Reproductive genetic carrier screening provides individuals and couples with information regarding their risk of having a child affected by an autosomal recessive or X-linked recessive genetic condition. This information allows them the opportunity to make reproductive decisions in line with their own beliefs and values. Traditionally, carrier screening has been accessed by family members of affected individuals. In recent years, improvements to accessibility and updates to recommendations suggest that all women planning or in early pregnancy should be offered reproductive genetic carrier screening. As uptake moves towards the population scale, how can the genetic counselling needs of such large-scale screening be met? A scoping review of the literature was performed to ascertain what the genetic counselling needs of reproductive genetic carrier screening are, and what future research is needed. Four broad themes were identified in the existing literature: (1) The offer—when and in what context to offer screening; (2) Information—the importance of and what to include in education, and pre- and post-test counselling; (3) Who and how—who the genetic counselling is performed by and how; (4) Personalization—how do we find the balance between standardized and individualized approaches? Based on the existing literature, we present a set of recommendations for consideration in implementing population-scale reproductive genetic carrier screening as well as suggested areas for future research. Research suggests that 300 million people worldwide are affected by rare diseases . GeneticCarrier testing has been used for many years by family members of individuals affected by a recessive genetic condition such as cystic fibrosis (CF), spinal muscular atrophy (SMA), or fragile X syndrome (FRAX) to determine their carrier status ,4,5. In Population-based carrier screening was first performed for genetic conditions with high carrier frequencies, particularly in ethnic populations, such as sickle cell anaemia or thalassaemia in Mediterranean populations, or Tay Sachs disease in the Ashkenazi Jewish population . Single It has been possible in Australia to access a basic reproductive genetic carrier screening (RGCS) panel inclusive of CF, SMA and FRAX since 2012 on a user-pays basis [More recently, the advances in genomic sequencing technology and its increasing affordability have made it possible to offer the large panel screening of 100 s to 1000 s of genes at once ,11. ThesOver recent years, professional bodies in a number of countries have released recommendations advising that all women should be offered genetic carrier screening when planning a pregnancy or in early pregnancy ,16,17,18In 2021 the American College of Medical Genetics (ACMG)  releasedThe increasing push to offer RGCS programs at the population scale raises questions relating to how the substantial genetic counselling requirements for such programs can be adequately met. What are the genetic counselling requirements specifically? Who should be involved in providing genetic counselling for RGCS programs and at which stages in the process? Are there alternative methods for performing genetic counselling? Where should the specialist skills and experience of genetic counsellors be focussed within the scope of RGCS service delivery at scale? To answer these questions the genetic counselling requirements of genetic carrier screening must be explored and understood. This scoping review was conducted based on the Arksey and O’Malley  frameworThis review was guided by the question, ‘What are the genetic counselling requirements for RGCS?’ where ‘RGCS’ refers to expanded gene panels and whole exome sequencing with targeted computational analysis  for autoA search was conducted in three electronic databases; MEDLINE/PubMed, EMBASE and CINAHL/EBSCO. The databases were selected to be comprehensive and to cover the relevant disciplines. No limits on date, language, or type were placed on the database search. The search query consisted of terms relating to three key areas: genetic counselling, carrier screening and reproductive health/behaviour. The search query tailored these three subject areas to the specific requirements of each database . UnpubliA two-stage screening process was used to assess the relevance of studies identified in the search. All citations were imported into the web-based bibliographic manager Mendeley Reference Manager for the subsequent title and abstract relevance screening. The first level of screening involved a review of only the title and abstract of citations to identify those studies that met the minimum inclusion criteria. Exclusion criteria included duplicate articles, unpublished articles or grey literature, ineligible article type , references to screening unrelated to RGCS , etc.), or where there were no genetic counselling outcomes. The second level of screening involved a full review of the article for exclusion or inclusion in the data characterisation phase. All citations deemed relevant after the title and abstract screening were obtained for subsequent review of the full-text article. Study characteristics such as publication year, research characteristics, participant type, and carrier screening type were extracted into a single spreadsheet for descriptive statistical analyses. The researcher adopted a narrative synthesis approach due to methodological heterogeneity across the reviewed studies. This allowed for the organisation, description, exploration and interpretation of the study findings relating to genetic counselling outcomes , each ofhttps://doi.org/10.17605/OSF.IO/MR8ZK, accessed on 21 June 2022) along with the protocol . Specific ethics approval for this scoping review was not required.The database search, article review, data characterisation and evidence synthesis were conducted by a single researcher. The scoping review was registered on Open Science Framework  adequate and consistent genetic counselling is provided, (2) the burden on genetic counselling resources is minimized, and (3) genetic counsellor skills are utilized for maximum effect:This scoping review was conducted by a single researcher and may thus be limited by a subjective interpretation of the data. The nature of a scoping review means that the available literature was not screened to exhaustion as a systematic review would have been. Thus, it is possible that potentially valuable studies may have been omitted by chance. Finally, the reviewed studies were conducted within varying politico-legal and cultural contexts, the specifics of which were not stated and thus assumptions may have influenced the formation of the recommendations. Implementing generalised recommendations into each specific regional context may be challenging but would offer a starting point on a potentially global journey to offering RGCS at scale.The review of the literature identified a distinct dearth of insight into pathology provider and policymaker views on genetic counselling requirements for RGCS. Pathology providers represent an important stakeholder group given that they perform the screening, variant curation and reporting of results. How does genetic counselling impact their processes? What are the roles of laboratory-based genetic counsellors? Similarly, standardized practices might be mandated by government regulatory bodies as they have been in Germany with the German Genetic Diagnostics Act , yet theWhilst it is recognized that RGCS offers based on positive family history result in the under-identification of carrier individuals and at-risk couples , family Further research is also needed into standardising and streamlining RGCS education for both the public and healthcare professionals. Evidence would suggest that healthcare professionals access RGCS education from a number of different sources, including local and regional conferences, commercial provider representatives, journal articles and colleagues. The provision of standardized RGCS education for the public would likely reduce the time burden on genetic counselling resources, as individuals would enter the pre-test counselling process with a greater knowledge base . SimilarThis scoping review of the literature identified the need for consistent standardized information during pre- and post-test counselling for RGCS and also highlighted the importance of personalized genetic counselling when necessary. Finding the right balance between standardisation and personalisation will be extremely important when implementing RGCS programs at scale. Further research into how this balance might be achieved is needed. One possibility may be integrating genetic counsellor support for both healthcare professionals and individuals/couples throughout the RGCS process, enabling the genetic counsellors to provide personalisation within a standardized system. Another area for investigation is interactive pre- and post-test counselling methods that provide an alternative to, and minimize the need for, traditional genetic counselling, e.g., decision aids, chatbots, online education, etc.The genetic counselling requirements for RGCS are not novel but are significantly more complex when implemented at scale. The genetic counselling requirements of smaller RGCS programs are evident and can provide insight. Yet the specific complexities of RGCS at scale appear under-researched and should be explored further in future studies. With the push for RGCS to be offered at scale and the potential conflict between commercial and patient agendas, it is imperative for a consensus of recommendations for the implementation of RGCS programs at scale from the wider genetics community in each country. The researchers put forward the recommendations set out in this scoping review as a starting point for developing consensus.


Neuromodulatory systems are generally known to play roles in complex behaviors such as the sleep/wake cycle, attention, arousal and learning  in unprecedented detail using viral vectors, tract tracing, and immunohistochemistry. Together they describe major output projections of cholinergic neurons in the pontomesencephalic tegmentum (PMT), which is not just a primary source of cholinergic output in the brain, but may serve as a hub for multiple neuromodulators.This topic's contents include what are likely to be enduring discoveries providing new insights into the anatomy of cholinergic circuitry influencing auditory nuclei. Instkerveli and Metherate demonstrated how activation of nicotinic signaling increases response gain and shortens response latency across populations of auditory cortex neurons. These changes improved response reliability across trials and countered adaptation to repeated stimuli which may be important for processing ongoing signals including speech. Similarly, Rivera-Perez et al. revealed the cellular mechanisms for nicotinic gain control in a genetically defined population of neurons in the IC. Specifically, they showed that α3β4 nicotinic receptors are primarily responsible for mediating a depolarizing inward current that both boosts membrane excitability and enhances summation of excitatory inputs. Together these papers show that modulation by acetylcholine can rapidly and consequentially enhance responses to sound in two major auditory centers.Several works appearing in this volume illustrate the power of investigating neuromodulatory physiology in the auditory system, where modulation of stimulus driven activity in real-time can be interpreted in its functional context. Polese et al. presented anesthetized mice with broadband vocalizations while recording IC neuron responses in the presence or absence of serotonin agonists. They showed that serotonin released in the context of mating has the potential to sharpen neural selectivity for specific vocalizations and call features.Auditory modulation is not limited to cholinergic input however, and several studies collectively demonstrated the wide variety of factors that are brought to bear on auditory computation. Serotonin is a neurotransmitter well known to be released in specific behavioral contexts such as mating. Goel et al. illustrate the many ways that mGluRs control both presynaptic release at excitatory synapses and simultaneously modulate the excitability of the postsynaptic cells in the sound localization circuitry of both birds and mammals. Kopp-Scheinpflug and Forsythe review how NO, synthesized in response to Ca++ entry during synaptic activity, can mediate a myriad of effects in both postsynaptic and presynaptic neurons. Importantly, NO as a soluble messenger has the potential to act on neurons away from its site of production and independently of synaptic connections, including non-auditory or multisensory neurons.Two review contributions highlight bodies of work that reveal complex and varied influences of metabotropic glutamate receptors (mGluRs) and nitric oxide (NO) signaling. Wollet and Kim. Activity-dependent BDNF release is known to influence synaptic plasticity during long term potentiation Xu et al.  mutant mice failed to develop normal frequency-dependent patterning of intrinsic neural properties. A second compelling example is presented by Pagella et al., who showed that Urocortin 3, a neuropeptide transmitter released during stress, and its receptor CRFR2, are expressed broadly in the auditory pathway by principal and interneurons, respectively, suggesting a reverse modulation from principal neurons to the canonical modulatory neurons. Urocortin 3 knockout animals have been shown to be particularly sensitive to noise damage suggesting that the auditory pathways express modulators that confer an auto-protective function (Fischl et al., Knipper et al. propose a model by which trauma induced hyperexcitability along the auditory pathway influences BDNF signaling to disrupt the balance of excitation and inhibition. They go on to propose that downstream changes to NO signaling may disrupt the neural-vascular interface which may cause deficits beyond the auditory system.Modulation may not necessarily derive from discrete clusters of functionally similar neurons releasing a canonical transmitter, nor is modulatory function temporally constrained to influence processing in time scales limited to stimulus duration or behavioral state. One example of modulatory release from principal auditory neurons themselves is provided by u et al. . Here, WThese studies open new lines of inquiry beyond investigations of excitation and inhibition in principal auditory neurons to include the additional layers of complexity provided by modulation from the ear to cortex and back again. Further, it is evident that modulatory circuitry shapes neural responses on time scales ranging from long-lasting developmental processes to immediate effects in the mature organisms. We hope the work presented here will stimulate further exploration into what promises to be a rich and emerging field of auditory neuroscience.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


RNA viruses have high mutation rates, with the majority of mutations being deleterious. We examine patterns of deleterious mutation accumulation over multiple rounds of viral replication, with a focus on how cellular coinfection and heterogeneity in viral output affect these patterns. Specifically, using agent-based intercellular simulations we find, in agreement with previous studies, that coinfection of cells by viruses relaxes the strength of purifying selection and thereby increases the rate of deleterious mutation accumulation. We further find that cellular heterogeneity in viral output exacerbates the rate of deleterious mutation accumulation, regardless of whether this heterogeneity in viral output is stochastic or is due to variation in the cellular multiplicity of infection. These results highlight the need to consider the unique life histories of viruses and their population structure to better understand observed patterns of viral evolution. RNA viruses have high mutation rates and undergo frequent population bottlenecks, making them particularly prone to the accumulation of deleterious mutations. As such, these populations can experience deleterious mutation loads, which is the burden on fitness that recurrent and persistent mutations have on populations . Indeed,Several processes reduce the accumulation of deleterious mutations in RNA viruses. One such mechanism is through the evolution of higher fidelity polymerase proteins, thus reducing deleterious mutation rates  also redThe effect of cellular MOI on the rate of deleterious mutation accumulation is particularly interesting to consider given its uniqueness to viral populations and that cellular coinfection is, in effect, a double-edged sword: providing an opportunity to purge deleterious mutations via viral sex, while relaxing selection on deleterious mutations by increasing the extent of phenotypic hiding. However, when phenotypic hiding dominates, the benefits of coinfection are greatly reduced for viruses that cannot recombine or reassort. Here, we develop a model to examine the effects of cellular coinfection on deleterious mutation accumulation in viral populations in the context of these opposing effects. We first show that the simplest version of the model recapitulates previous findings in the literature ominance . We makeV progeny virions for the next generation from across the set of infected cells. Each progeny virion is drawn independently, with the probability that the virion comes from cell c proportional to c, each of its y gene segments is drawn randomly from the parental virions that infected the cell. As such, a high-fitness gene segment is as likely to be drawn from a cell as a low-fitness gene segment, reflecting our assumption that cellular fitness depends on the aggregate of shared viral proteins that have been produced in a cell. Once all parental gene segments have been chosen, the mutations are added as described above. We repeat this full process for t discrete generations. We next describe several extensions of this base model that allow us to examine the effects of cellular heterogeneity in viral output that stem from 2 distinct sources: differences in intrinsic cellular characteristics (see Heterogeneous Cellular Output Stemming from Differences in Cellular Characteristics) and differences in cellular multiplicities of infection (see Heterogeneity in Cellular Output Stemming from Differences in Cellular Multiplicity of Infection). In Alternative Fitness Functions, we describe alternative cellular fitness functions that we consider a sensitivity analysis to the incomplete dominance fitness function described above.At the end of each generation, we draw the cellular output c, the cellular fitness c is no longer proportional to cW and is instead proportional to k. The gamma distribution’s probability density function is given by:Viral output from cells can be affected by host cell characteristics such as size, cell type, and cell cycle stage , so there is no reassortment. One key finding from the field of population genetics is that reducing population size increases the rate of deleterious mutation accumulation due to an increased rate of genetic drift, particularly in asexual populations . At later times, reassortment results in a slower “clicking” of the ratchet , it can substantially slow Muller’s ratchet , consistHeterogeneous Cellular Output Stemming from Differences in Cellular Characteristics, we implement the effect of heterogeneity driven by host cell characteristics by combining individual cell heterogeneity with virus-driven differences in cellular fitness using draws from a gamma distribution, parameterized with dispersion parameter k. As expected, simulations with Phenotypic Hiding Relaxes Selection that do not incorporate stochastic heterogeneity  and variance eV, because the Poisson approximation to the binomial offspring distribution allows 1 individual to have more offspring than there are total virions in the population. Even when the number of virions is large, our formula also overestimates the variance if MOI is large, because the noise in cellular output is shared among coinfecting virions. However, this effect only becomes appreciable when the number of cells is very small.The effect of stochastic cellular heterogeneity on mutation accumulation can be better understood by quantifying the effective viral population size, ribution . We can eV is indeed the relevant predictor for evolution  was also loosely based on influenza virus  exhibit within-host spatial structure (reviewed in The second key feature of natural infections that we do not incorporate into our models but that likely impacts viral evolutionary dynamics is the dramatic change in viral population sizes over the course of a natural acute infection. While our model assumes a constant viral population size, viral populations in natural infections often expand from a small founding population, reach population sizes in the millions, and then decline again until the host immune response clears the viral infection. Population expansion is known to increase the number of segregating deleterious mutations in a population but also decreases the per-individual number of deleterious mutations , if a viBeyond our models’ simplifying assumptions of a constant viral population size over generations and free mixing of virions, our models also assume that the fitness effects of deleterious mutations are independent of one another. One possible genetic extension of our model would be to include epistasis among mutations. Positive epistasis would result in additional mutations accumulating because the fitness effect of adding a new mutation decreases with each subsequent mutation. Negative epistasis would have the opposite effect: selection would be more strict and thus fewer mutations would accumulate. However, neither form of epistasis should have much effect on mutation accumulation at high MOI where phenotypic hiding renders mutations effectively neutral.Phenotypic hiding can be seen as an example of social interactions between viruses at the intracellular level. The emerging field of “sociovirology” examines how such interactions between viruses, including during cellular coinfection, can have an impact on the evolution of viral populations . The impiyac127_Supplementary_DataClick here for additional data file.


Dear Editor,Road traffic accidents have emerged as a pandemic of the modern world. Chest trauma, being commonly associated with these road traffic accidents, becomes the leading cause of morbidity and mortality. Management of pain is essential in these trauma patients as, along with patient comfort, it also decreases patient’s respiratory complications.Mid-point transverse process to pleura (MTP) block is a recently described ultrasound-guided novel technique that involves injecting the drug at mid‐point between the transverse process and the pleura.Management of acute pain is highly advantageous for enhanced recovery after trauma to prevent the neuroendocrine stress response and thus combat the cascade of events occurring after activation of the sympathetic nervous system and catecholamine release. Therefore, we believe that MTP block can be a game-changer for the chest trauma patients due to an easy approach and reduced risk profile.The authors declare no conflicts of interest.


Behavioural reactivity to potential threat is used to experimentally refine models of anxiety symptoms in rodents. We present a short review of the literature tying the most commonly used tasks to model anxiety symptoms to functional recruitment of bed nucleus of the stria terminalis circuits (BNST). Using a review of studies that investigated the role of the BNST in anxiety-like behaviour in rodents, we flag the certain challenges for the field. These stem from inconsistent methods of reporting the neuroanatomical BNST subregions and the interpretations of specific behaviour across a wide variety of tasks as ‘anxiety-like’. Finally, to assist in interpretation of the findings, we discuss the potential interactions between typically used ‘anxiety’ tasks of innate behaviour that are potentially modulated by the social and individual experience of the animal. Some concepts of emotional experience used in the neuroscientific literature are both intuitive but nonetheless difficult to experimentally define, including stress, fear and notably anxiety. What are diagnosed as anxiety disorders have fluctuated over time, remarkably in light of what neurobiological experimental evidence has demonstrated to be associated with particular symptoms that characterise specific spectra of disorders of anxiety, such as trauma, stressor-related or obsessive-compulsive disorders . AttemptStudies indicated that the Bed Nuclei of the Stria Terminalis (BNST) may be preferentially recruited over the amygdala nuclei for anxiety-evoking rather than fear-evoking stimuli . Both asIn laboratory rodents, the BNST is posited to have a role in innate responses to a stressor, such as brightly lit environment or predator odour  and in cA significant challenge remains the limited resolution of the subnuclei of the BNST in human functional magnetic resonance imaging . The BNSTo illustrate the array of terms that were used to describe the anatomical subdivisions of the BNST and the variety of tasks used to investigate anxiety mechanisms, we performed a systematic literature review of recent rodent studies. Searches were conducted using PubMed, the Cochrane Library and pre-print archives  using the Boolean operators:(((BNST) OR ) AND ((anxiety) OR (‘sustained fear’)) AND ((‘rodent’) OR (‘mice’) OR (‘rat’))”“This generated 584 unique literature records (published from 1997–2021). Pre-prints were included as the purpose was to sample what terms and tasks are being actively used in recent studies of the BNST. Two authors screened the papers with exclusion criteria that articles should be open access, in English, and have investigated a functional role of the BNST in tasks proposed to model anxiety-like behaviour see . A totalThe validation of a task for translational potential is complex, including for pharmacological predictive validity for complex neuropsychiatric disorders like anxiety . AnotherFor over a century, scientists have used mazes to study rodents’ behaviours. Mazes provide key insights into both innate and learned behaviours, as they show how a rodent instinctively navigates through a space, and additionally aids in an understanding of the mechanisms behind learning and memory. One of the first rodent mazes detailed by Willard Small in 1901 was rooted in a desire to understand Darwinian-inspired innate behaviours, and ‘to make observations upon the free expression of the animal's mental processes' .The Elevated Plus Maze (EPM), pioneered by Pellow and colleagues , has beeAnother common and ethologically relevant test is the Open Field Maze (OFM), pioneered by Hall . It is pWhile mazes offer some advantages over operant behavioural testing as the rodents perform more natural behaviours, there exist numerous limitations and challenges ,45. A poAlthough mazes provide one aspect of studying ethological measures, in order to fully understand the development of anxiety-like behaviour it is useful to study acquired aversions . In so-cThese type of classical conditioning protocols are used to typically measure conditioned freezing . This haIn rodents, a variety of aversive tasks, mazes or associative training, can evoke overlapping behavioural responses including avoidance, elevated heart rate, freezing, hypoactivity, suppressed food consumption, and increased vigilance . In addiUSVs may not be simply reflexive alarm calls, as rats seem to use them for communication as well, with 22 kHz calls typically being admitted in aversive situations and 55 kHz calls being admitted during appetitive situations . DemaestRodents are social animals, and direct exposure to a threat is not the only way for an animal to learn and escape from danger. Thus, fear learning often occurs within a social context as an individual observes fear in other individuals. Social fear learning (SFL), otherwise known as vicarious fear learning, is a phenomenon in which an individual learns transmission of threat information through observation of a conspecific . In rodeWhen housed together, rodents naturally form inter-individual hierarchies, which are important to consider when studying behavioural responses to potential threats. As early as 1938, scientists have observed and documented the presence of different social hierarchies in male mice . Social Recent studies have underscored differences between the anxiety-like behaviour of dominant and submissive cage-mates in both mice and rats. A meta-analysis and systematic review of the relationship between social dominance status and common behavioural phenotypes in male laboratory mice found little evidence for systematic phenotypic differences between dominant and submissive male mice . NonetheIt should be noted that we have discussed literature from both mice and rats herein, and there are noted differences in their behaviour and biology that is related to anxiety . MoreoveMazes that measure exploration of novel aversive contexts continue to be widely utilised as a valid approach to assess behaviour akin to anxiety despite certain limitations . It is wFrom a recent analysis of the literature, it becomes clear that the number of reports of interventions targeting the BNST to modulate anxiety-related behaviour is growing. Given the variability in reporting of sites of manipulation or regions where activity is correlated to behaviour, there is a need for consensus on how to refine the reporting of neuroanatomical locations. Despite these technical difficulties in comparison across studies, the BNST has certainly come more to the forefront as a target region for investigation in anxiety research .Neuroanatomical evidence ties the BNST to anxiety-driven responses, but the role of distinct subregions remains unclear.Conceptions of how to experimentally define anxiety-like behaviour have evolved over time and many different tasks are used in rodents.Performance in typical tasks to measure ‘anxiety’ like behaviour may be sensitive to individual and social experience of the animal.


The computational power of the software is increased by including calculations of higher order reaction mechanisms, and the program includes defining additional organ and tissue structures for a more physiologically relevant analysis of tissue oxygenation in complex co-cultured systems, or in vivo. AngioMT may serve as a digital performance measuring tool of vascular networks in microcirculation, experimental models of vascularized tissues and organs, and in clinical applications, such as organ transplants.Measuring the capacity of microvascular networks in delivering soluble oxygen and nutrients to its organs is essential in health, disease, and surgical interventions. Here, a finite element method-based oxygen transport program, AngioMT, is designed and validated to predict spatial oxygen distribution and other physiologically relevant transport metrics within both the vascular network and the surrounding tissue. The software processes acquired images of microvascular networks and produces a digital mesh which is used to predict vessel and tissue oxygenation. The image-to-physics translation by AngioMT correlated with results from commercial software, however only AngioMT could provide predictions within the solid tissue in addition to vessel oxygenation. AngioMT predictions were sensitive and positively correlated to spatial heterogeneity and extent of vascularization of 500 different vascular networks formed with variable vasculogenic conditions. The predictions of AngioMT cross-correlate with experimentally-measured oxygen distributions  Efficient removal of reaction biproducts and toxins plays an important role in maintaining homeostasis or when they are altered in various diseases including cancer, type 1 diabetes, and physical injuries like hemorrhage4. The ability of microvascular networks to effectively penetrate regions of deep lying tissues helps the body maintain oxygen saturation and prevents instances of hypoxia5. On the other hand, various pathological conditions affect the extent of vascularization or barrier function7, and hence it becomes important to evaluate the mass transport characteristics between the vasculature-tissue regions in order to develop effective therapeutic strategies.The microvascular niche functions as the mediator of transport of soluble factors and nutrients from the cardiovascular system to deep tissuesin vivo enabling digital reconstruction of microvasculature9. Like image-based visualization methods, previous methods of studying oxygen delivery in vivo have employed complex imaging setups that use optics like FLIM, phosphorescence decay etc., however, these methods require specialized membranes or materials for real-time imaging and are seldom non-invasive10. Hence non-invasive vascular imaging coupled with computational techniques is a powerful alternative to traditional oxygen measurement methods.Imaging techniques like fluorescence, radioisotope tracking, OCT etc. have been employed in clinical research to visualize the spatial distribution of microvascular networks in vivo vasculature, these techniques do not demonstrate oxygen delivery to the tissue. Commercially available software packages like Ansys, COMSOL etc. pose serious limitation in terms of modeling multiphase transport, and they do not possess native ability to process imaging data as input geometry. In cases where these aforementioned packages are able to read 3D reconstruction of images in the form of meshed files , generating solid objects requires manual clean-up of the imported geometry which might not be possible for high throughput datasets. Hence, there is a need to develop computational tools built specifically for studying multi-phase mass transport within the microvascular niche using imaging data.Although computational tools have been developed previously to map the distribution of in vivo and it predicts tissue and vessel oxygenation in proportion to the extent of vascularization of a network. Finally, we demonstrate that AngioMT also possesses the ability to include the contributions of multicellular solid tissues with variable metabolic parameters for a more physiologically relevant prediction of oxygen transport. Therefore, this software is expected to serve as a digital non-invasive sensor of a native or model of a vascularized organ under conditions of health, disease or surgical treatments.In this study, we present AngioMT (Angio Mass Transfer), a finite element method based computational solver, for investigating vascular and surrounding tissue oxygen transport phenomena. This mass transport solver uses built-in image processing capabilities to independently detect and mesh multiple complex domains consisting of vessel networks, tissue and disconnect networks. AngioMT follows by implementing the Galerkin finite element formulation to solve the mass transport over a digital mesh. This image-to-physics software is validated against experimentally-measured oxygen in a microvascular network Z-stack images of microvascular networks formed in the organ-chip were obtained using fluorescence microscopy and were flattened along the z-axis. A total of 500 images were acquired spanning various experimental conditions including growth factor, matrix stiffness, cell density variations. The images were exported as 16-bit TIFF files respectively.16-bit grayscale images were acquired from fluorescence microscopy and were then binarized using a threshold value of 0.2 for all images. Binarization resulted in vascular networks displayed as white pixels (intensity = 1) and hydrogel regions being displayed as black pixels (intensity = 0).Im2mesh developed by Jiexian Ma et al11. This script reads greyscaled images with different grayscale values and meshes the image into distinct domains based on the number of greyscale value. Since our images had three distinct grayscale values; 1 for connected vessels, 0.5 for disconnected vessels and 0 for surrounding tissue regions (fibrin hydrogel in our case), the obtained mesh had three distinct domains.The segmented and recolored images were then meshed using an open-source Delaunay Triangulation scheme called 16, or they can be acquired by microscopy, we were inspired to start our analysis using binarized images of microvascular networks  and force matrices (Felement) were then mapped to the global stiffness  and force matrices  respectively. The final solution was calculated by performing the matrix inversion operation:Once nodal and element information was obtained from the meshing, we generated the element stiffness and force matrices in AngioMT according to the Galerking finite element formualtion of After nodal concentration values were obtained at all nodes of the region of interest, the program calculates auxiliary variables like flux vectors, area-averaged mass transport etc .As described above, we applied the image labeling algorithm to the segmented images in AngioMT, extracted vascular domains , and meshed them into distinct triangular mesh regions . Since t19. Triangular meshes are one of the most common element types used in numerical modeling schemes to discretize surfaces into elements20. To evaluate whether the mesh we generated using the Im2mesh routine in AngioMT was able to generate high quality triangular meshes, we calculated the Element Quality parameter for each element according to the following relation21:Next, we set out to test the quality of the mesh as low-quality elements may cause poor interpolation of the computed results as well as introduce numerical inaccuraciesA is the area of the triangular element and ||EdgeRMS||is the root mean square edge length 21. We calculated the element quality for the meshes generated using Delaunay Triangulation and found that the average quality scores were greater than 0.9 when a gradient limit of 0.2 was chosen for meshing . The experimentally observed oxygen distribution values suggest that blood vessels act as the sole oxygen source and the transport of oxygen is into the tissue . We fed the brightfield imaging data into our AngioMT software, which successfully generated a binarized image of the microvasculature , while bulk oxygen distribution may provide a first-order metric of tissue health, granular information, such as, vascular oxygen potential and oxygen delivered to the tissue, may expand our knowledge of how a vascular network specifically regulates the microenvironment. Therefore, we next computed several ancillary transport metrics with AngioMT that are representative of vascular network connectivity and mass transport in the context of microvascular networks, but typical commercial solvers may not provide. For example, we could easily compute total oxygen flux that represents the rate of oxygen delivery of a vascular network . CorrespWe next set out to examine the oxygen content specifically within the vascular domain of the network, and oxygen delivered specifically to the solid tissue domain, as these two measures are physiologically-relevant and may predict the health of a vascularized tissue. We first calculated “Vascular Oxygen” as the area averaged oxygen concentration  within the vessel domain and is indicative of the network’s capacity to carry oxygen . Unlike We then also computed ‘Oxygen Delivery’, which is the area averaged oxygen concentration in the tissue/hydrogel domains  in the vessel representative of the directional end-to-end transport of oxygen when only one inlet is assumed (left edge). This measurement correlates to applied experimental techniques like dextran perfusion capacity, or connectivity measurements reported in experimental literatureonnected . Althoug35, we also assessed these metrics when we arranged the vascular network samples based on variation of these parameters , Positron Emission Tomography (PET), Nuclear Magnetic Resonance (NMR) etc., can measure oxygen tension in a tissue in 3D with higher depth resolution, relative to a more classical technique that we have utilized for validation of computed oxygen profiles in AngioMT. However, it is important to note that the more recent approaches to visualize oxygen transport are mostly restricted to a local region, and literature is very sparse which shows oxygen distribution maps over large tissue regions that can be successfully used to validate the model with higher rigor. Additionally, since 2D imaging is easy and faster to perform in vivo, and 2D data is still a gold standard in a variety of clinical decision-making, we have strategically created AngioMT as a 2D image-to-physics solver first. However, with current computational hardware and available speeds, AngioMT can be extended to 3D systems as well for modeling more complex, three dimensional imaging datasets, when needed.In this study we demonstrate AngioMT, a spatially-informed computational mass transport solver to evaluate tissue and vascular oxygentaion levels in microcirculation. Currently, there are no dedicated programs/ codes for studying mass trasnport through microvascular networks that utlize the actual spatial geometry and heterogeneity of microvascular networks. Using images obtained through either 37. Therefore, for simplicity, the current version of the program does not include the effect of fluid convection through the microvsacular networks. However, convenction phenomena may be incorporated into the finite element formulation, and may strengthen the analysis of mass transport in more complex microvasculature like the tumor microenvironment, where convection may also become a determinant in oxygen distribution. Similarly, temporal analysis of mass transport process may be performed later by incorporating time stepping schemes.Microvascular transport is typically diffusion driven, as dimensionless Peclet numbers (ratio of convective to diffusive flux) range between 0.1 to upto 10 within the microvascular nicheOverall, our analysis describes the use of AngioMT as a computational tool which may enable vascular scientists and engineers to visualize nutrient or drug transport through complex, heterogenous microvascular networks. Furthermore, unlike commercailly available packages, the AngioMT procedure can be automated to analyze large amount of data. Automatation of this computational methodology is possible with minimal interventions for screening high throughput data, which can eventually be incorporated with next generation machine learning techniques. This analytical approach has potential to enable clinicians and scientists to assess whether the extent of vascularization is enough for delivering and retreiving important factors like oxygen, growth factors etc, which can ultimately affect the success of clinical procedures like pancreatic grafts etc.


Memory impairment following an acquired brain injury can negatively impact daily living and quality of life—but can be reduced by memory rehabilitation. Here, we review the literature on four approaches for memory rehabilitation and their associated strategies: (1) the restorative approach, aimed at a return to pre-morbid functioning, (2) the knowledge acquisition approach, involving training on specific information relevant to daily life, (3) the compensatory approach, targeted at improving daily functioning, and (4) the holistic approach, in which social, emotional, and behavioral deficits are addressed alongside cognitive consequences of acquired brain injury. Each memory rehabilitation approach includes specific strategies such as drill and practice (restorative), spaced retrieval (knowledge acquisition), memory aids (compensatory), or a combination of psychotherapy and cognitive strategies (holistic). Past research has demonstrated mixed support for the use of restorative strategies to improve memory function, whereas knowledge acquisition strategies show promising results on trained tasks but little generalization to untrained tasks and activities of daily living. Compensatory strategies remain widely used but require intensive training to be effectively employed. Finally, the holistic approach is becoming more widespread due to improvements in psychosocial wellbeing, yet there are considerable resource and cost requirements. Several factors can influence rehabilitation outcomes including metacognition and emotional disturbances. Considerations for future research to improve the applicability of strategies for memory rehabilitation include assessing memory impairment severity, examining memory needs in daily life, and exploring the long-term effects of memory rehabilitation. An acquired brain injury (ABI) involves an insult to the brain resulting in neuronal activity changes and can be derived from traumatic brain injuries (TBI) such as accidents or falls, or from non-traumatic internal events such as stroke  is to improve memory to a level similar to that of pre-morbid functioning  without the expected improvement in general memory functioning , control participants produced significantly more words without requiring letter cues than did the memory-impaired patients  compared the restorative and compensatory approaches with a self-help approach. Individuals who reported memory problems due to ABI engaged in training sessions with a researcher and completed homework exercises. The restorative and compensatory experimental groups learned about internal memory aids and errorless learning techniques. The compensation group was additionally taught how to employ external memory aids. The restoration group was taught how to encode information using “Who,” “What,” “Why,” “When,” “Where,” and “How” questions, attention retraining exercises, and letter and number cancellation tasks. The self-help group was taught relaxation and coping techniques but no memory strategies. The restoration and compensation groups had significantly higher scores on the Internal Memory Aids Questionnaire at five and seven-month follow-up points. However, there were no significant differences between any groups on the primary outcome measure, the Everyday Memory Questionnaire. In general, the restoration and compensation groups showed similar outcomes. The authors reported that the restoration group also developed the use of some compensatory strategies on their own, but without a baseline test of the External Memory Aids questionnaire, this can only be considered an observation by the researchers. Based on the few significant outcomes, limited sample size, and heterogeneity of the sample, the authors concluded that concrete assertions are not warranted regarding the effectiveness of the memory rehabilitation approaches in this study (das Nair and Lincoln 2012).Withiel et al.  comparedcombining different approaches or strategies within an approach. CACR has been found to have some positive effects on cognitive task outcomes when combined with traditional cognitive therapy (Ressner et al. Rather than comparing approaches, some studies have examined the outcomes of There is little conclusive evidence indicating whether one type of rehabilitation approach is more effective for memory rehabilitation because the appropriate approach may vary based on a multitude of patient characteristics and availability of resources (Barman et al. Systematic reviews of cognitive rehabilitation over the past two decades have consistently recommended a combined use of internal and external compensatory strategies as memory training for patients with mild to moderate memory impairments (Cicerone et al. In addition to cognitive impairments following ABI, deficits in metacognition and emotional disturbances including depression and anxiety can be present and impact daily functioning and community participation such as returning to work (Afsar et al. Higher levels of metacognitive skills such as self-awareness are correlated with better task and functional outcomes, whereas impaired self-awareness is linked to poorer functional outcomes (Al Banna et al. Emotional disturbances such as anxiety and depression can be prevalent and persist following ABI (Gould et al. Alternatively, rehabilitation may positively influence emotional adjustment to the impact of memory impairment (Chouliara and Lincoln There are several general limitations to current memory rehabilitation approaches. Early restorative strategies such as drill and practice are relatively ineffective, whereas CACR has limited generalization effects at the current stage (López and Antolí There is a wide variety in the structure of memory rehabilitation programs. There is no consensus for the treatment method, frequency, or duration of treatment with some ranging from two weeks to several months and each session lasting between 30 min and two hours (das Nair et al. 2016; Dou et al. Across some studies, the differences between the three approaches have been confounded. Schacter and Glisky  stated tThe overlaps in function and strategies may be related to the development of the approaches throughout the years. Following the negative outcomes of restoration attempts with drill and practice based on the mental muscle idea, there was a shift to focus on alleviation of memory deficits and learning specific knowledge or skills relevant to a particular domain in life (Hunkin and Parkin Based on the outcomes from previous studies, some considerations for future research arise. Firstly, consideration might be given to whether etiologies should be mixed or examined separately, followed by the severity, variety, and chronicity of memory impairment within the target group (Exner et al. Qualitative studies examining patient perspectives have demonstrated the importance of group settings for social interaction, feedback from others, and ability to compare progress in a motivational or appreciative capacity (Chouliara and Lincoln Finally, follow-up studies should be conducted to examine the lasting impact of memory rehabilitation. For example, recent studies have found that rehabilitation following ABI had positive impacts on community integration, quality of life, and continued use of external aids for several years beyond termination of the program (Shany-Ur et al.


Monkeypox was declared a public health emergency of international concern by the World Health Organization (WHO) on 23 July 2022. Between 1 January and 23 July 2022, 16,016 laboratory confirmed cases of monkeypox and five deaths were reported to WHO from 75 countries on all continents. Public health authorities are proactively identifying cases and tracing their contacts to contain its spread. As with COVID-19, PCR is the only method capable of being deployed at sufficient speed to provide timely feedback on any public health interventions. However, at this point, there is little information on how those PCR assays are being standardised between laboratories. A likely reason is that testing is still limited on a global scale and that detection, not quantification, of monkeypox virus DNA is the main clinical requirement. Yet we should not be complacent about PCR performance. As testing requirements increase rapidly and specimens become more diverse, it would be prudent to ensure PCR accuracy from the outset to support harmonisation and ease regulatory conformance. Lessons from COVID-19 should aid implementation with appropriate material, documentary and methodological standards offering dynamic mechanisms to ensure testing that most accurately guides public health decisions. From 1 January through 22 July 2022, 16,016 laboratory confirmed cases of monkeypox and five deaths were reported to the World Health Organization (WHO) from 75 countries [PCR is a rapidly deployable and versatile approach, but it must be applied with care if it is to accurately identify patients and aid contact tracing. The versatility of PCR can also be its weakness; notwithstanding the immense public health benefits of the rapid development and roll-out of PCR in the face of a new pandemic, there were certain challenges in the early response to COVID-19, such as contamination problems  and varyPoxviridae family, however, as these other family members are generally rare their undesired detection is not likely to impact on test specificity. Members of the Poxviridae family have very large genomes with many potential PCR targets of varying specificities: assays can range from pan-Orthopoxvirus detection [Orthopoxvirus-specific diagnostic PCR. The most likely adverse effect of a poorly designed and/or poorly optimised monkeypox PCR assay will be on the limit of detection (LOD), leading to false-negative results and reduced analytical sensitivity. Unlike severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in early 2020, monkeypox virus is not a new virus and several published assays are already available -11, someetection  to specietection  Table);;PoxviridTo mitigate against false-negative results associated with design or optimisation, laboratories should always evaluate published protocols by using control materials and/or clinical specimens with their own reagents and instruments; it is not appropriate to simply repeat the published protocols verbatim. Analytical performance evaluations, such as the LOD, should be performed with accurately quantified control materials that are ideally similar to the clinical specimen of choice . Given the current state of the art, it can be expected that, once the pre-analytical factors are accounted for, the PCR amplification step of the procedure will be capable of detecting small amounts (copy numbers) of DNA per reaction. Well-designed and optimised assays could be expected to demonstrate near single-copy performance. False-positive results will primarily be caused by contamination. In viral diagnostic PCRs, there are two broad sources of potential contamination: specimens with high titres and synthetically derived template. Consequently, when specimens from positive and negative patients are processed together, cross-contamination from the former to the latter is a potential risk. Contamination with synthetically derived templates, such as amplicons, is an inherent weakness of PCR and can be mitigated by physically separating specimen preparation from analysis .Other synthetically derived contamination can occur if demand increases for synthesised oligonucleotides as positive controls , which iThe clinical accuracy of monkeypox PCR assays will be determined by the clinical community and influenced by how they are used in terms of disease stage and specimen choice. Specimens typically comprise swabs of skin lesions and oropharynx but may extend to a range of other specimens for research purposes . Pre-anaPositive monkeypox virus controls are important in providing test confidence, but can also be used to determine analytical performance, such as LOD. However, they come in a range of different formats that vary in suitability for such assessments. In the short term, synthetic molecules (mentioned above) provide quick-access positive controls for the PCR step, but they cannot control for other parts of the diagnostic process, such as the extraction step used to purify nucleic acids, and are therefore of limited value when exploring factors like the LOD of a diagnostic pipeline. With time, whole-viral control materials, or virus-like materials such as pseudo-viruses, typically become established to provide a more comprehensive route for harmonising clinical diagnostic pipelines. Such materials are therefore preferred for a more accurate assessment of analytical performance characteristics like LOD, once widely available. While cultured virus is preferable to purified nucleic acid, it is less suitable for controlling for differences in specimen type, heterogeneity or sampling. As noted during the COVID-19 pandemic, routes to better standardise diagnostic protocols that use disparate and heterogenous specimens remain an important yet challenging area of research and development. Work on this topic will hopefully improve diagnostic test accuracy for diseases that are more far-reaching than monkeypox or COVID-19. As PCR detection of SARS-CoV-2 RNA became more established and as the pandemic evolved, the importance of sequencing to identify and track variants of concern became clear. This was epidemiologically important but also predicted the impact of genetic changes on assay performance and potential false-negative results. While monkeypox virus may not evolve as quickly as SARS-CoV-2, it should not be assumed to be slow evolving  and its While quantitation of monkeypox virus DNA may not be used to direct clinical decisions, quantitative considerations are required to evaluate analytical performance, such as LOD, and facilitate demonstration of competencies to meet regulatory requirements and in response to industry target product profiles. Furthermore, monkeypox virus genome quantities, and how they change during the course of infection, will probably be used to attempt to understand disease dynamics and infer disease stage or infectiousness. Such assessments of virus quantities and their epidemiological or clinical implications must rely on adequate methodology, taking into consideration the impact of pre-analytical factors outlined above and careful standardisation . q) ), but uncalibrated Cq should not be used as a unit to estimate quantities, set thresholds or report LOD [q for quantification. However, users need to select appropriate standard materials for determining copies as available positive control materials may not have been accurately certified for quantitation if they are not intended for use as calibrators. Reference materials that have been comprehensively characterised in terms of quantity, stability, batch variability etc. are key for ensuring accurate traceable evaluation of test performance in support of harmonisation and determination of characteristics such as LOD. Quantitative PCR (qPCR) is automated and consequently a popular diagnostic format. It provides a crude quantitative output in the quantification cycle  can act as a reference as they can provide high quantitative accuracy without calibration . In its The WHO interim guidance (published in May 2022) is very timely for the efforts of harmonising monkeypox diagnostics . The guiThe WHO guidance further suggests that controls should provide information about sample and nucleic acid quality as well as an evaluation of the success of the diagnostic pipeline (to ensure that processes such as nucleic acid extraction have worked properly). While the latter may be readily addressed by including internal process controls, metrics for specimen and nucleic acid ‘quality’ are not easy to deliver. The monkeypox protocol from the United States Centers for Disease Control and Prevention (CDC), published in June 2022 , is adapA human target can control for specimen and nucleic acid quality as well as diagnostic pipeline, yet how well this can control diagnostic performance is often undefined and will vary with specimen type and quality. Alternative internal positive controls, such as spiked cultured viruses, control for processes like lysis and nucleic extraction, but not specimen quality, are preferable to pure nucleic acid spike controls. Nucleic acid internal controls, while popular, cannot control for preanalytical steps such as viral lysis. The appropriateness of different internal controls can be evaluated experimentally by comparing their performance with positive clinical specimens or contrived samples. This can be achieved by simulating poor extraction, inclusion of inhibitors, etc. although based on current literature such assessment of internal control suitability is seldom performed. q rarely dropped below cycle 20 [q can vary 1,000-fold [9 genome copies/mL) easily detected even with lower-sensitivity test formats, a lower titre (≤ 106 genome copies/mL) making increased analytical sensitivity more important, or somewhere in between. Future studies exploring the quantities of monkeypox virus DNA must provide an estimation of how a given Cq value corresponds to viral genome copies if this work is to be used to aid the diagnostic discussion. Moreover, the correlation between virus quantity in various specimens and infectiousness needs to be established if PCR is to be used for risk assessment and not only virus detection. The Cq value alone provides limited information on which to make such assessments.The importance of the considerations specified by the WHO in terms of clinical relevance may vary for monkeypox diagnosis with different specimen types. The quantity of virus in clinical specimens will impact on how easily a test protocol can detect viral genomes, with high amounts simplifying detection but concomitantly increasing the risk of false-positive results because of cross contamination. Reports on quantities of monkeypox virus in specimens are limited. In a study of seven patients by Alders et al. 2022 the PCR Ccycle 20 . As Cq c000-fold , it is dWider guidance on how to approach and deploy diagnostic PCRs has also been provided in a number of documentary standards and technical specifications from the International Standards Organisation (ISO) technical committees 212  and 276 (Biotechnology) which directly address molecular measurement. Examples include technical specifications on SARS-CoV-2  and moreA notable legacy of the COVID-19 pandemic is the application of diagnostic PCR at an unprecedented global scale and an enhancement of molecular diagnostic capacity. The infrastructure, expertise and important experience gained during the COVID-19 response are likely to result in rapid development and adoption of molecular assays for other emerging or re-emerging threats. Should the monkeypox epidemic require testing to be deployed even more widely and sustainably, a focused effort to standardise the laboratory response and implement the lessons learned from COVID-19 should be prioritised. Such a proactive response will not hinder any diagnostic response; the opposite is more likely. COVID-19 has taught us that it is not acceptable to assume detection is simple because the viral load (and thus genome copies) can be high. A cautious approach to deploying PCR tests for monkeypox is likely to improve their impact and ensure the trust of the scientific community in this approach is sustained. This will empower those tasked with responding to the epidemic with the most accurate information to guide decisions across the whole spectrum from patient management to public health policy.


Tension pneumocephalus is a neurosurgical emergency that occurs when air is trapped in the intracranial cavity, leading to brain compression and causing severe neurological symptoms such as decreases in motor function, sensory function, and consciousness. Most cases of pneumocephalus require conservative treatment; however, because of the possible fatal complications, rapid diagnosis and appropriate treatment are important. Here, we present the case of an 81-year-old male patient who had undergone head trauma three hours prior to being admitted to our emergency room (ER) because of mental cloudiness. The radiologic findings showed tension pneumocephalus caused by an ethmoidal roof fracture. Emergency reconstruction of the ethmoidal roof with craniotomy was performed to remove the intracranial air using normal saline irrigation. By sharing our experience with this case, we hope to provide an option for the treatment of such cases.


Cancer treatment related infertility (CTRI) affects more than one third of young women undergoing anti-cancer protocols, inducing a premature exhaustion of the ovarian reserve. In addition to ovarian suppression by GnRHa, oocyte and cortex cryopreservation has gained interest in patients with estrogen-sensitive tumors for whom the hormonal burst to prompt the multiple follicular growth could provide a further pro-life tumor pulsing. On the other hand, cortex reimplantation implies a few drawbacks due to the unknown consistency of the follicles to be reimplanted or the risk of reintroducing malignant cells. The capability of ovarian stem cells (OCSs) from fresh ovarian cortex fragments to differentiate in vitro to mature oocytes provides a tool to overcome these drawbacks. In fact, since ovarian cortex sampling and cryopreservation is practicable before gonadotoxic treatments, the recruitment of OSCs from defrosted fragments could provide a novel opportunity to verify their suitability to be expanded in vitro as oocyte like cells (OLCs). Here, we describe in very preliminary experiments the consistency of an OSC population from a single cryopreserved ovarian cortex after thawing as well as both their viability and their suitability to be further explored in their property to differentiate in OLCs, thus reinforcing interest in stemness studies in the treatment of female CTRI. The incidence of cancer during a woman’s procreative life is apparently increasing with age, and it rises to 0.003% in the nearly 45-year aged female population . It is eIt is well known that at least one third of women under 45 years undergoing anti-cancer protocols including gonadotoxic therapies with alkylating agents, small molecules and radiotherapy, or their combination, often develop irreversible side-effect damage to the reproductive system ,7,8,9, wA primary approach to prevent ovarian desertification in young patients undergoing anti-cancer protocols was based on GnRH agonist (GnRHa) administration during gonadotoxic treatments to protect the OR ,11 in reCurrently, in young women with cancer, a well-established practice for fertility preservation (FP) is provided by oocyte recruitment and cryopreservation for fertilization after the cancer. However, in the presence of hormone-sensitive cancer in prepuberal age, several concerns related to the possibility to reinforce the cancer activity should advise caution in using this method that, on the other hand, is inappropriate for the scheduled time when anti-cancer treatments are urgently needed. Therefore, further strategies concerning the collection of immature oocytes at the germinal vesicle-stage for a subsequent in-vitro maturation (IVM) and fertilization (IVF) ,18,19 asKawamura and co-workers proposed the in vitro stimulation of biologic activity in primordial multiple follicles (PMF) in the context of a poor OR by upregulating the PI3K/Akt pathway through in vitro administration of molecular inducers to neutralize the disruption of Hippo pathway resulting from ovarian cortex fragmentation ,24. HoweNovel information related to potential application in the FP field in 2004 reported the existence of mitotically active germ cells in postnatal mouse ovaries which was also confirmed in other mammalian species, including humans. While this discovery opens new avenues for implementing neo-oogenesis and folliculogenesis in adult life and counteracting all forms of ovarian failure ,28,29,30Additional investigation by Guo and co-workers using genetic mouse models provided more knowledge on the OSC fate in vivo, including the postnatal oogenesis in adult females, and they confirmed the occurrence of germ cell meiotic entry into a common neo-oogenesis program during reproductive life ,50. ThesEmployment of OSCs to induce neo-oogenesis in FP programs and particularly in CTRI is thus of great interest since the potential utilization of these cells to induce neo-oogenesis avoids additional oncogenic risk related to folliculogenesis induction. In this context, several studies are in progress to optimize their suitability to undergo mature oocyte differentiation in vitro and subsequent cryopreservation. On the other hand, the possibility to derive OSCs from ovarian cortex strips already cryostored from cancer wo-men waiting for their disease resolution before undergoing FP approaches needs to be investigated to offer alternative options.Here, we review several aspects of the biologic properties of OSCs along with preliminary data on the viability assessment and the functional patterns of the same cells derived from a cryostored cortex. Though initial, our results suggest that even from cryopreserved fragments of ovarian cortex, it is possible to isolate aliquots of OSCs that could apparently be suitable to differentiate to mature oocytes.Based on the demonstration of the existence of OSCs in mammalian ovaries, several studies focused their phenotype and their molecular characterization. Original observation in mice described on these cells the expression of Ddx4 (DEAD box polypeptide 4) , a germlHowever, there is an open debate on the realistic existence of OSC in mammalian ovaries. To this regard, several investigators claim the role of the Ddx4 molecule as a marker of oogonial cells , as wellIn this regard, the debate on mammalian neo-oogenesis arose within the scientific community by several studies published by independent groups of investigators, namely Pacchiarrotti J , BukovskIn this contest, there was an additional question regarding the use of Ddx4 as a germline marker for sorting OSCs , which i+OSCs has been clearly demonstrated to express stemness markers such as Oct4 and SSEA4, thus providing evidence in support of active germ cells in an adult mouse ovary [On the other hand, Ddx4se ovary . The expse ovary . Similarse ovary . In factse ovary .Nevertheless, several aspects need to be better defined in relation to the obtainment of homogeneously expanded populations of well-differentiated oocytes in vitro. In this regard, an intensive investigation is in progress to identify specific oogenic factors enrolled in the final differentiation stage of mature oocytes. Ongoing research from major investigative groups postulate that undifferentiated granulosa cells, capable of interaction with newly generated oocytes and formation of the primordial follicle, prime the final maturation of OLCs in relation to the expression of stemness markers and genes . HoweverThese results validate the hypothesis that OSCs are capable of regenerating the oogenesis and that they are suitable to be further investigated for a future enrollment in FP programs in women with POF or CTRI. Particularly in young patients with cancer, the utilization of OSCs to promote the neo-oogenesis will be safe and easily affordable since their recruitment and their process to obtain OLC progenies provides the advantage to select the most viable OLCs to be cryopreserved without oncogenic risks due to hormone stimulation with the advantage of selecting the best eggs to be used.Although not highly diffused in the majority of cancer FP centers, the biobank of cryopreserved ovarian tissue provides a considerably source of biological material to investigate the OSC population of women. Here we revisit major aspects useful to obtain OSCs from cryostored strips.Cortical ovarian tissue isolation and cryopreservation—Ovarian tissue cryopreservation is an immediate, alternative FP procedure for female cancer patients urgently requiring oncological treatments. The recovery of cortex strips of approximately 1 cm by 4–5 mm [y 4–5 mm  is genery 4–5 mm  to minimy 4–5 mm . Suggesty 4–5 mm , as welly 4–5 mm .The cryopreservation of ovarian tissue is usually performed by two techniques: the slow freezing and the vitrification, as reported in The vitrification of the ovarian tissue is considered the most suitable option for the FP in prepubertal girls undergoing gonadotoxic treatments. The procedure utilizes solutions at high viscosity along with high concentrations of cryoprotective agents . EthylenOvarian cortex cryopreservation in cancer patients—Cryopreservation is indicated for adolescents and women whose cancer treatment is urgently required and cannot be delayed. The first birth obtained after ovarian tissue cryopreservation and auto-reimplantation to a prepubertal patient was reported in 2015 [ in 2015 . In a re in 2015 .Drawbacks in using the cortical graft—Several surgical techniques are currently being used for frozen-thawed ovarian tissue auto-reimplantation. However, to be successful at least two points need to be respected, namely a well vascularized orthotopic transplantation site and joining ovarian tissue by stitches whose edges are fixed with fibrin glue. Suitability of orthotopic sites for auto-transplantation is pivotal for ovarian activity renewal with described efficacy for restoring fertility in over 40% of patients and exponential success rates in terms of pregnancies [gnancies .Safety issues surrounding ovarian tissue reimplantation from cancer patients have represented a few concerns. Several studies investigated the risks of reintroducing malignant cells together with frozen-thawed ovarian tissue, which could induce a recurrence of the primary tumor . In the Based on these considerations, the cryostorage and reimplantation of ovarian tissue for the FP includes both advantages and drawbacks, particularly in oncology. Whilst it has been improved in technical methodology for acquiring viable follicles as well for improving the grafting of implanted ovarian biopsies, on the other hand the selection of follicles is based only on their ultrasound detection and the quality of oocytes to be reimplanted is usually not investigated. Although the risk to regenerate certain tumors in relation to the presence of cancer cells in the cryopreserved ovarian cortex can be limited by selecting the best time for the biopsy, the procedure cannot be considered completely safe at least in patients with hematologic and a few solid malignancies.Despite encouraging results on the potential of OSCs from freshly derived strips of ovarian tissue to generate OLCs and their subsequent differentiation to mature eggs, at present very few information is available on both viability and maintenance of biological properties of the same cells after freezing for cryopreservation and thawing as reported for the cortex slices reimplanted in ovaries. This information is particularly needed due to the large availability of cryopreserved cortex specimens in biobanks of institutional centers deputed to FP programs, which could indeed take considerable advantage of deriving mature oocytes from cryostored ovarian samples.Therefore, here we describe our preliminary experience in verifying both occurrence and viability of OSCs from ovarian cortex fragments after thawing.During the past two years, we collected ovarian cortex biopsies from 11 non-menopausal women age 20–43 years) with the purpose of expanding our previous studies on OSCs by verifying their suitability after cryopreservation. The patients recruited were affected by urogynecology tumors with no ovarian involvement and before abdominal surgery, chemo-radiotherapy, with serum anti-Müllerian hormone (AMH) levels higher than 1.4 ng/mL and antral follicle count (AFC) greater than four by ultrasound evaluation. The cortical biopsies were performed mostly by laparoscopy during the abdominal surgery or by specifically needed laparotomy in single instances and all patients agreed to the cortex sampling by dedicated informed consent to donate their biological material for research purposes. Once obtained, the cortex pieces were quickly prepared for the cryopreservation within one hour and their freezing in liquid nitrogen was performed according to the ‘slow freezing’ protocol [–43 yearsThe cortex piece was derived from a 28-year-old woman, with AMH 3.56 ng/mL and AFC 28 follicles values, submitted to left annexiectomy for a 7 cm benign ovarian cyst. To perform our test, the ovarian sample was originally maintained in Leibovitz L-15 medium in ice and the medulla was gently scraped by a scalpel to isolate the cortical tissue. Thus, the cortical parts were subsequently fragmented in strips of 5 by 5 by 1–2 mm, then maintained in the same medium added with 4 mg/mL human serum albumin and 1.5 mmol/L DMSO, and ultimately frozen by a controlled slow-freezing procedure and then stored in liquid nitrogen. Thus, after the cryopreservation period, the vials containing the cortical strips were thawed in a warm water bath at 37 °C for 2 min and the strips were then carefully washed three times with fresh medium to remove cryoprotectant.® tissue homogenizer. The recruited cell suspension was then stratified on a Ficoll gradient and centrifuged; then, it was recovered and incubated at 4 °C with rabbit polyclonal anti-Ddx4  followed by further incubation with anti-rabbit IgG-FITC antibody . The OSC suspension was then incubated with 7-amino-actinomycin D labelled with phycoerythrin-5  as viability dye, and it was ultimately evaluated by flow cytometric analyses . The analysis was performed in technical triplicates to obtain standardized values.To better protect cell viability, isolation of OSCs from the strips was performed by an enzyme-free method since energic enzymes as collagenase may damage the cell membrane proteins. Briefly, the strips were further fragmented and then placed in 35µm of disposable disaggregating medium  containing a phosphate buffer was added by the MedimachineAs shown in Although preliminary and completed on a single sample, our results are in accordance with the viability data of a previous study completed by Wagner  who descThus, our results on Ddx4-positive cell viability from thawed strips along with data from Wagner , althougRecent advances in the field of regenerative medicine have focused an increasing in-terest concerning the employ of stemness technology as a possible novel application in the treatment of cancer-related-infertility for young women at risk for CTRI. Since a number of studies proved the ability of cultured OSCs to differentiate into OLCs, their future utilization is to be necessarily considered a promising practice not only to treat infertility in women affected by POF or to restore the hormonal balance in post-menopausal age, but a safety alternative to oocyte and ovarian cortex cryopreservation in patient candidates for FP programs and particularly in young women at risk for CTRI.Their major suitability in this field concerns the possibility to design an FP program based on OSC recruitment from fresh ovarian cortex fragments before gonadotoxic treatments, differentiation in mature oocytes, selection of populations of most viable eggs, and cryopreservation. Thus, the main advantage of this approach includes the availability of a large population of oocytes from each patient, the full absence of risks for the estrogenic stimulation or of reintroducing malignant cells, and immediate applicability to pre- and post-pubertal patients.The possibility to isolate OSCs viable up to 95% from cryostored ovarian cortex, a percentage similar to fresh samples, also suggest that this cellular population is apparen-tly resistant to the temperature stress for freezing and thawing thus maintaining excellent viability and perhaps the capability to generate mature and fertilizable oocytes in vitro. This is exactly what we will explore in a next study by verifying the expression of oocyte molecular markers in cultured OSCs from cryopreserved samples.


Hemispheric Asymmetries in the Auditory Domain, a topic which has given rise to a huge amount of scientific work utilizing behavioral, neuropsychological, electrophysiological, and neuroimaging techniques . In their fMRI study, Kazimierczak et al. looked for modulation of brain activity by top-down effects of forced-attention during a dichotic listening paradigm . The author emphasizes that sex differences in hemispheric asymmetry, namely planum temporale asymmetries, might be an important factor for the female advantage in verbal learning.In an opinion paper, Moulin showed that the word-level contextual influences during speech perception in hearing-impaired patients were stronger on the right than on the left ear. This asymmetry between the ears increased with increasing right-ear advantage and decreased with age, mainly due to an increasing contextual influence on the left ear. This age effect is consistent with reduced hemispheric asymmetry with age  were similar between presentation modalities whereas the right hemispheric involvement was different between modalities, e.g., more right frontal involvement during the visual than auditory condition.Based on a visual language paradigm, Han et al. start from the fact that unilateral deafness reduces the ability to localize binaural sounds and leads to a substantial change in auditory cortical processing. To unveil the underlying mechanisms, authors compared primary auditory cortical activity and hemispheric asymmetries of normal hearing, unilaterally deaf (right or left), and simulated hearing loss participants while listening to speech sounds delivered from different locations in the azimuth plane. The right ear hearing-loss group revealed slower reaction times for sound localization and prolonged neural latency compared to the left ear hearing-loss group. In addition, individuals of the right ear hearing-loss group with better sound localization showed increased responses in the hemisphere ipsilateral to the hearing ear. Conversely, the left ear hearing-loss group showed contralateral enhanced activity related to right ear stimulation. Thus, the authors demonstrated that right ear unilateral deafness can rely on better plastic reorganization compared to left ear deafness. This paper evidences the presence of potential plasticity targets, which should not be neglected in auditory rehabilitation.In their paper on asymmetries in unilateral deafness, Kim et al.; Tanaka and Kirino). Tanaka and Kirino deal with a very interesting topic concerning musical imagery. They exploit the common condition of subjects who lie in an fMRI scanner and hear the typical machine noise using this condition as a baseline. During the musical imagery condition, in which musicians imagined to perform a music piece, they found enhanced right-lateralized connectivity of the auditory cortical areas with other brain regions devoted to cognitive, memory, and emotional processing. This outcome demonstrates that auditory cortex is involved with other areas in imagined music performance  whereas connectivity from the right to the left superior temporal gyrus was enhanced for the most ambiguous condition (dominant-submediant). Thus, they showed that syntactic irregularity and perceptual ambiguity elicit dissociated processing between bilateral auditory areas.Hemispheric Asymmetries in the Auditory Domain embrace competences that arise from various scientific fields whose interaction can bear fruit for cognitive neuroscience and for the other subject areas ranging around brain science. Exploiting the knowledge acquired on auditory hemispheric and functional asymmetries will generate further studies facing the question at different levels of analysis to disentangle the intriguing processes underlying such complex psychobiological functions.The overall message that this Research Topic conveys is that All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Plasmids are a major driver of horizontal gene transfer in prokaryotes, allowing the sharing of ecologically important accessory traits between distantly related bacterial taxa. Within microbial communities, interspecies transfer of conjugative plasmids can rapidly drive the generation genomic innovation and diversification. Recent studies are starting to shed light on how the microbial community context, that is, the bacterial diversity together with interspecies interactions that occur within a community, can alter the dynamics of conjugative plasmid transfer and persistence. Here, I summarise the latest research exploring how community ecology can both facilitate and impose barriers to the spread of conjugative plasmids within complex microbial communities. Ultimately, the fate of plasmids within communities is unlikely to be determined by any one individual host, rather it will depend on the interacting factors imposed by the community in which it is embedded. Current Opinion in Microbiology 2022, 68:102152Environmental MicrobiologyThis review comes from a themed issue on Paul Hoskisson and John BruceIEdited by Environmental Microbiology”For complete overview of the section, please refer to the article collection, “Available online 2nd May 2022https://doi.org/10.1016/j.mib.2022.102152http://creativecommons.org/licenses/by/4.0/).1369-5274/© 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license  occurs extensively among prokaryotes and is a major driver of bacterial evolution and diversification, with large proportions of bacterial genomes consisting of horizontally acquired genes Modern advances in sequencing technologies have unearthed a huge diversity of plasmids within neutral microbial communities Here, I summarise the latest advances in our understanding of how community context, that is, species diversity together with interspecies interactions, can impact the dynamics of conjugative plasmids by facilitating or limiting plasmid transmission in natural microbial communities. This perspective focuses predominately on the persistence and spread of conjugative plasmids within communities. While the factors altering the stability of non-mobilisable plasmids may be different, due to interspecies and multitrophic interactions potentially altering host–plasmid properties (such as fitness costs), it remains likely that community context will also impact the persistence of non-mobilisable plasmids.Enterobacterales, causing multispecies outbreaks of carbapenem-resistant infections in situ within infant gut microbiota has also shone a light on the importance of conjugative plasmid transfer in the spread of virulence factors and antibiotic resistance genes between coexisting bacterial lineages Diverse multispecies microbial communities are considered hotspots for HGT; in such communities, conjugative plasmids are capable of rapidly spreading between diverse taxa and persisting for long periods of time The host range of a conjugative plasmid is a key factor determining its ability to spread to a diverse subset of a community. While some plasmids are restricted to a single or closely related subset of species due to limitations in the ability to form mating pairs or incompatibility between the plasmid replication system and their hosts Microbial communities are rightly considered hotspots for HGT; however, barriers to plasmid-mediated HGT exist both at the single cell level  can vary for the same plasmid in different host backgrounds P. fluorescence through conjugation while imposing high fitness costs, whereas the same plasmid was lost from monoculture Pseudomonas putida populations, despite imposing a lower fitness burden, due to significantly lower intraspecies conjugation rates. However, in coculture, P. fluorescence was able to maintain pQBR57 in less the permissive P. putida at low frequency due to interspecies conjugation. Interestingly, positive selection for mercury resistance led to the mercy resistance gene being transferred to the chromosome of P. putida in single-species populations. Subsequently, the plasmid was lost from these population, being replaced by chromosomal resistance mutants. Although this alternative route to mercury resistance clearly benefited P. putida in single-species populations, during mercury treatment of coculture populations chromosomal resistant mutants were less common and source-sink transfer maintained the plasmid within P. putida. Within more complex multispecies mouse gut communities, Ronda et al. Conjugation rates can differ greatly between different genetic backgrounds Acinetobacter, Pseudomonas, and Enterobacteriaceae, in which plasmid carriage either incurred no cost, or was beneficial. However, the survival of the plasmid within the community was dependent upon the abiotic environment. Cultivation of the community in anoxic conditions and specific nutritional environments lead to the loss of the plasmid from the community as the permissive phylotypes were selected against, suggesting the fitness effects were dependent upon the environmental context.Plasmids often cause physiological changes to their hosts that reduce the competitive fitness plasmid bearing bacteria A similar diversity of plasmid fitness effects was also observed in co-occurring enterobacteria strains isolated from the gut microbiota of hospital patients in vitro populations. However, it is becoming increasingly evident that the plasmid dynamics within natural microbial communities are much more complex. Communities are likely to contain a high diversity of plasmid–host interactions, ranging from completely incompatible hosts to highly proficient hosts. However, the dynamics of conjugative plasmid transmission and persistence within a community will not be solely dependent upon any one host, rather it will dependent upon the ecology context in which the plasmid is set. Importantly, the ecological properties of a community that can facilitate the persistence of plasmids, can in different contexts also limit the spread of the plasmid within the community. For example, while increased diversity of host permissiveness can increase stability via spill over transmission from proficient reservoir donors, the presence of non-permissive hosts can also dilute and destabilise the spread of purely infectious plasmids. Multi-tropic interactions, such as predation and parasitism from protists and phage, can promote conjugation plasmids, but equally drive plasmids to extinction.Our understanding of the dynamics of conjugative plasmids has historically focused on controlled relatively simple single-species in vitro multispecies and multitrophic communities (such as The relative importance of different variables and how they interact to facilitate or limit the spread of plasmids within natural communities, are major outstanding questions. The complexity of microbial communities will make it challenging to answer such questions, in part due to the diversity of hosts — differing in their ability to receive, maintain and disseminate plasmids — as well as the diversity of interspecies and multitrophic interactions that are present within natural communities. A combination of approaches including the use of more representative  such as , 56, inc such as , 65••, a such as  will allThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.