqueur génétique très puissant qui permet l’identification rapide des animaux transgéniques et qui permet l’enrichissement des populations transgéniques en présence de l’antibiotique, facilitant ainsi la maintenance des lignées. Ce système ne nécessite aucun contexte génétique particulier pour fonctionner et est donc compatible avec des lignées receveuses mutantes, ainsi que des lignées transgéniques ayant été transformées avec d’autres marqueurs génétiques. De plus, le gène neo est sous le contrôle du promoteur du gène de C. elegans rps-27, codant pour une protéine ribosomale dont la séquence est hautement conservée entre les nématodes. Nous avons utilisé ce gène comme marqueur génétique pour la transgénèse de l’espèce Caenorhabditis briggsae, ce qui suggère que le système neo pourrait aussi être utilisé pour d’autres espèces de la famille Caenorhabditis. Finalement, nous avons aussi montré que le système neo peut être utilisé dans le contexte des techniques d’ingénierie génétique basées sur le transposon Mos1. En conclusion, la sélection en présence de G-418 offre des nouvelles possibilités d’expériences pour la transgénèse de C. elegans et d’autres espèces proches. Les avantages du système neo devraient ainsi contribuer à développer des techniques de transgénèse du ver plus flexibles et efficaces.The generation of transgenic animals has been instrumental to study many biological aspects of Caenorhabditis elegans biology. Transgenic animals can be obtained by either microinjection of the exogenous DNA into the syncitial gonad of the hermaphrodite or by bombardment of a population of worms with DNA coated microparticles. Both techniques rely on the use of genetic markers to facilitate the recovery of transformed animals and the maintenance of transgenic lines. We developed a nematode expression vector carrying the neomycin resistance gene (neo) as a selection marker. This gene confers resistance to G-418, an antibiotic that normally inhibits protein synthesis in eukaryotes and is lethal for wild-type nematodes. We showed that the neo marker is a potent tool that allows a clear-cut selection of transgenic animals and hands-off maintenance of non-integrated populations on G-418 plates. This system does not imply any prerequisite on the original genotype of the recipient strain and can therefore be used on mutants lines as well as transgenic strains obtained with common markers. Moreover, we placed the neo gene under the control of the C. elegans rps-27 promoter, a highly conserved ribosomal protein throughout the nematode phylogeny. We were able to provide resistance to Caenorhabditis briggsae using this vector; this likely indicates that neo can be used in any species from the Caenorhabditis family. Finally, we demonstrated that this powerful selection system can be used in the context of Mos1 transposon excision-repair methods. Therefore, the neo system offers a wide range of new possibilities for transgenesis both in C. elegans and in other related species. We therefore believe that the benefits of the neo system should contribute to the development of more flexible and efficient techniques for nematode transgenesis

Development of a novel genetic marker for nematode transgenesis

La construction d’animaux transgéniques est une technique clef qui a permis l’étude de nombreux aspects de la biologie du nématode Caenorhabditis elegans. Les animaux transgéniques peuvent être construits soit en injectant l’ADN exogène dans les gonades syncitiales de l’hermaphrodite adulte, soit en bombardant une population de vers avec des microbilles enrobées d’ADN. Dans les deux cas, l’utilisation de marqueurs génétiques est indispensable pour l’identification des individus transgéniques et la maintenance des lignées. Nous avons développé un vecteur d’expression pour les nématodes contenant le gène de résistance à la néomycine (neo), qui fonctionne comme marqueur génétique. Le gène neo confère la résistance au G-418, un antibiotique qui inhibe la synthèse de protéines chez les eucaryotes et qui est létal pour les nématodes sauvages. Nous avons montré que le marqueur neo est un marqueur génétique très puissant qui permet l’identification rapide des animaux transgéniques et qui permet l’enrichissement des populations transgéniques en présence de l’antibiotique, facilitant ainsi la maintenance des lignées. Ce système ne nécessite aucun contexte génétique particulier pour fonctionner et est donc compatible avec des lignées receveuses mutantes, ainsi que des lignées transgéniques ayant été transformées avec d’autres marqueurs génétiques. De plus, le gène neo est sous le contrôle du promoteur du gène de C. elegans rps-27, codant pour une protéine ribosomale dont la séquence est hautement conservée entre les nématodes. Nous avons utilisé ce gène comme marqueur génétique pour la transgénèse de l’espèce Caenorhabditis briggsae, ce qui suggère que le système neo pourrait aussi être utilisé pour d’autres espèces de la famille Caenorhabditis. Finalement, nous avons aussi montré que le système neo peut être utilisé dans le contexte des techniques d’ingénierie génétique basées sur le transposon Mos1. En conclusion, la sélection en présence de G-418 offre des nouvelles possibilités d’expériences pour la transgénèse de C. elegans et d’autres espèces proches. Les avantages du système neo devraient ainsi contribuer à développer des techniques de transgénèse du ver plus flexibles et efficaces.The generation of transgenic animals has been instrumental to study many biological aspects of Caenorhabditis elegans biology. Transgenic animals can be obtained by either microinjection of the exogenous DNA into the syncitial gonad of the hermaphrodite or by bombardment of a population of worms with DNA coated microparticles. Both techniques rely on the use of genetic markers to facilitate the recovery of transformed animals and the maintenance of transgenic lines. We developed a nematode expression vector carrying the neomycin resistance gene (neo) as a selection marker. This gene confers resistance to G-418, an antibiotic that normally inhibits protein synthesis in eukaryotes and is lethal for wild-type nematodes. We showed that the neo marker is a potent tool that allows a clear-cut selection of transgenic animals and hands-off maintenance of non-integrated populations on G-418 plates. This system does not imply any prerequisite on the original genotype of the recipient strain and can therefore be used on mutants lines as well as transgenic strains obtained with common markers. Moreover, we placed the neo gene under the control of the C. elegans rps-27 promoter, a highly conserved ribosomal protein throughout the nematode phylogeny. We were able to provide resistance to Caenorhabditis briggsae using this vector; this likely indicates that neo can be used in any species from the Caenorhabditis family. Finally, we demonstrated that this powerful selection system can be used in the context of Mos1 transposon excision-repair methods. Therefore, the neo system offers a wide range of new possibilities for transgenesis both in C. elegans and in other related species. We therefore believe that the benefits of the neo system should contribute to the development of more flexible and efficient techniques for nematode transgenesis

Selectable genetic markers for nematode transgenesis

The nematode Caenorhabditis elegans has been used to study genetics and development since the mid-1970s. Over the years, the arsenal of techniques employed in this field has grown steadily in parallel with the number of researchers using this model. Since the introduction of C. elegans transgenesis, nearly 20 years ago, this system has been extensively used in areas such as rescue experiments, gene expression studies, and protein localization. The completion of the C. elegans genome sequence paved the way for genome-wide studies requiring higher throughput and improved scalability than provided by traditional genetic markers. The development of antibiotic selection systems for nematode transgenesis addresses these requirements and opens the possibility to apply transgenesis to investigate biological functions in other nematode species for which no genetic markers had been developed to date

Axonal fusion: an alternative and efficient mechanism of nerve repair

Injuries to the nervous system can cause lifelong morbidity due to the disconnect that occurs between nerve cells and their cellular targets. Re-establishing these lost connections is the ultimate goal of endogenous regenerative mechanisms, as well as those induced by exogenous manipulations in a laboratory or clinical setting. Reconnection between severed neuronal fibers occurs spontaneously in some invertebrate species and can be induced in mammalian systems. This process, known as axonal fusion, represents a highly efficient means of repair after injury. Recent progress has greatly enhanced our understanding of the molecular control of axonal fusion, demonstrating that the machinery required for the engulfment of apoptotic cells is repurposed to mediate the reconnection between severed axon fragments, which are subsequently merged by fusogen proteins. Here, we review our current understanding of naturally occurring axonal fusion events, as well as those being ectopically produced with the aim of achieving better clinical outcomes

Applying antibiotic selection markers for nematode genetics

Antibiotic selection markers have been recently developed in the multicellular model organism Caenorhabditis elegans and other related nematode species, opening great opportunities in the field of nematode transgenesis. Here we describe how these antibiotic selection systems can be easily combined with many well-established genetic approaches to study gene function, improving time- and cost-effectiveness of the nematode genetic toolbox

Disruption of RAB-5 increases EFF-1 fusogen availability at the cell surface and promotes the regenerative axonal fusion capacity of the neuron

Following a transection injury to the axon, neurons from a number of species have the ability to undergo spontaneous repair via fusion of the two separated axonal fragments. In the nematode , this highly efficient regenerative axonal fusion is mediated by epithelial fusion failure-1 (EFF-1), a fusogenic protein that functions at the membrane to merge the two axonal fragments. Identifying modulators of axonal fusion and EFF-1 is an important step toward a better understanding of this repair process. Here, we present evidence that the small GTPase RAB-5 acts to inhibit axonal fusion, a function achieved via endocytosis of EFF-1 within the injured neuron. Therefore, we find that perturbing RAB-5 activity is sufficient to restore axonal fusion in mutant animals with decreased axonal fusion capacity. This is accompanied by enhanced membranous localization of EFF-1 and the production of extracellular EFF-1-containing vesicles. These findings identify RAB-5 as a novel regulator of axonal fusion in hermaphrodites and the first regulator of EFF-1 in neurons. Peripheral and central nerve injuries cause life-long disabilities due to the fact that repair rarely leads to reinnervation of the target tissue. In the nematode , axonal regeneration can proceed through axonal fusion, whereby a regrowing axon reconnects and fuses with its own separated distal fragment, restoring the original axonal tract. We have characterized axonal fusion and established that the fusogen epithelial fusion failure-1 (EFF-1) is a key element for fusing the two separated axonal fragments back together. Here, we show that the small GTPase RAB-5 is a key cell-intrinsic regulator of the fusogen EFF-1 and can in turn regulate axonal fusion. Our findings expand the possibility for this process to be controlled and exploited to facilitate axonal repair in medical applications

An antibiotic selection marker for nematode transgenesis

We have developed a nematode transformation vector carrying the bacterial neomycin resistance gene (NeoR) and shown that it could confer resistance to G-418 on both wild-type Caenorhabditis elegans and C. briggsae. This selection system allows hands-off maintenance and enrichment of transgenic worms carrying non-integrated transgenes on selective plates. We also show that this marker can be used for Mos1-mediated single-copy insertion in wild-type genetic backgrounds (MosSCI-biotic)

EFF-1-mediated regenerative axonal fusion requires components of the apoptotic pathway

Functional regeneration after nervous system injury requires transected axons to reconnect with their original target tissue. Axonal fusion, a spontaneous regenerative mechanism identified in several species, provides an efficient means of achieving target reconnection as a regrowing axon is able to contact and fuse with its own separated axon fragment, thereby re-establishing the original axonal tract. Here we report a molecular characterization of this process in Caenorhabditis elegans, revealing dynamic changes in the subcellular localization of the EFF-1 fusogen after axotomy, and establishing phosphatidylserine (PS) and the PS receptor (PSR-1) as critical components for axonal fusion. PSR-1 functions cell-autonomously in the regrowing neuron and, instead of acting in its canonical signalling pathway, acts in a parallel phagocytic pathway that includes the transthyretin protein TTR-52, as well as CED-7, NRF-5 and CED-6 (refs 9, 10, 11, 12). We show that TTR-52 binds to PS exposed on the injured axon, and can restore fusion several hours after injury. We propose that PS functions as a 'save-me' signal for the distal fragment, allowing conserved apoptotic cell clearance molecules to function in re-establishing axonal integrity during regeneration of the nervous system