Articulography and functional neuroimaging are two major tools for studying the neurobiology of speech production. Until recently, however, it has generally not been possible to use both in the same experimental setup because of technical incompatibilities between the two methodologies. Here we describe results from a novel articulography system dubbed Magneto-articulography for the Assessment of Speech Kinematics (MASK), which we used to derive kinematic profiles of oro-facial movements during speech. MASK was used to characterize speech kinematics in two healthy adults, and the results were compared to measurements from a separate participant with a conventional Electromagnetic Articulography (EMA) system. Analyses targeted the gestural landmarks of reiterated utterances /ipa/, /api/ and /pataka/. The results demonstrate that MASK reliably characterizes key kinematic and movement coordination parameters of speech motor control. Since these parameters are intrinsically registered in time with concurrent magnetoencephalographic (MEG) measurements of neuromotor brain activity, this methodology paves the way for innovative cross-disciplinary studies of the neuromotor control of human speech production, speech development, and speech motor disorders. While it is relatively straightforward to measure the acoustic consequences of speaking with audio recordings, measuring, and characterizing the physical movements (motor behaviors) that produce acoustic speech signals presents some more formidable challenges. These challenges are due to the inaccessible nature of many of the components of the vocal tract, which are completely or largely hidden from direct view within the laryngeal cavity, the pharynx, the nasal cavity, and the oral cavity. One approach is to simply limit measurements to line-of-sight movements of the lips and jaw, which can be readily characterized with optical (video) tracking and facial capture systems with added precision from placement of reflective –4 or actElectromagnetic articulography  was developed to image within the oral cavity by tracking movements of marker coils placed on the tongue . MovemenCommercial EMA systems, the Carstens AG series , the recently discontinued NDI Wave  and othRecent advances in our understanding of speech motor control indicate that it would be advantageous to have access to both types of information in studies of speech production. Most notably, a study by Chartier et al.  used ultThe recent development of a magnetoencephalograpic (MEG) scanner-compatible speech tracking system  finally The current study extends the description of MASK motion tracking capabilities by Alves et al.  with a dThree healthy adults with typical developmental histories participated in this study: E1 ; M1 ; M2 . E1 participated in the EMA experiment at the Oral Dynamics Lab at the University of Toronto; M1 and M2 participated in the MASK experiment at Macquarie University. All procedures were approved by the University of Toronto and Macquarie University Human Research Ethics Committees.Time-aligned audio and EMA position signals were recorded using the AG501 system  with a large helmet size and automated calibration. EMA coils were attached on the mid-sagittal vermilion border of the upper and lower lip, tongue tip (1 cm from the apex), tongue body (2 cm from the tongue tip), and tongue dorsum (4 cm from the tongue tip) using surgical glue . Three additional coils were placed at fiducial points on participant's left and right preauricular points and nasion for reference purposes , 21]. AfMASK tracking data and neuromagnetic brain activity were recorded concurrently with a KIT-Macquarie MEG160  whole-head MEG system consisting of 160 first-order axial gradiometers with a 50-mm baseline , 25. MEGTime-aligned speech acoustics were recorded in an auxiliary channel of the MEG setup with the same sample rate as the MEG recordings. An additional speech recording was obtained with an optical microphone  fixed on the MEG dewar at a distance of 20 cm away from the mouth of the speaker; and digitized using a Creative sound blaster X-Fi Titanium HD sound card with 48 kHz sample rate and 8-bit quantization precision. The higher sample rate acoustic recordings were time-aligned off-line with the 4,000 Hz auxiliary speech channel to bring them into time register with the neuromagnetic data.Three non- word productions were used as experimental stimuli: Two disyllabic sequences with a V1CV2 structure /ipa/ and /api/; and one trisyllabic sequence /pataka/. The di- and tri-syllabic non-words were selected for measuring intra- (between single articulator movements) and inter- (between consonant and vowel gestures) gestural coordination within a single task . The samParticipants were presented with a fixation cross on a display screen and instructed to take a deep breath. The stimulus non-word then appeared on the screen for 12 s. For the normal rate production, participants were required to utter productions at a normal, comfortable rate as they would do while conversing with a friend, until the stimulus non-word disappeared from the screen. For the faster rate, they were instructed to produce the stimuli as fast as possible while maintaining accuracy . FollowiMagneto-articulography for the Assessment of Speech Kinematics coil position and orientation data initially localized in the MEG sensor frame of reference at a sample rate of 25 Hz was transformed off-line to the occlusal plane and low pass filtered at 6 Hz. These coil locations, orientations, signal magnitudes strength were imported to EGUANA software , 21. AllComputation of kinematic parameters  were performed for the opening and closing movements of the BC and TB gestures:Movement amplitude (with units of mm) refers to the maximum displacement from a peak to a valley and vice versa.Movement duration (ms) refers to the time needed for the gesture to move from a peak to a valley and vice versa.Peak velocity (mm/s) refers to the maximum velocity achieved by the gesture while moving from a peak to a valley and vice versa.Stiffness (1/s) refers to the slope of the relationship between peak velocity and amplitude .* duration , is the stiffness duration .Relative phase analysis was used to quantify two types of speech coordination , 35:Intra-gestural coordination refers to coordination between two individual articulators, in which the coordination of their movement is controlled by the same gesture while.Inter-gestural coordination refers to movements controlled by two separate gestures.As a first step, the power spectra of the BC and TB signals of each trial were computed with the Fourier transform using a frequency resolution of 0.1Hz. The frequency component with greatest power provides a good estimate of the dominant influence on movement patterning over time [; see alsThe productions /ipa/ and /api/ provide a useful contrast in their mirrored positionings of the tongue and lips and the contrasting positionings are clearly observed in both the MASK and EMA measurements of tongue and lip gestures. In * duration scaled in arbitrary units) is a numerical index of the shape of the velocity profile of a speech movement, whose value varies as a function of the shape of the basis velocity function. As such, velocity profiles have application in motor control both for determining the shape of the potentially underlying control variable ; and for determining if a control parameter pertains or changes across linguistic conditions. Velocity profile parameter , and that the MASK and EMA experiments were carried out in separate laboratories, these results further support the interpretation that these kinematic profiles reflect relatively stable properties of speech motor control.The preceding analyses have focussed on kinematic properties of individual speech movements: we now turn to the matter of coordination of articulator movements within and between speech gestures. In studies of speech motor control coordination is often defined in terms of relative timing as indexed by relative phase between two articulators or two gestures [, 28, 39;We would expect the relative timing of the UL and LL to be consistent across different speaking rates to maintain intelligibility, and indeed intra-gestural coordination of UL and LL in the three speech tasks (As noted above (raw tracking results) the gestural movements of /ipa/ and /api/ can be seen as mirror images, where the relative motions of tongue body and BC gestures are reversed. This reversal is readily apparent in the inter-gestural timing plots of A key characteristic of human speech is that speech goals are consistently achieved in the face of a truly remarkable amount of variation in the conditions under which they must be expressed. The design of the present study captures some aspects of these variable demands: speech movements were measured from participants with widely varying speech acquisition backgrounds, in different laboratories in different countries using different tracking equipment, in upright vs. supine speaking positions, and at different speaking rates. The orderly patterns of speech behaviors that emerge in the present results are thus suggestive of parameters that play key roles in the motor control of human speech.Such findings conform well to concepts within state speech motor control models such as Articulatory Phonology (AP) and the associated Task Dynamics framework (TD), which hold that articulators create functional relationships to cause local vocal tract constrictions . The absa point-attractor system which acts similarly as a dynamical, damped mass-spring-system, i.e., movement of a mass attached to a spring moving toward to an equilibrium position, which produces and releases constrictions of the end-effectors that are being controlled  , 53 is aComparable RSA analyses have been successfully applied to speech movements in several recent fMRI studies. Carey et al.  construcThe present results show that MASK provides the capability, for the first time, for deriving subject-specific articulatory contrast matrices, based on well-established and robust motor control parameters, in the same experimental setup as the brain recordings and in temporal and spatial co-register with the brain data.A reviewer of a previous version of this manuscript has noted that it would potentially be of considerable interest to have the capability to record MEG neural activity at a sampling rate comparable to that used in acoustic analyses of speech, i.e., 16 kHz or greater, allowing researchers to probe MEG data for brain activities associated with high frequency acoustic features such as fricatives, in addition to the lower frequency speech movement signals addressed in the present study. This is likely to be possible in the future with ongoing advances in digital storage and processing capacities, but maximal sampling rates of current commercial MEG systems are typically in the range of circa 4 kHz. For the purposes of the present study the acoustic data serve as markers of where events have occurred and the 4 kHz sampling rate is sufficient for lower frequency features such as formants. The time-aligned high sample rate audio signal is used where a more detailed inspection of the acoustic signals is required .The problem of how humans develop speech is a central, unanswered question of neurolinguistics. The topic has been and remains conspicuously under-studied . StudiesThe neural control of speech is also highly relevant to acquired apraxias, and to the burgeoning fields of speech prosthetics and brain computer interfaces . It bearThe present results demonstrate that the MASK technique can be used to reliably characterize movement profiles and kinematic parameters that reflect development of speech motor control, while simultaneously measuring the brain activities that provide this control. MASK brings articulography into the brain itself and thereby bridges a crucial methodological gap between the fields of speech science and cognitive neuroscience. The importance of this gap has recently been emphasized by invasive ECoG studies which have demonstrated that speech motor cortex operates by encoding and computing speech kinematic parameters that can be derived only with detailed measurements of the movements of individual articulators, including non-line-of-sight measurements of the oral cavity. This new capability sets the stage for innovative cross-disciplinary efforts to understand the neuromotor control of human speech production.The impacts of such research flow from the fact that articulography, the current state-of-the-art for studies of speech motor control, measures only the final output of the brain's speech production system. Concurrent MEG neuroimaging powerfully extends the state-of-the-art into the brain itself. In turn, concurrent articulography promises to dramatically improve the precision and inferential power of MEG measures of speech-related brain activity. These studies can therefore facilitate a shift in the current focus of the field and set the stage for new collaborative efforts across a number of disciplines including linguistics, kinesiology, developmental psychology, neuroscience and speech pathology. The results will bear on and eventually inform diagnostic methods and interventions for speech fluency and other motor speech disorders, which are the most common developmental disorders encountered by families, speech-language pathologists, pediatricians, and general practitioners.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Macquarie University Human Subjects Ethics Committee and by the Health Science Research Ethics Board at the University of Toronto. The patients/participants provided their written informed consent to participate in this study.IA conducted the experiment. IA and BJ analyzed the data. All authors conceived, designed the experiment, discussed the results, wrote, and edited the manuscript.This work was supported by a Child Development Fund Research Grant from the Waterloo Foundation (Ref. no. 2532 – 4758) and a Discovery Project Grant from the Australian Research Council (DP170102407).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.


During the year 2020, the COVID-19 pandemic rapidly became a severe health emergency worldwide. In order to contrast the spread of the novel SARS-CoV-2, many countries implemented extraordinary restrictive measures, such as a strict lockdown and school closures. The pandemic had a great impact on children and adolescents’ daily life, leading to a much more sedentary lifestyle, to larger use of electronic devices and to an increase in stress-related symptoms. These conspicuous changes acted as disruptors of children’s normal development. Since the beginning of the pandemic, many studies reported an increase in the number of precocious puberty cases as well as a faster progression rate of puberty itself, if compared to the pre-pandemic years. In this review, our aim was to evaluate the incidence of new cases of early and precocious puberty during the COVID-19 pandemic, analyzing variations in the timing of puberty and in pubertal progression rate, and to investigate the role of environmental and lifestyle factors during the pandemic in modulating the physiopathology of pubertal development. While a direct effect of SARS-CoV-2 infection remains, at the moment, a remote hypothesis, both physical and psychological factors related to the pandemic seem to have a role in triggering GnRH pulsatile secretion leading to earlier pubertal onset. It is indeed important to stress the need to clarify the exact role of COVID-19 in early pubertal onset comparing data from all over the world; long-term comprehensive studies are also pivotal to explain whether this phenomenon will continue while we resume pre-pandemic habits. School closure and stay-at-home policy resulted in increased psychological stress .Since the beginning of 2020, several pediatric endocrinology centers observed an increase in referrals for suspected precocious puberty and in the number of central precocious puberty (CPP) and rapidly progressive puberty cases, mainly in girls. Many of those children needed to start therapy with GnRH analogs.This new trend was first reported in November 2020 in Meyer Children’s University Hospital, Florence ; many reIn the following paragraphs, after describing the mechanisms underlying pubertal development and the main causes of central and peripheral precocious puberty, the results of these studies will be examined to assess the influence of many aspects potentially linked with precocious puberty and rapidly progressive puberty. Both direct effects of SARS-CoV-2 infection and physical and psychological changes related to the pandemic will be investigated as potential underlying causes.2Great advantages in understanding the physiology of puberty have been made in the last decades. Puberty can be defined as the transition period between childhood and adulthood. Development of secondary sexual characters and gonadal maturation happen during this phase, thus leading the individual to the achievement of reproductive capacity . PubertyA transient activation of the hypothalamic-pituitary-gonadal axis happens however from the first week of life and achieves its peak during the first 3-6 months of life leading to high gonadotropin and sex steroid levels. This postnatal activation of the axis, called “minipuberty”, allows for sexual organs maturation in both boys and girls . The traAs for the reactivation of the axis, it is probably due to a dense interaction between genetic, epigenetic, and neuroendocrine factors . NutritiPubertal development is the result of increased secretion of hypothalamic gonadotropin-releasing hormone (GnRH), gonadotrophins and, subsequently, gonadal sex steroids. Gonadotropins  are released in a pulsatile way from the pituitary gland as a response to GnRH stimulation. Studies demonstrate that what increases at puberty is GnRH pulse amplitude rather than its frequency , 11. ParAfter the increase in amplitude of GnRH pulse, the rise in LH and FSH levels enhances the production of sex hormones by the gonads. Sex hormones, mainly estrogens, also mediate effects on growth rate, central nervous system functioning, bone mineralization .Although there can be variations in the timing and tempo of puberty in boys and girls, the underlying mechanisms are the same. To assess pubertal development, physicians use detectable changes in secondary sexual characteristics to represent the degree of pubertal maturation. These modifications, described by Marshall and Tanner , 17, conIn females puberty usually starts between age 8 and age 13. Even though puberty pattern can vary, in most cases the first event is the thelarche , followed by pubarche (the first appearance of pubic and axillary hair). In the meanwhile there is an increase in linear growth that reaches its peak in stage 4. At this point, the menarche can happen. In some studies, it is debated if pubarche without breast development may represent, in girls, the true onset signal of pubertal development . In male3Precocious puberty is traditionally defined as the appearance of secondary sex characters before 8 years of age in girls and 9 years of age in boys . PrecociBased on the nature of its etiology, precocious puberty can be defined central or peripheral.CPP derives from earlier activation and maturation of the hypothalamic-pituitary-gonadal axis. CPP happens through the same stages as normal pubertal development and is a disease with female preponderance. While the idiopathic etiology is the most common cause in girls, underlying tumors or injuries of the central nervous system (CNS) are more common in boys , 27. Rec- CNS tumors;- CNS injuries;- Genetic variants and syndromes;- Familial forms of CPP.Peripheral precocious puberty (PPP) is less frequent than CPP and results from sex steroid exposure without the activation of GnRH pulsatile secretion. PPP can be acquired or congenital. Some important causes of PPP are –30:- Congenital adrenal hyperplasia (CAH);- McCune Albright syndrome;- Familial male-limited precocious puberty (FMPP or testotoxicosis);- Sex steroid secreting tumors;- Exogenous exposure to sex steroids;- Van Wyk Grumbach syndrome .In CPP, treatment with GnRH agonist is considered safe, without significant adverse effects, and can be administered based on the progression of puberty and on the age of the child to stop the development of secondary sex characteristics and to preserve final adult height. Late onset of treatment , advanced bone age, shorter duration of treatment are some of the factors linked with less success in preserving adult height .In PPP the clinical presentation and the treatment of choice depend on the underlying cause. The primary purpose of therapy in PPP is to remove the extra source of sex steroids thus restoring prepubertal values of sex hormones .Precocious puberty has been, in the past decades, a matter of concern as the decline in age at puberty has been associated in both boys and girls with several health risks and adverse behaviors. Changes in pubertal timing are related to rapid growth and reduced final adult height  and to ath century to approximately 13 years in the mid-20th century  study, showed a decline in the age of breast and pubic hair development. As a reply to this research, in 1999 the Lawson Wilkins Pediatric Endocrine Society proposed new guidelines to define precocious puberty , suggestAfter the PROS study, European –45 and A4During the past two and a half years, multiple centers globally recorded an increase in CPP and rapidly progressive puberty diagnoses Table 1.The first reports of this trend came from Italy. A retrospective study on patients referred to the Auxo-endocrinology and Pediatric Gynecology Unit of the Meyer Children’s University Hospital was published in November 2020. The Authors reported an increase in CPP cases during the first lockdown (March – July 2020); the number of new diagnoses was significantly higher if compared to the mean from the same period of time in the previous five years. They also collected data concerning the rate of pubertal progression and found that some patients who had previously received CPP diagnosis moved from a slowly progressive puberty to a rapidly progressive puberty during the first lockdown. As for the reason of this new trend, the first hypotheses concerned the increase in BMI and the augmented use of electronic devices during the first lockdown. In both CPP and rapidly progressive puberty group BMI increased significantly during the lockdown and families reported an augmented use of electronic devices .A different Italian study conducted in the Endocrinology Unit of Bambino Gesù Children’s Hospital in Rome compared the first 2020 months with the same months in 2019 and described a significant increase in outpatient consultations for precocious puberty. In this case, however, no significant differences in BMI were reported between the first and the second group .In the outpatient clinic of pediatric endocrinology of the University of Campania Luigi Vanvitelli, CPP incidence rate was 2.5-fold higher from April 2020 to April 2021 than from 2017 to 2020. While sleep disturbance was a frequently associated comorbidity, BMI was found to be similar between the two groups. CPP cases diagnosed during the pandemic had higher levels of LH, FSH and 17 beta estradiol than CPP cases diagnosed before the pandemic .A significant increase in CPP diagnoses in girls in 2020 if compared to 2019 (+16%) was also found in Trieste, Italy. No increase in BMI was found in the 2020 group. Conversely, CPP diagnoses decreased in boys (-75%): the Authors traced this trend back to the fact that clinical findings such as testicular enlargement often went unnoticed and family pediatrician visits were missed during the first lockdown .Similar results promptly came from research conducted all over the world. In a study among school-aged girls in Shanghai, the incidence rate of precocious puberty in 2020 was higher than that in 2016-2019; serum concentrations of MKRN3  were lower and GnRH concentrations were higher in girls from the 2020 group. These girls had several risk factors related to lifestyle changes that happened during the first lockdown such as weight gain , augmented screen time, reduced physical activity . An analIn a report from Turkey, the Authors highlighted how the number of CPP and rapidly progressive early puberty diagnoses increased three times in the pandemic period (April 2020 – June 2021) if compared to the pre-pandemic period (January 2019 – March 2020). Among these girls, 85% of the ones diagnosed with CPP and 67% of the ones diagnosed with rapidly progressive early puberty needed to start treatment with GnRH analogs. Interestingly, the Authors noticed that 28% of CPP and 69% of rapidly progressive early puberty cases were obese or overweight and were diagnosed during the pandemic period .However the correlation with an augmented BMI was not described in other Turkish reports. In the first one, the Authors reported an increase in both CPP cases and in rapidly progressive puberty during the first months of the pandemic, with earlier age at the diagnosis and at the beginning of treatment. The role of environmental factors was considered; BMI SDS (standard deviation score), however, was similar between the two groups . A diffeData from a tertiary level pediatric endocrinology center in Western India are consistent with data from other countries. The Authors compared a pre-lockdown and a lockdown group of boys and girls and showed a three-fold increase in the referrals for precocity. A higher increase was reported in girls. Mean weight and BMI were not significantly different between the pre-lockdown and the lockdown group. In most cases a rapidly progressive puberty was observed with a more advanced bone age .Analyzing data from the past two and a half years, some limitations arise. Most studies considered a girls only population. Male precocious puberty is less common and more often linked to organic causes. However, the role of environmental factors on male pubertal timing is still not studied and understood enough.Many studies had a retrospective nature and did not define how many patients had a confirmed SARS-CoV-2 infection thus preventing the Authors to assess a possible direct effect of COVID-19.While nutritional status and BMI are known factors that affect pubertal timing, not all the studies conducted during the COVID-19 outbreak reported a correlation between precocious puberty and an augmented BMI. Furthermore, the potential risk factors for precocious puberty taken into account in the studies were not homogeneous.On a side note, it has to be considered that some of the patients that received a diagnosis during the pandemic had pubertal onset prior to that time.5Puberty is characterized by a broad range of physical and psychological changes necessary for the achievement of full reproductive maturity. These changes parallel modifications within the CNS : in a peTaking a step back, GnRH neurons in the hypothalamus share common embryonic origin with the olfactory bulb (OB): they develop outside the brain, in the olfactory placode, and then migrate to the hypothalamus during embryogenesis. Then, when this process is complete, GnRH neurons project their axons to the median eminence and from there they release GnRH . Some auAlthough there is no current proof of neuroinvasiveness of SARS-CoV-2 and neuroimaging during the acute infection is rare due to disease control and safety concerns, there is evidence for abnormal brain findings in autopsies and viral spread from the olfactory epithelium to the OB has been demonstrated . HoweverInflammatory cytokines may play a crucial role also stimulating N-methyl-D-aspartate (NMDA) receptors which promote GnRH pulsatile secretion through inputs from neurotransmitters such as glutamate , 68, 69.6In the first months of the COVID-19 pandemic, when vaccines were still unavailable, containment measures such as lockdown and social distancing were effective in limiting viral transmission thus reducing case incidence and growth rate .On the other hand, these measures had negative effects on both physical and psychological health in adults as well as in children and adolescents , 72. ThePsychological factors such as fear and anxiety could have played a part in the outbreak of precocious puberty cases mediating an effect on CNS mediators such as NMDA  and glutThe main lifestyle changes regarded the reduction of both indoor and outdoor physical activities during the first pandemic wave , 81. ThiChildhood obesity has been associated with the secular trend of puberty anticipation, especially in girls, observed over the last few decades , 88. SomThere is also a positive correlation between hyperinsulinemia, alone or in conjunction with adiposity, insulin resistance and precocious puberty , 91. It Though playing an important role in the rise in precocious puberty diagnoses during the pandemic, weight gain and BMI have probably acted together with other environmental factors.As a result of the pandemic, screen time increased in children as well as in adults. In prepubertal age, particularly, screen time increased both due to online school lessons and to the reduction of social interactions and outdoor activities. Studies reported increases in both total and leisure screen time , with moAnother condition observed during the first months of the pandemic was a decline in vitamin D levels related to the reduction of time spent outdoors and of sun exposure because of the restrictions . VitaminIn the past decades, sleep changes as well as melatonin levels have been linked to pubertal development and, although studies on this topic are limited, data suggest that high melatonin levels in prepubertal age have an inhibitory effect on pubertal progression . In anotThe increase in the use of disposable items could have also contributed to a greater exposure to endocrine disrupting chemicals such as bisphenol A (BPA) and phthalate esters, used as additives in the making of plastic , 101. Se7During the first phase of the COVID-19 pandemic, many governments applied social distancing measures and strict lockdowns to prevent the spread of the disease. As a result, changes in children’s physical and psychological health were recorded. An interesting finding was an increase not only in precocious puberty cases, but also in pubertal progression rate.It is still not sure if SARS-CoV-2 infection can play a direct role in this event through direct OB inflammation, blood-brain barrier disruption or cytokine storm. There is instead more research on lockdown effects. Changes in lifestyle could likely have functioned as precocious puberty triggers. Changes in BMI, glucose and insulin metabolism, screen time, sleep quality, vitamin D levels, endocrine disruptors are all factors potentially linked to sex hormones modifications and early onset of puberty as well as faster pubertal progression rate Table 2.To further validate these hypotheses and better explain the exact role of SARS-CoV-2 infection and of the pandemic, more results from specialized centers from all over the world are expected in the near future. In the last analysis, two remaining questions arise. The first one regards parents’ role, as, maybe, the increase in precocious puberty referrals could be linked to closer monitoring of children during the pandemic. Parents working from home may have spent more time with their children and this may have helped in identifying in advance pubertal changes. The second one is about the duration of this phenomenon and whether it will continue or not while we resume pre-pandemic habits.The authors confirm contribution to the paper as follows: SP and FC contributed to conception and design of the review. SP drafted the manuscript. FC edited and revised the manuscript. Both authors contributed to the article and approved the submitted version.


Drosophila midgut epithelium is derived from a group of stem cells called adult midgut precursors (AMPs) that are specified during the migration of the endoderm in early embryogenesis. AMPs are maintained and expanded in AMP nests that lie on the basal side of the larval midgut throughout the larval development. During metamorphosis, the larval midgut undergoes histolysis and programmed cell death, while the central cells in the AMP nests form the future adult midgut and the peripheral cells form the transient pupal midgut. Here we review what is known about how cells polarise in the embryonic, larval, pupal and adult midgut, and discuss the open questions about the mechanisms that control the changes in cell arrangements, cell shape and cell polarity during midgut development.The adult  Drosophila intestine is composed of several different cell types, including epithelial cells, muscle cells, neurons, and trachea cells , Laminins , Nidogen and Perlecan , except for the invaginations of the basal labyrinth, which do not appear to have any ECM in their lumens . IntegriPerlecan   can divide and differentiate into both ECs and ee cells in the adult midgut. The signals and mechanical cues that regulate ISC division and differentiation have been extensively characterised and have been summarised in many excellent reviews of this topic , Par-6, atypical protein kinase C (aPKC), Crumbs (Crb), Stardust (Sdt), Discs large (Dlg), Lethal (2) giant larvae (Lgl) or Scribble . Insteadpolarity .Drosophila midgut epithelium is derived from AMPs, which are specified during early embryogenesis and segregated from the cells of the larval and pupal midgut during development , the Glycipans Dally and Dally-like and Syndecan (Sdc) . Integrito AMPs, .Ecad/shg mutant embryos, it is therefore unclear whether their delamination and translocation is a passive cell-sorting event or an active migration process  expands ventrally and dorsally to form the circular muscle fibres and the endodermal layer follows this movement to form a closed chamber. Although the early specification of the endoderm into distinct PMEC, ICP, pLee and AMP cell types does not depend on interaction with the mesoderm, VM induces the further specification and development of future larval midgut epithelium after the midgut rudiments fuse, including the formation of the three midgut constrictions during stages 14–16 and the specification of the middle midgut region and proventriculus .Between stage 16 and 17, the future larval ECs change their morphology from short cuboidal cells to tall columnar cells and develop elaborate cellular junctions and an apical brush border . Smooth Drosophila embryonic midgut formation takes less than 9 h, between stage 10 when PMG starts EMT and stage 15 when the midgut migration finishes. Both EMT and MET happen gradually, whereas cell polarity changes dramatically and rapidly during this process. The apical polarity factor Crb disappears early on, the original apical-lateral junctions dissolve, giving rise to a group of mesenchymal migratory cells connected by limited spot AJs. These cells later re-polarise forming smooth SJs rather than pleated SJs at the apical/lateral side of the cell-cell junctions. Embryonic midgut development also demonstrates the importance of the sustained basal signalling from the mesoderm much like the polarisation of the EBs in the adult midgut epithelium. It is still unclear, however, what lies downstream of basal ECM and their receptors to induce epithelial polarisation, and whether apical extracellular LanA and apically localised αPS3/βν integrins plays any role in polarising the embryonic midgut epithelium. Past work has focused on the morphological development of the midgut and the genetic control of endoderm formation and differentiation  and Su(H)GBE > GFP/lacZ -positive peripheral cells, which elongate and surround the inner mass of small, round central cells. Peripheral cells are post-mitotic with bigger nuclei than the central cells. They function as a niche to maintain the stem-cell state of the central AMP cells until metamorphosis , the larval midgut shortens, bringing the scattered AMP nests together. The outer peripheral cells contact each other first and by 6h APF become squamous and join together to form a multi-layered sheet called the transient pupal midgut (tPMG). Central cells also change their shape, flattening longitudinally and expanding laterally, to form a continuous layer of presumptive adult midgut (AMG) in a process that is thought to be MET . At thisThe separation between larval midgut/tPMG and the AMG is essentially the delamination of larval midgut epithelial cells and the detachment between peripheral cells and central cells of the AMP nests. This results in the reorganisation of the tissue into three layers with spot AJ still present among them . The reoAMPs are specified at an early embryonic stage, delaminate apically but remain attached to the PMECs via spot AJs and stay in the apical lumen of the migrating midgut primordium. They then translocate across the newly formed epithelium at the end of midgut development and remain basally after the embryo hatches. The mesoderm is not involved in the AMP specification and delamination, whereas cell-cell adhesion is proposed to play an important role in both the delamination and translocation. These processes are accompanied by the migration and repolarisation of the midgut primordium to form the future larval midgut epithelium. The migration and repolarisation require secreted Laminins from both germ layers, LanW at the basal side from the mesoderm and LanA at the apical side from the endoderm. The LanW from the basal side interacts with integrins receptors to activate downstream signaling pathways that are proposed to provide the cue that polarises the midgut epithelium and induce further polarised trafficking. The smooth SJs and the apical brush border microvilli form as the last step of polarisation in the epithelium. The polarised membrane features in the embryonic midgut epithelium are different from the steady state adult midgut epithelium, where integrin signalling components are only found basally and the lateral cell-cell junctions are clearly separated into apical-lateral sSJs and basal-lateral AJs. However, similar transcription factors control adult ISC maintenance and differentiation and embryonic midgut morphogenesis . MoreoveDuring larval development, the AMPs expand, differentiate and form a nest containing peripheral cells and central cells. The peripheral cells are polarised to form sheath that surrounds and presumably isolates the central cells from the larval epithelial cells. It is not clear what type of cell-cell junctions form in the AMP nest and between the nest and larval epithelial cells. The peripheral cells later separate from the central cells to form the tPMG and delaminate with the larval epithelium at the start of pupation. By contrast, the central cells remain in contact with the basement membrane while adhering with each other to form the future AMG. Although both peripheral cells and central cells originate from AMPs, the peripheral cell-derived tPMG will develop pleated SJs instead of smooth SJs. During the separation and reorganisation, spot AJs are found connecting the larval midgut, tPMG and AMG. This raises the possibility that cell-cell adhesion dynamics regulate the separation. After the visceral muscle and ECM layer reform at the basal side, the central cells start to polarise. Little is known about how polarised domains form in the AMG, except that aPKC localises to the apical domain, Dlg and FasIII occupy the apical-lateral junction and Arm/Ecad are localised at the basal-lateral domain . Since bDrosophila midgut during embryonic, larval, pupal and adult development have paved the way for investigations into how cells are specified at each stage and how their polarity is controlled. Elucidating the roles of cell-cell interactions and signals from the ECM in the control of cell fate, cell shape and cell polarisation, will advance our understanding of how the gut epithelium develops and functions under healthy conditions, and how this is perturbed in diseased states such as cancer.In summary, studies on the behaviour of stem cells and the stem cell niche in the bioRxiv.org.By the time of submitting this review paper, these two research papers  are stil


According to the design of the experiment (DoE), multivariate analysis models were used to optimize the critical process parameters combined with multi-objective optimization.Amomi fructus but also reduces energy demands and CO2 emissions.Based on the optimized operating conditions, the MILT-HD method not only enhances the extraction efficiency from Based on the density functional theoretical (DFT) and molecular dynamics (MD) simulations, the mechanisms for ionic liquids (ILs) to improve the extraction efficiency of essential oil was comprehensively revealed.Amomi fructus (Latin) was used to explore the mechanism of ionic liquids (ILs) in improving the extraction efficiency of essential oils. Microwave assisted ionic liquid treatment followed by a hydro-distillation (MILT-HD) process for isolating Amomi fructus essential oil was optimized by multi-objective optimization. Under optimum operating conditions, the IL-assisted extraction method not only enhances extraction efficiency but also reduces energy demands and CO2 emissions. Since the hydrogen bond structure network of cellulose in the cell wall is an important reason for hindering diffusion of essential oils, the mechanism of ILs was explored by density functional theoretical (DFT) and molecular dynamics (MD) simulations. According to DFT calculations, ILs can facilitate the cleavage of cellulose chains and have strong non-covalent interactions with cellulose. Based on the MD simulations, the degree of destruction of the cellulose hydrogen bond structure was explored. According to the DFT and MD simulations, the ILs can significantly destroy cellulose structure, thereby promoting essential oil release from the plant. These results were confirmed by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). This work is conducive to better understand the MILT-HD process for isolating essential oil and comprehensively understand the mechanism of ILs in the extraction process.In this paper,  Amomi fructus, as one of the top four authentic Traditional Chinese Plant Medicines Cl mass concentration at a given irradiation power. It is likely to improve the dissolving capacity of the cellulose by increasing the mass concentration of ILs. It may be conducive to enhance the permeability of cell walls, resulting in a higher release of essential oils Cl and water were shown in Ecoul) and the Lennard-Jones potential (EL-J). Ecoul and EL-J refer to electrostatic interaction and van der Waals interaction between solvent and cellulose, respectively. The results are listed in To quantitatively characterize the interaction, the interaction energies of cellulose with [C4mim]Cl and water were calculated. The interaction energy is the sum of the Coulomb force  decreased apparently, which was in good agreement with the results of molecular dynamics simulation. It revealed that IL could destroy the network of hydrogen bonds between the carbohydrate hydroxyl protons Cl was shown to be an efficient ionic liquid used in the MILT-HD process. DoEs and MVA were used to comprehensively evaluate the process of extracting essential oils. Furthermore, the operating parameters determined by multi-objective optimization accelerated the isolation of essential oil from However, there are still some limitations in this study. In this study, only the cellulose model was used for molecular dynamics and DFT studies, and other chemical components in the cell wall were ignored. In the future, a cell wall model containing lignin and other chemical components can be constructed for further research.


The main aim of the present research is to analyze the predictive value of individual characteristics such as online self-efficacy, adaptability to uncertainty, and sources of stress during online learning on learning engagement. We also aimed to highlight if these relationships could be mediated by the online self-regulated learning strategies, during the COVID-19 pandemic. The participants were 529 university students and the design was cross-sectional. The results showed significant associations of the sources of stress in online learning with self-efficacy, leaning engagement and self-regulated learning strategies. Self-regulated strategies—task strategies and goal setting represent mediators of stressors perceived by the students under the conditions of the sudden shift to online activity and online learning engagement. The most relevant self-regulation strategies seemed to be goal setting and task strategies, which confirm the need for a clear structure of learning in the context of online activities. The implications of this study reside in the increased awareness regarding how learning engagement in online learning can be predicted by individual characteristics. In the context of the COVID-19 pandemic, which forced much if not all academic experiences to move to a virtual environment, exploring the possible antecedents of students’ engagement in online learning became a focal point of attention for teachers and researchers. The sudden change in the learning environment has differently influenced the way students engage in learning. For some of them, change was perceived as a challenge, getting involved in academic tasks by showing willingness to invest effort, through concentration and perseverance, even in difficult conditions . As we acknowledge that it does tend to show particular features in an online setting, learning engagement remains a strong factor in predicting academic performance , positivLearning engagement is a key piece in trying to understand students’ academic behavior. The growing research interest surrounding learning engagement is well justified. As As core part of the overall academic experience, learning engagement can be considered students’ willingness and behavioral commitment to participate in learning activities related to their academic role. Therefore, commitment to educational goals , willingStudent engagement refers to behavioral , emotional (interactions with teachers and classmates and a sense of belonging) and cognitive dimensions, including self-regulated learning . KnowingWell-designed and structured online content and activities enhance student engagement in the learning process . In a stOnline self-efficacy will positively predict learning engagement (H1)  .Discussing predictive relations between relevant individual variables and learning engagement, for the specific purpose of our research, we selected the self-concept  and learning adaptability. Around the concept of individual adaptability, In our present study, the I-ADAPT theory was used to explain the prediction value of the individual adaptability on learning engagement and also support our hypothesis regarding the mediating action of self-regulation  on the effects of learning characteristics and sources of stress in relation to learning engagement.Learning adaptability will positively predict learning engagement (H2).Adaptability was investigated in many recent articles. Higher levels of academic adaptability are associated with lower levels of burnout and higher levels of learning engagement and academic performance . StudiesThe COVID-19 pandemic brought many challenges for students also on the academic dimension of their lives, university life has become more stressful than usual for many students . PreviouSources of stress will negatively predict learning engagement (H3).The transition from in-person to remote learning brought many challenges, such as the requirement to move off campus with little notice, inadequate learning spaces for learning while moving to their homes, technological difficulties, health- or economic-concerns, etc. . We expeSelf-regulated learning (SRL) can be seen as a form of intentional learning in which the learner has control over the process, steering, and directing cognitive and motivation processes to achieve the learning goal . In thisThe relation between academic achievement and self-regulated learning is also discussed by The SRL model that Building upon the understanding that self-regulation in learning is associated with academic performance, it is noted that self-regulation is contextualized, meaning that the specific skills and strategies employed by the student are highly dependent on the learning context . TherefoSelf-regulated learning strategies will mediate the effects of learning characteristics and sources of stress on learning engagement (H4).The shift to the remote emergency learning context generated many academic stressors that students had to manage. In order to maintain their involvement, they activated their self-regulated learning strategies which could act as mediators between the personal characteristics , academic stressors and learning engagement. More specifically, while online self-efficacy and adaptability will sustain the efficient use of self-regulated learning strategies which in turn, will lead to a higher learning engagement, the sources as stress will act as triggers for enabling SRL strategies as mechanism to cope with the stress in order to maintain higher levels of learning engagement. Therefore, we proposed the following hypotheses: age = 23.76), attending various study domains  from several universities in Romania, for different educational levels: first year students (N = 52.4%), second (N = 27%), third and fourth year of study (N = 20.6%).The participants were 529 university students , the instrument which measures students’ perceived sources of stress associated with online learning. The 28 items are grouped into 6 dimensions: Inadequacy of teaching methods and teaching style—13 items (α = 0.92), Lack of social support—4 items (α = 0.81), Technical difficulties—3 items (α = 0.73), Role conflict—2 items (α = 0.60), Time constraints—2 items (α = 0.77), Diversity of techniques—2 items (α = 0.75), Inflexibility—2 items (α = 0.50). The measuring scale is a 5-point Likert scale . Cronbach’s Alpha for the global SSOLS is 0.93. The Inflexibility scale was not included in the study given the low Cronbach’s Alpha value.Self-regulated learning strategies were measured with the Online Self-Regulated Strategies Scale (OSLQ) . AccordiLearning engagement was measured with the UWES Learning Engagement Scale . UWES asA cross-sectional design was used. The previously described instruments were administered online during the second academic semester (May–June 2020), as self-report measures.Our study followed the principles of the Declaration of Helsinki regarding ethical aspects on human subjects. We did not collect any data that could lead to the identification of the participants. The participants gave their informed consent, while the participation at the study was voluntary and no incentives were offered. The study was reviewed and approved by the Council of the Faculty of Psychology and Education Sciences, Transilvania University of Brasov, No 87/5.05.2020.The normality assumption was met for all the numeric variables. The statistical design followed the cross-sectional research guidelines. Several significant associations between variables  and the The Pearson coefficient correlations showed significant associations of the sources of stress in online learning with self-efficacy, leaning engagement, and self-regulated learning strategies .Including SRL strategies as mediators between the sources of stress and engagement, lead to a significant prediction. The model explained 54% of the total variance of learning engagement. The total variances explained for the self-regulated learning strategies were the following: Goal setting 49%, Environmental structuring 32%, Task strategies 34%, Time management strategies 35%, Help seeking 6%, and Self-evaluation 26%.The mediation model showed several significant direct and indirect effects of personal factors and sources of stress on learning engagement, confirming the hypothesis of a partial mediation . The mosThe results of the study must be interpreted in the context in which at the beginning of the COVID-19 pandemic, universities from Romania decided unanimously to move didactic activities synchronously online and the use of ZOOM, Google Meet, Teams, and Skype communication platforms type, as well as the online educational platform Moodle type. The students participating in the study are enrolled in programs of study developed traditionally on-site, their experience being limited in asynchronous or hybrid online learning. In the context of COVID-19 pandemic, online learning engagement becomes an indicator of educational quality and a predictor of academic performance and of the dropout rate .The study analyses the relation between individual characteristics  and sources of stress and learning engagement in the online context through the forced transfer to online learning due to COVID-19 pandemic, as well as the role of online self-regulated learning strategies. According to Online self-efficacy reflects students’ perception to use technical skills in order to achieve academic tasks as being a good predictor of learning engagement . The resThe positive association of online self- efficacy with learning engagement can be explained through the mechanism of the perceived behavioral control—a construct related to the individual’s perception of the availability of knowledge, resources, and opportunities required to perform the specific behavior ; the intThe results of the study reveal positive associations between online self-efficacy and SRL strategies in the context of online learning, advocating the idea that self- efficacy promotes SRL behaviors , such asIn our study, students filled out the research instruments at the beginning of their online learning experience caused by the pandemic. Given the novelty of the context (students did not experience online learning before) and based on that, from a SRL point of view, they were entering the first phase of the self-regulating process, as described by The speed and amplitude of the transition to online learning highlighted students’ adaptability to the new learning environment, this being an essential characteristic which allowed the use of new instruments and learning methods which therefore enabled online learning engagement. Online learning environment used by students during the COVID-19 pandemic can be described in terms of novelty, uncertainty and variability by also autonomy, which represented a real challenge for them . The onlThe results of our study highlights a direct positive relation between students’ capacity for adaptability and online learning engagement , and alsThe study demonstrates that adaptability represents a personal key resource , a facilThe whole context determined by COVID-19 pandemic, characterized by insecurity and unpredictability represents a potential source of stress . The sudAnother objective of our study proposes to identify which sources of stress associated to online learning are predictors of online learning engagement and to which extent self-regulated learning strategies mediate the relation. Between the measured sources of stress and online learning engagement we identified significant negative relations with different levels of intensity. Between sources of stress associated to the use of technical tools and online learning engagement significant negative relations have been measured, but are of low intensity. The category of technological difficulties refers to the constraints caused by the infrastructure offered by universities and its functioning, the devices used by students, the limited/difficult access to internet sources and the category of diversity of techniques refers to simultaneous use of different tools specific to digital learning . Technical difficulties, respectively, diversity of techniques represent sources of stress which can interfere with online learning activity, through a rise in the number of disruptions, which may generate a rupture in the cognitive focus, cognitive interferences, setbacks in processing information and in increasing effort, preventing thereby the progress in learning tasks . Thus, tInstruction environments, respectively, online learning environments are considered instruments, which, designed efficiently, offer numerous gains both in teaching and in learning. Students have the possibility to engage actively in learning, reflecting critically upon the information, which facilitate the acquisition of knowledge and metacognitive abilities, while teachers have the possibility to address to a large community of students and to transform teacher-centered passive instruction, to active instruction, student-oriented, through reconsidering the instructional design and the teaching style. The sudden transition to online instructional environment obliged teachers to adapt their design and teaching style rapidly to the virtual environment instructional activity.Significant relative relations have been found between time constraints and online learning engagement. Research on time constraints in online learning are parochial. The perception of time constraints represents a source of stress, which affects learning activity, task engagement, and task performance. Students who perceive a lower pressure of time have are lucky enough to be self-determined in order to engage in learning tasks. The results of our study point out a significant negative relation between inadequacy of teaching methods and teaching styles and online learning engagement, relation supported by current studies developed in similar contexts . Among tThe significantly negative relation, proved by the study between the lack of social support and online learning engagement is also supported by other studies carried out in the online context . Social Role conflict appears as an effect of the tensions created between personal life and professional life. Role conflict occurs when an individual perceives incompatible time, strain, or behavior-based demands between work and non-work roles. Role conflict appears as a source of stress associated to learning engagement. In the specialty literature the role conflict associated to online learning context is insufficiently studied as compared to working context. The studies showed a significant positive association between distance working done at home and work-to-family conflict. Difficulty separating work and family spheres and fully unplugging from work can increase role conflict for teleworkers . Online Self-regulated strategies—task strategies and goal setting represent mediators of stressors perceived by the students under the conditions of the sudden transition to online activity and online learning engagement. Task strategies represents the regulating mechanisms which diminish the negative effect of the analyzed sources of stress—inadequacy of teaching methods and teaching styles, lack of social support, technical difficulties, role conflict, time constraint, and diversity of techniques), and goal setting intervenes additionally diminishing the effect time constraint over online learning engagement. The regulation of the learning environment plays an important role in students’ learning , thus inThe findings of this study should be seen in light of some limitations. A first limitation refers to the low Cronbach’s Alpha values for some of the Sources of Stress in Online Learning Scale, specifically for the Role conflict scale, a possible explanation being the small number of items included in this scale. We also dropped the Inflexibility scale which had a low reliability. Further studies should investigate alternative factor structures of the instrument and focus also on other psychometric properties such as predictive validity. A second limitation concerns the use of self-report measures which imply the possibility of biased answers because of the participants’ report on their own experiences and the cross-sectional design  which do not allow to draw causal conclusions. A follow-up study as a future research direction could offer a dynamic view on the evolution of students; perceptions, use of self-regulation strategies and management of the sources of stress and their impact on learning engagement.A third limit refers to the convenience sampling that was used in this study. Participants were not randomly selected to take part in the study, thus the sample may lack proper representation. Given the nature of the psychosocial context at the time of the study , getting students to thoroughly participate in our research was not as easy as to make us comfortable in a position of probabilistically selection. However, we do acknowledge the possible biased results stemming from not reaching participants based on balanced representation but based on voluntary choice. We are also aware of the limitation of not being able to generalize our present results beyond the studied sample.The implications of this study reside in the increased awareness regarding how learning engagement in online learning can be predicted by individual characteristics such as online self-efficacy and learning adaptability, with statistically discernible effects of several sources of stress such as inadequacy of teaching methods and styles, lack of social support, and role conflict, and the mediating influence of self-regulated strategies such as Task strategies, Goal setting on the relationship between individual characteristics and learning engagement. Therefore, from a pedagogical standpoint, it can prove useful for an educator to consider using teaching strategies and designing learning opportunities that foster specific learning practices in students enrolled in university programs, learning practices that relate to setting feasible yet challenging learning goals and striving toward them, and systematically self-assigning and performing learning tasks pertaining to their learning goals. From a psychological point of view, studying the relationship between various perceived sources of stress related to the academic experience, such as lack of social support or role conflict, and learning related behaviors (ex. Task strategies) can be a step forward in better understanding the coping mechanisms that young adults—with different levels of self-efficacy and learning adaptability- employ in dealing with the specific challenges they face.The main aim of the present research is to analyze the predictive value of individual characteristics such as online self-efficacy, adaptability to uncertainty and sources of stress during online learning on learning engagement. We also aimed to highlight if these relationships could be mediated by the online self-regulated learning strategies, during the COVID-19 pandemic.The use of efficient self-regulated learning strategies, namely goal setting, and task strategies could lead to a higher effect of self-efficacy and individual adaptability on engagement. Furthermore, the use of efficient self-regulated strategies could reduce the negative effects of sources of stress on learning engagement. The most important self-regulation strategies seemed to be goal setting and task strategies, which confirm the need for a clear structure of learning in the context of online activities. Our study explores possible antecedents of online learning engagement, stress factors and self-regulated mechanisms in the context of the sudden change of the learning environment during COVID-19 pandemic.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Council of the Faculty of Psychology and Education Sciences, Transilvania University of Brasov, No 87/5.05.2020. The patients/participants provided their written informed consent to participate in this study.IRT and DVN developed the study concept. MMS conducted the data collection and drafted the manuscript. A-MC performed the data analysis. All authors took part in result interpretation, reviewed, edited several versions of the manuscript, provided critical revisions, and approved the final version of the manuscript for submission.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.


With the aim to offer personalized treatment regimens to patients undergoing PCI, much effort has been devoted in the last decade to improve the identification of patients at increased risk of bleeding complications. Several clinical scores have been developed and validated in large populations of patients with coronary artery disease (CAD) and are currently recommended by guidelines to evaluate bleeding risk and individualize the type and duration of antithrombotic therapy after PCI. In clinical practice, these risk scores are conventionally computed at the time of PCI using baseline features and risk factors. Yet, bleeding risk is dynamic and can change over time after PCI, since patients can worsen or improve their clinical status and accumulate comorbidities. Indeed, evidence now exists that the estimated risk of bleeding after PCI can change over time. This concept is relevant, as the inappropriate estimation of bleeding risk, either at the time of revascularization or subsequent follow-up visits, might lead to erroneous therapeutic management. Serial evaluation and recalculation of bleeding risk scores during follow-up can be important in clinical practice to improve the identification of patients at higher risk of bleeding while on DAPT after PCI.Dual antiplatelet therapy (DAPT) with aspirin and a P2Y Sinceications . With thications . With thications .The aim of this review is to sum up current evidence on DAPT in patients with ACS and CCS, provide a comprehensive overview of available bleeding risk scores and algorithms, and highlight recent findings that have the potential to improve the identification of patients at higher risk of bleeding while on DAPT after PCI.12 inhibitor. The choice of a P2Y12 inhibitor depends on the clinical presentation  and both the thrombotic and bleeding risks of any given patient. Importantly, evidence now exists that short- and long-term adverse events in patients treated with DAPT are closely associated with both bleeding and ischemic events [The standard of care for treating patients undergoing PCI is DAPT ,2. Afterc events .The current recommendations of the European Society of Cardiology (ESC) regarding DAPT are shown in 12 inhibitor monotherapy and stop aspirin after 1 to 3 months of DAPT.Similarly to the ESC recommendations, the latest clinical guideline from the American College of Cardiology and American Heart Association provides an evidence-based approach to the treatment of patients undergoing coronary revascularization . Of noteStratification of the individual risks of ischemia and bleeding is of paramount importance for a personalized approach to DAPT after PCI . To thisIdentification of patients with a high bleeding risk that might benefit from a shorter duration of DAPT can now be achieved by using standardized risk scores or algorithms. Bleeding predictors primarily consider the patient’s characteristics, the complexity of the procedure and the potency of the antithrombotic regimen .For years, no standardized tool has been available to predict long-term bleeding risk for post-PCI patients on DAPT. Among the existing models that have been tested and validated, all were originally developed for alternative purposes and applied to DAPT patients empirically ,16,17,18http://www.precisedaptscore.com (accessed on 17 June 2022)) was incorporated into the 2017 ESC focused update on DAPT in CAD patients [In the last decade, dedicated risk scores have been developed and validated to address the proper identification of bleeding risk in post-PCI patients  19,20,2,24,25. Ipatients  and has patients ,28 or inpatients ,30. HoweDespite several bleeding prediction scores being currently available and recommended by international guidelines to characterize patients undergoing PCI, the identification of patients at high bleeding risk remains challenging in clinical practice . Indeed,These observations are in keeping with the results of the RE-SCORE study, a multicenter registry that sought to investigate the possible prognostic implications of changes in the PRECISE-DAPT score at long-term follow-up ,34. The Overall, the results of the RE-SCORE registry showed that the external validation of the PRECISE-DAPT score calculated at baseline was barely satisfactory, whereas the performance of the score improved when it was reassessed regularly. These findings support the concept that the risk of bleeding is not static and that the PRECISE-DAPT score can do well in the real world only if the score is recalculated over follow-up. Apart from age and previous spontaneous bleeding, three determinants of the score can fluctuate with time. Creatinine clearance can decrease after PCI, as renal function might progressively deteriorate as a consequence of acute kidney injury , post-prThe findings from the RE-SCORE investigators underscore the concept that patients who are given DAPT after PCI are potentially at high risk and therefore require attention and close surveillance over time by clinical and laboratory reassessment . To thisSeveral risk scores are currently recommended by the guidelines to evaluate bleeding risk and individualize the type and duration of antithrombotic therapy after PCI. In clinical practice, these risk scores are generally assessed at the time of PCI using baseline features and risk factors. Yet, bleeding risk is dynamic and can significantly change over time. This concept is relevant, as the inappropriate estimation of bleeding risk, either at the time of revascularization or subsequent follow-up visits, could lead to erroneous therapeutic management. With this respect, recent findings shed light on a novel predictive strategy that might enhance the proper identification of patients at higher risk of bleeding. The evidence that bleeding scores are dynamic underscores that the estimated risk of bleeding after PCI can change with time and should therefore be regularly reassessed. Frequent evaluation and recalculation of bleeding scores should be implemented in clinical practice to potentially improve the identification of high bleeding patients after PCI.


Patients with metastatic colorectal cancer (mCRC) frequently experience epidermal growth factor inhibitors (EGFRI)-induced skin side effects. Antibiotic treatment with doxycycline is often required in order to manage the skin and mucosal toxicity. Since these patients already have significant gut dysbiosis, the long-term antibiotic treatment may destabilize their gut microbiome.ObjectivesThe assessment of intestinal dysbiosis in patients undergoing treatment with EGFRI, who require antibiotic treatment with doxycycline in order to manage adverse skin effects.MethodsWe conducted a prospective pilot study between 2020 and 2021 involving 10 patients with mCRC. These patients were undergoing treatment with EGFRI and required either short-term or long-term treatment with doxycycline in order to manage skin toxicity.ResultsThe patients with mCRC who were treated with doxycycline for 8 weeks showed overexpression of Escherichia coli, Candida, and Geotrichum species compared to the patients who only received doxycycline treatment for two weeks.ConclusionsThe elevated levels of Escherichia coli and Candida species in the patients who received doxycycline for eight weeks compared to the patients who received the treatment for two weeks could provide a starting point for the development of a standardized guideline regarding the use of pre-active or reactive antibiotic treatment. We also highlight the importance of analyzing the intestinal microbiome of these patients. The identification of overexpressed species, as well as the deficiency of certain protective species, could guide the administration of probiotics to cover and repair the affected intestinal flora. The emergence of molecularly targeted therapies, in particular epidermal growth factor inhibitors (EGFRI), has been an important upgrade in oncological treatments. Although they are characterized by unique adverse effects, being dominated by the occurrence of cutaneous toxicity, particularly the papulopustular rash, they have much lower systemic adverse effects compared to classical chemotherapy .Tetracycline treatment is usually chosen to treat post-EGFRI papulopustular rashes -3. The lHowever, since colorectal cancer (CRC) patients already have significant gut dysbiosis, the use of long-term antibiotic treatments may further destabilize their gut flora .The human microbiome is in a permanent symbiotic relationship with the host, playing an important role in the maintenance of the metabolic homeostasis, human physiology, digestion, and detoxification processes, as well as in the development of the immune system .Intestinal dysbiosis occurs as a consequence of the qualitative and quantitative changes in the gut microbiota -8. The cThe disturbances of the microbiota may be promoters in the development of colorectal cancer; however, the ecosystem imbalances may also be a consequence of the anticancer therapy .Given the important role played by the Fusobacterium nucleatum in the physiology and pathology of colorectal cancer, the use of antibiotics whose spectrum targets this bacterial species may benefit CRC patients significantly. Although there are no official guidelines for treating Fusobacterium nucleatum infections, according to the Sanford 2020 guidelines, the oral use of tetracycline-type bacteriostatic agents is extremely useful in the treatment of chronic Fusobacterium spp infection. Therefore, the use of doxycycline in patients under EGFRI treatment has a dual role: management of therapy-induced cutaneous rash and action against Fusobacterium .The aim of this study is to assess the impact of the doxycycline-based antibiotic treatment on the microbiome of colorectal cancer patients undergoing EGFRI therapy. We also analyzed and compared, for the first time, the gut dysbiosis between two groups of colorectal cancer patients, who received short- or long-term doxycycline treatment, respectively.Case selectionThe study was conducted in agreement with the local ethics committee and in accordance with the Helsinki Declaration. This study included 10 colorectal cancer patients from St. Nectarie Medical Centre undergoing EGFRI treatment (Cetuximab and panitumumab), as well as doxycycline-based treatment, for the management of EGFRI-induced skin toxicity between 2021-2022.The 10 patients were divided into two study groups; patients from the first group received doxycycline treatment for less than two weeks while patients from the second group received doxycycline treatment for longer periods (between six and eight weeks).In this study group of 10 patients, we identified dermatological side effects such as papulopustular rashes Figures  more fr, prurituFeces cultureThe fecal samples (minimum 1 g) were collected in a container without a transport medium and transported to the laboratory at 2-8° C in order to be cultured. The samples were secured and transported to Germany  for gut flora analysis by fecal culture and identification of the dysbiosis degree.Only patients who had not received antibiotic or probiotic treatment in the last 14 days prior to collection and patients who had no dietary changes were included in the study.Following the laboratory analysis, we performed cultures for putrefactive microbiome flora , protective flora , and fungi .In order to determine intestinal dysbiosis degree in the two groups under study, the analysis of the intestinal microbiome consisted of the analysis of the putrefactive flora, the protective flora, and the identification of the presence or absence of fungi in the feces.In terms of the pH values of feces, there were no significant changes between patients receiving short (less than two weeks) Figure  and patiGroup 1: patients undergoing EGFRI treatment and doxycycline for less than two weeksPatients who received doxycycline treatment for less than two weeks showed changes in the putrefactive flora for only two bacterial species: Escherichia coli in one out of five patients and Clostridium species in three out of 12 patients Figure .Regarding the protective flora of patients who underwent doxycycline treatment for less than two weeks, we recorded changes in Bacteroides species in two out of five patients, Bifidobacterium species in four out of five patients Figure , LactobaRegarding the fungal composition, only one patient who received doxycycline treatment for less than two weeks showed changes in Geotrichum species Figure .Group 2: patients undergoing EGFRI treatment and doxycycline for more than eight weeksWe observed changes in the putrefactive flora of patients undergoing EGFRI treatment and doxycycline for more than eight weeks. All patients showed overexpression of E. coli Figure , one outRegarding the composition of the protective flora, patients who received doxycycline treatment for more than eight weeks experienced value changes of Bacteroides species in one out of five patients, Bifidobacterium species in one out of five patients Figure , LactobaIn terms of the fungal composition, we recorded overexpression of Candida albicans species in one out of five patients Figure , other CThe current study aimed to identify the totality of compositional and diversity changes in the gut microbiome, as well as the assessment of dysbiosis, following antibiotic (doxycycline) treatment in patients with colorectal cancer and skin toxicity undergoing EGFRI treatment.At the moment, there is no specific guideline for the management of dermatological reactions occurring in patients undergoing EGFRI treatment. The use of antibiotic treatment is either prophylactic or reactive.Patients with CRC often present imbalances in the intestinal flora. In addition to genetic factors, environmental factors, such as a high-fat diet, fiber deficiency, red meat consumption, lack of physical activity, infectious agents, surgery, or antibiotics, are involved in the etiology of CRC. The presence of these environmental factors ultimately leads to changes in the gut microbiome .The use of antibiotic treatment causes many changes in the intestinal flora. Most commonly, these changes consist of a decrease in the beneficial, protective bacteria accompanied by overexpression of the pathogenic or putrefactive flora .In this study, we highlighted the variations in the intestinal bacterial and fungal flora found in cancer patients undergoing EGFRI treatment and doxycycline for short or long periods of time. All patients treated with doxycycline for eight weeks showed increases in the Escherichia coli (E. coli) values.The occurrence of the inflammatory processes in the gastrointestinal tract as a result of infections, antibiotic treatment, or dietary changes leads to intestinal dysbiosis characterized mainly by an increase in enterobacterial species .The Enterobacteriaceae group consists of species such as E. coli, Enterobacter, Klebsiella, Proteus, Citrobacter, Pseudomonas, Serratia, Morganella, Hafnia, and Yersinia. High values associated with these species in the environment may justify their presence in the feces of healthy people following food consumption. However, elevated values of these species (more than 10Ù5 KBE/g feces) indicate disturbances in the intestinal flora.Increased levels of Escherichia coli can result in the release of considerable amounts of metabolites. When an increase in the carbohydrate release occurs, flatulence and meteorism are observed. In case of increased protein substrate release, E. coli causes changes in the form of amines  and ammonia, which have toxic effects on the liver.Overexpression of Escherichia coli was also recorded in the colorectal cancer patients who were treated with doxycycline in this study. All patients undergoing long-term (eight weeks) doxycycline treatment showed dysbiosis phenomena of the putrefactive flora. This event is characterized by the overexpression of E. coli and was correlated to the long-term use of doxycycline since only one patient from the short-term (two weeks) group showed a slight increase in E. coli levels.Patients with a longer duration of the antibiotic treatment (group 2) also experienced overexpression of fungal populations. More specifically, we recorded increased levels of Candida albicans, other Candida species, and Geotrichum species in four out of five patients. Similar to the E. coli analyses, this event was correlated to the prolonged use of the antibiotic, with only one patient from the short-term doxycycline treatment group showing overexpression of the Geotrichum species.The protective flora is made up of a high proportion of bacterial species such as Bifidobacterium, Bacteroides, Lactobacillus, and Enterococcus. Most of the bacterial species belonging to the protective flora are included in the composition of probiotics, underlining their importance in maintaining good health .The presence of normal values of Bifidobacterium species has been associated with the occurrence of T-cell-mediated anti-tumor response. Among the subspecies with these properties, Bifidobacterium adolescentis, Bifidobacterium breve, and Bifidobacterium longum, which are known to have a strong effect on immunity, have been identified .Numerous studies highlight the ability of Bifidobacterium species to modulate and stimulate the host immune response, including both the immune defense and the acquired immunity .Alongside Bifidobacterium, certain Bacteroides species, known as "anti-cancer probiotics," have now been identified, and they form the bulk of the colonic flora. Bacteroides species have a beneficial, protective effect by metabolizing oligosaccharides and polysaccharides, with the purpose of producing vitamins and nutrients needed by the host .In adults, the number of Bacteroides species can also be influenced by environmental factors, antibiotic treatment, and the host’s diet .Another extremely important component of the protective flora is Lactobacillus, so the low levels of Lactobacillus species increase the risk of putrefactive bacteria and external agents overgrowth. Lactobacilli also play an important role in acidifying the intestinal flora .Another marker of intestinal dysbiosis is the decreased levels of Enterococcus, with normal values of Enterococcus species representing a marker for assessing the stability of the protective flora. Its deficiency increases the external risk of the development of infections and colonization with pathogens -21.Both groups of patients included in this study showed acute dysbiosis of the species defining the protective flora.In the group of patients with long-term antibiotics treatment (8 weeks), a greater deficit of Enterococcus species was observed compared to patients who underwent antibiotic treatment for only two weeks.The main limitation of this study is the relatively small number of patients. Another limiting factor is represented by the lack of microbiome analysis before the start of the antibiotic treatment, which would have enabled us to dynamically highlight the totality of changes in the gastrointestinal microbiome.This analysis is a personal contribution to the study of the gut microbiota in colorectal cancer patients undergoing EGFRI treatment and experiencing skin toxicity. We identified the bacterial and fungal species that cause intestinal dysbiosis and highlighted dysbiosis following treatment with doxycycline, which is widely used in these patients. Understanding these data will facilitate the choice of the prophylactic or reactive treatment with doxycycline and therefore the duration of the antibiotic treatment in patients with skin toxicity undergoing EGFRI treatment.The use of the antibiotic treatment has profound and sometimes persistent effects on the gut microbiota by depleting beneficial species and raising the levels of pathogenic species, especially in colorectal cancer patients with already altered gut flora.The overexpression of Escherichia coli and candida species in patients who received doxycycline for a longer period of time compared to patients who received it for two weeks could represent the basis for the development of a standardized guideline for the use of pre-reactive or reactive antibiotic treatment. We also highlight the importance of analyzing the intestinal microbiome of these patients. The identification of overexpressed species, as well as of the deficiency of specific protective species could guide the administration of probiotics to cover and repair the affected gut microbiota.