COVID-19 has dramatically affected the mental health and work environment of the educational sector. Our primary aim was to investigate preschool teachers’ psychological distress and work engagement during the COVID-19 outbreak, while examining the possible protective role of participating in a mindfulness-based intervention geared to foster compassion  and emotion regulation. The prevalence of emotional distress, work engagement, and COVID-19 concerns were evaluated in 165 preschool teachers in the early stages of the COVID-19 outbreak in Israel through questionnaires. The findings showed that preschool teachers experienced increased emotional distress. Teachers who had participated in the C2C-IT intervention six months before the pandemic outbreak (N = 41) reported lower emotional distress, higher use of adaptive emotion regulation strategies, and higher work engagement, compared to their counterparts that had not participated in the intervention (N = 124). Emotion regulation strategies mediated the link between participating in CTC-IT intervention and emotional distress and work engagement. Teaching is a highly demanding occupation, especially during a pandemic, thus making it important to invest resources in empowering this population. The findings here suggest that the implementation of a mindfulness-based intervention during the school year can enhance teachers’ well-being, even during stressful events such as the COVID-19 pandemic. Starting in December 2019, coronavirus (COVID-19) spread rapidly and turned into a global pandemic, disrupting the functioning of societies and posing a mental health threat of great magnitude around the world, for both individuals and communities . Even beThese stressors have had a detrimental effect on teachers’ mental health . For exaMindfulness refers to the capacity to devote one’s complete attention to the experiences occurring in the present moment, in a nonjudgmental accepting way . There iContemporary mindfulness interventions often include components of compassion. Compassion defined as sensitivity to suffering in the self and others, with a commitment to try to alleviate it . Some thSeveral mechanisms are considered to underlie the salutary effects of mindfulness, a mechanism that has consistently proposed as a central process is emotion regulation (for a review see ). EmotioMindfulness can also lead to more adaptive ER in a variety of ways. Generally speaking, mindfulness triggers processes that optimize psychological functioning, such as the ability to manage emotions . This isIn recent years, MBIs have been adapted and applied in the teaching context . For teaThe Call2Care-Israel for Teachers (C2C-IT) is a mindfulness and compassion-based intervention geared to foster self and other compassion/care . This inThe main objective of the present study was to investigate preschool teachers’ psychological distress and work engagement during the early stages of the COVID-19 outbreak, while examining the possible protective role of participating in the C2C-IT intervention and the mediating role of ER strategies. To the best of our knowledge, no study has examined the relationship between participating in a mindfulness-compassion-based intervention and emotional distress and work engagement in times of crisis. Hence, the research aims were as follows: (1) to evaluate the extent of preschool teachers’ exposure to the COVID-19 pandemic during the first quarantine in Israel (May 2020) and to evaluate the extent to which preschool teachers experienced COVID-19 related concerns, emotional distress, work engagement during the first quarantine in Israel. (2) To explore the differences between teachers who participated in the C2C-IT intervention before the COVID-19 outbreak and teachers who did not participate in the intervention, on measures of emotional distress, work engagement and ER tendencies during the pandemic. (3) To examine whether ER strategies mediated the association between participation in the CTC-IT intervention and emotional distress and work engagement. The hypotheses were as follows. (1) Preschool teachers will report increased emotional distress symptoms and reduced work engagement. (2) Preschool teachers who participate in the intervention will report higher work engagement, enhanced use of ER strategies, and fewer symptoms of emotional distress, compared to teachers who did not participate in the intervention program. (3) Based on the Coffey et al.  model, ECross-sectional data collected online through Google forms, during April–May 2020. At the time, Israel experienced a surge in the number of daily new cases. The ongoing rise in the numbers of diagnosed cases affected the public’s estimations of the severity and controllability of the virus  and led The C2C-IT intervention first administered in May–June 2019, 6 months before the pandemic. The intervention is composed of 20 sessions that address three modes of care: receiving care, self-care, and extending care . We further asked participants to indicate the last five digits of their ID, for purposes of cross-referencing with participants in the C2C-IT intervention.COVID-19 concerns questionnaire. Aimed at assessing the level of concerns arising from the COVID-19 pandemic . Participants responded to this questionnaire on a 5-point scale ranging from 1  to 5 (very much). This questionnaire was based on a measure of teachers’ stress about the COVID-19 outbreak administered by  than the control group. The C2C-IT group, as compared to the control group, reported lower emotional distress , and higher levels of work engagement . The groups also differed in their habitual use of all emotion regulation strategies. Specifically, the C2C-IT group used more reappraisal  and acceptance , and engaged to a lesser extent in rumination  and catastrophizing  compared to the control group.To evaluate preschool teachers’ exposure to the COVID-19 pandemic, levels of COVID-19 related concerns, ER tendencies, emotional distress, and work engagement, as well as to explore the differences between teachers who participated in the C2C-IT intervention before the COVID-19 outbreak and teachers who did not participate in the intervention on these measures, independent sample T-tests and chi-square analyses conducted . The thrp < 0.05, 95% CI ), catastrophizing . However, the indirect effect of group type and emotional distress via acceptance  and rumination  was not significant; see To determine whether ER strategies mediated the relationship between participation in the C2C-IT course and emotional distress, mediation models were conducted for each strategy separately see a. Becausp < 0.05, 95% CI ), catastrophizing , and acceptance  whereas the mediation effect of rumination was not significant .Mediation models conducted for each strategy separately to test the indirect effects of the C2C-IT intervention on work engagement through ER strategy see b. The toThe present study had three aims including: (1) Investigating preschool teachers’ psychological distress and work engagement during the early stages of the COVID-19 outbreak; (2) Explore the possible protective role of participating in the C2C-IT intervention on preschool teachers’ mental health and work engagement; (3) Examine whether ER strategies mediate the relationship between participation in the C2C-IT intervention and mental distress and work engagement. The findings suggest that the intervention group used more effective ER strategies, had fewer symptoms of mental distress, and reported on higher work engagement compared to the control group. In addition, reappraisal and catastrophizing found to mediate the relationship between participation in the CTC-IT intervention and mental distress and work engagement. Findings discussed below and should be examined further in future research.Regarding the first aim of the current study, we found that during the first lockdown in Israel, there was low actual exposure to the virus among teachers, but great uncertainty and concern. The most worrying concern reported by the preschool teachers had to do with becoming infected or infecting family members. This finding is consistent with other studies that have found that fear of infection is one of the most significant causes of anxiety among teachers, which probably stems from teachers’ close interaction with children daily ,63. The Studies have shown that mindfulness and compassion-based interventions have positive outcomes on teachers’ mental health during routine periods (for a review see ) and durMany interventions aim to enhance teachers’ coping abilities with mental distress , yetThe third hypothesis based on the Coffey et al. model , which pTaken together, the findings point to the positive, long-lasting effects of a C2C-IT intervention on emotional distress and work engagement of preschool teachers, which maintained even in the high-intensity negative emotional situation of the COVID-19 outbreak. The current findings suggest that through the practice of mindfulness, individuals may be able to develop an expanded, nonjudgmental state of present-moment awareness that facilitates positive reappraisal and acceptance of stressful life events while reducing a tendency to catastrophize these events, which may finally lead to reduced emotional distress and increased work engagement.This study has a few limitations that should be acknowledged. First, it consisted of a cross-sectional exploration of post-intervention implications. Although this method has been applied in other studies ,74, it iTeachers’ work environment, which includes physical contact with many children, little opportunity to maintain hygiene and wear masks as recommended, may result in psychological distress, anxiety, and depression. Therefore, it is of utmost importance to find protective factors to shield against the mental distress of this population. The findings here showed that teachers who participated in a mindfulness and compassion intervention six months before the pandemic outbreak showed lower symptomology rates of emotional distress during the COVID-19 outbreak compared to teachers who did not participate in the intervention. This study thus provides preliminary evidence that participating in a C2C-IT intervention may help teachers sustain psychological wellbeing, even during a major crisis. The results suggest that the associations between mindfulness and compassion intervention and psychological distress and work engagement mediated by applying more adaptive ER strategies, thus suggesting that ER is one of the mechanisms underlying the deployment of teachers’ mindfulness. The COVID-19 pandemic is entering its third year; hence, mental difficulties may accumulate and the commitment to work may decrease, especially among preschool teachers. Reality cannot be controlled, but the interpretation of reality, and consequently the effect on the state of mind is to some extent something that can be practiced. Therefore, the Ministry of Education and other labor organizations should consider mindfulness-based interventions as a practical approach to improving well-being and work engagement, even during stressful events such as the COVID-19 pandemic. In addition, the relevant bodies should allocate resources to the rehabilitation of teaching staff at the end of the crisis, as they were found to be experiencing high emotional distress. From a scientific point of view, longitudinal studies are required to examine which intervention programs and protective factors maintain their effectiveness over time. Future studies can build on this work by examining the longitudinal role of mindfulness and compassion-based interventions on other related variables, such as burnout and resilience.


Mutually inhibitory populations of neurons, half-center oscillators (HCOs), are commonly involved in the dynamics of the central pattern generators (CPGs) driving various rhythmic movements. Previously, we developed a multifunctional, multistable symmetric HCO model which produced slow locomotor-like and fast paw-shake-like activity patterns. Here, we describe asymmetric features of paw-shake responses in a symmetric HCO model and test these predictions experimentally. We considered bursting properties of the two model half-centers during transient paw-shake-like responses to short perturbations during locomotor-like activity. We found that when a current pulse was applied during the spiking phase of one half-center, let’s call it #1, the consecutive burst durations (BDs) of that half-center increased throughout the paw-shake response, while BDs of the other half-center, let’s call it #2, only changed slightly. In contrast, the consecutive interburst intervals (IBIs) of half-center #1 changed little, while IBIs of half-center #2 increased. We demonstrated that this asymmetry between the half-centers depends on the phase of the locomotor-like rhythm at which the perturbation was applied. We suggest that the fast transient response reflects functional asymmetries of slow processes that underly the locomotor-like pattern; e.g., asymmetric levels of inactivation across the two half-centers for a slowly inactivating inward current. We compared model results with those of in-vivo paw-shake responses evoked in locomoting cats and found similar asymmetries. Electromyographic (EMG) BDs of anterior hindlimb muscles with flexor-related activity increased in consecutive paw-shake cycles, while BD of posterior muscles with extensor-related activity did not change, and vice versa for IBIs of anterior flexors and posterior extensors. We conclude that EMG activity patterns during paw-shaking are consistent with the proposed mechanism producing transient paw-shake-like bursting patterns found in our multistable HCO model. We suggest that the described asymmetry of paw-shaking responses could implicate a multifunctional CPG controlling both locomotion and paw-shaking. The existence of multifunctional central pattern generators (CPGs), circuits which control more than one rhythmic motor behavior, is an intriguing hypothesis. We suggest that the cat paw-shaking response could be a transient response of the locomotor CPG. Our general prediction is that this CPG is multifunctional, and in addition to the locomotor rhythm, it can generate a transient, ten-times faster, paw-shake-like response to a stimulus. In our multistable half-center oscillator (HCO) CPG model, we applied perturbations to the locomotor pattern which resulted in a transient paw-shake-like pattern that eventually returned back to the locomotor pattern. We showed that the inactivation of the slow inward current that drives the locomotor rhythm produced asymmetry of the transient flexor and extensor activity in a symmetric HCO model. To test predictions from our model about the transient nature of the paw-shake response, we compared burst durations (BDs) and interburst intervals (IBIs) of the model half-centers in consecutive cycles of paw-shake-like responses with the BD and IBI of electromyographic (EMG) activity bursts of cat hindlimb flexors and extensors recorded during a paw-shake response. In both cases, we found similar asymmetric trends in the BD and IBI throughout a paw-shake response. We engage in many types of rhythmic motor behaviors in our everyday lives, such as walking, breathing, chewing, etc. Rhythmic behaviors like these are generally controlled by interneuronal networks called central pattern generators (CPGs) –4. Many In principle, multifunctional CPGs could produce multiple rhythms of different frequencies. Relatively slow long-lasting rhythmic behaviors could be controlled by a stable, steady-state rhythm of a multifunctional CPG until an input signal changes or halts its rhythmic activity. A short-duration, non-steady-state rhythmic behavior, on the other hand, could result from transient neural dynamics of the same CPG triggered by a perturbation, e.g., a sensory input signal. These transient neural dynamics could generate a short-duration rhythmic behavior as a reliable response to certain environmental stimuli, such as transient responses seen in insect olfaction processing –16.ICaS) and slowly inactivating sodium current (INaS). The locomotor-like rhythm in our model is largely generated and controlled by ICaS, while the paw-shake-like rhythm is largely generated and controlled by INaS. Although ICaS and INaS both inactivate relatively slowly, the time constant of inactivation of ICaS is much larger than the time constant of inactivation of INaS. The slow calcium current in our model deinactivates at voltages that are more hyperpolarized than the minimum voltage reached during the stable paw-shake-like rhythm. Therefore, ICaS is completely inactivated during the paw-shake-like rhythm, allowing INaS to control the paw-shake-like rhythm and maintain a much higher frequency than the locomotor-like rhythm. During the locomotor-like rhythm, however, ICaS and INaS are both active, but the kinetics of ICaS bring the rhythm to a much slower frequency. With a short perturbation (a pulse of conductance) to one or both half-center neurons, the model can be switched from the locomotor-like rhythm to the paw-shake-like rhythm or vice versa. We have shown that a neuromechanical model of cat hindlimbs controlled by this multifunctional CPG and motion-dependent somatosensory feedback could reproduce not only distinct rhythms of cat locomotion and paw-shaking but also the typical reciprocal activation of flexors and extensors during locomotion and the atypical reciprocal activation of anterior and posterior muscles during paw-shaking [We have recently developed a multifunctional CPG model that generates reciprocal rhythmic activity between cat hindlimb antagonists and exhibits multistability of slow, locomotor-like and fast, paw-shake-like steady-state rhythms ,18. The -shaking ,19. In a-shaking ,18. We aThus, the first goal of this study was to investigate the mechanism by which neural transient dynamics could be produced in the multistable, half-center oscillator CPG model. The second goal was to investigate temporal characteristics of EMG activity of cat hindlimb antagonistic muscles  in consecutive paw-shake cycles evoked during locomotion and compare them with those of the model. We tested the hypothesis that the temporal characteristics of recorded EMG activities during cat paw-shake responses would be consistent with the model transient dynamics.All surgical and experimental procedures of this study were in compliance with the “Guide for the Care and Use of Laboratory Animals. Eighth Edition”  and were approved by the Institutional Animal Care and Use Committee of the Georgia Institute of Technology (protocol number A13063).INaF), a slowly inactivating sodium current (INaS), a potassium current (IK), a low-voltage-deinactivated, slowly inactivating calcium current (ICaS), and a leak current (Ileak). Currents INaF and IK were modified from another mammalian locomotion CPG model [Ex is the reversal potential; mx and hx are the activation and inactivation variables, respectively, of some current Ix. The activation and inactivation variables range from 0 to 1, where the current is fully deactivated when mx = 0 and fully activated when mx = 1; the current is fully inactivated when hx = 0 and is fully deinactivated when hx = 1 [mSynPre is the synaptic activation variable of the presynaptic neuron which is governed by the corresponding presynaptic membrane potential VPre. The capacitance C in this model is 0.001 nF.We used our previously published Hodgkin-Huxley style model ,18 to inPG model ,21. The n hx = 1 . The var−5 seconds, an absolute tolerance of 10−8 and relative tolerance of 10−9. Values of the model parameters are provided in Tables We integrated these equations using the 8th order Runge-Kutta integration method and GSL ODE solver software, with a maximum step size of 10IE with an excitatory reversal potential EE = 0 mV, as a square pulse of conductance, gE. This pulse of modulatory current was applied to both neurons in the model when the model was engaged in a slow locomotor-like rhythm. It was applied at a specified time, tpul, corresponding to a specific phase, ppul, and it continued for a specified duration, dpul. The fast rhythmic activity that results from this pulse may be either transient or stable. Many cases of transient paw-shake-like activity were collected by varying the time of pulse onset, tpul, and the pulse duration, dpul. The phase ppul = 0% was defined as the instant of the first spike peak in each burst of neuron 2. We varied ppul from 0% to 100% with a step of 0.25% of the cycle, and for each of these phases, we varied pulse duration dpul from 200 ms to 1200 ms with a step of 5 ms.The transient fast rhythmic activity was elicited in our model by applying a pulse of an additional excitatory modulatory current, For each episode of transient paw-shake-like activity in the model, we measured the temporal characteristics of bursting activities of the two neurons comprising the half-center oscillator CPG for each subsequent activity cycle. These characteristics included bursting cycle period (CP), burst duration (BD), and interburst interval duration (IBI). CP of each neuron was calculated as the time between the peak of the first spike of a burst and the peak of the first spike of the next burst. BD was calculated as the time between the peak of the first spike of a burst and the peak of the last spike of the same burst. IBI was defined as (CP–BD). Duty cycle (DC) was defined as 100*BD/CP. Paw-shake-like rhythms were defined as rhythms with CPs < 210 ms. The progressions of BD and IBI throughout each episode of transient activity were measured for each neuron and each phase of pulse onset. Linear regression analysis was used to perform a linear curve fit in order to evaluate the rate of change of BD and IBI over time during transient activity. We excluded the very first burst after the pulse from the linear curve fit, because there were some simulations where the first burst was very short, sometimes consisting of only 2 or 3 short spikes. For each 0.25%-phase increment of pulse onset, we obtained a simulation of transient paw-shake-like activity. Among all collected responses, we selected and used for further analyses those which contained between 5 and 9 paw-shake-like burst cycles, and disregarded simulations with fewer or greater. We applied linear curve fits to BD and IBI versus time for each collected simulation, determined the slopes of these fits for each simulation, and used a sliding averaging of the slopes with a window width of 1.25% of the cycle duration. That gave us the mean slope values of BD and IBI for all simulations corresponding to each perturbation phase. We also investigated more closely the mean slopes of BD and IBI in the phase range of 20% - 40% which approximately corresponds to the locomotor cycle phases in which paw-shaking is actually initiated in cats.hCaS model”. In each neuron of the reduced model, the hCaS state variable was held constant and its corresponding equations were removed from the CPG model. We separately varied hCaS1 and hCaS2 as two parameters, producing a simulation of the model for each combination of values in a certain domain. Each simulation was integrated for 1000 s to ensure that the activity had reached a steady state. We obtained the mean values of BD and IBI for neurons #1 and #2 separately by averaging over the last 20 bursts for each simulation case. We measured the difference of DC between the two model neurons and plotted this difference versus the difference in hCaS of the two neurons, and we performed a linear curve fit on these data.We hypothesized that the asymmetry of the linear trends of BD and IBI throughout time in our model could be explained by the functional antiphase relationships between the slowest state variables of the two half-centers. To test this hypothesis, we built a reduced version of the model, which we called the “constant tibialis anterior  and iliopsoas  and extensors soleus , medial gastrocnemius , vastus lateralis and vastus medialis , and anterior biceps femoris .We analyzed EMG recordings from 12 adult female cats (mass 3.56±0.73 kg) that were part of our previous studies –26. DetaFor each of these muscles, we measured the progression of EMG burst characteristics of paw-shake cycles throughout each paw-shake episode. In order to measure these burst characteristics, we first full-wave rectified the EMG signal and slightly smoothed it using a zero-lag, moving average with an 8-ms window. For each channel, we then took a random time interval with no muscle activity that was at least 200 ms wide and determined the mean and standard deviation of the raw EMG signal within this selection. The standard deviation was used as a measure of the EMG noise level in each trial. We set the activity burst threshold to be 3 times this standard deviation. We defined the EMG paw-shake cycle period (CP) for each muscle as the time between the EMG burst onset in a given cycle and the burst onset in the next cycle. The EMG burst duration BD was defined as the time between the burst onset and offset, and EMG interburst interval IBI was defined as the time between the burst offset and next burst onset. The duty cycle DC was calculated as 100*BD/CP.We analyzed changes in BD and IBI in all consecutive cycles of individual paw-shake episodes for each muscle: 28 episodes for MG, 28 for SO, 12 for VA, 12 for BFA, 21 for IP, and 12 for TA . Each paIn our model of a half-center oscillator, two steady-state oscillatory regimes–slow locomotor-like and fast paw-shake-like, coexist , as prevs at the same time. Looking back at the example simulation in 2) grew faster than the BD of neuron #1 (BD1), and the IBI of neuron #1 (IBI1) grew faster than the IBI of neuron #2 (IBI2)  with identical burst characteristics for the steady-state slow regime and the fast regimes and that both neurons received the same perturbation2 (IBI2) . For eac2 slope became greater than BD1 slope, and at around phase = 55% (near the burst onset of neuron #1), BD1 slope became greater than BD2 slope. On the other hand, at around phase = 15%, IBI1 slope became greater than IBI2 slope, and at around phase = 60%, IBI2 slope became greater than IBI1 slope.We then collected all simulations from the analysis in s for neurons #1 (-0.0015) was negative, while the DC slope for neuron #2 was positive (0.003) (We then looked more closely at a range of pulse application phases from 20% to 40% where behaviors of BD and IBI in the model and cat experiments were similar (see next section) and where actual paw-shake responses during cat locomotion begin, i.e. in the flexor (swing) phase of a locomotion cycle assuming neurons #1 and #2 represent the extensor and flexor half-centers, respectively. Within this range, the pulse was applied around the middle of the spiking activity of neuron #2, which would represent the swing phase of a cat’s locomotion cycle. We produced 286 simulations in this phase range by modifying the phase of pulse onset and the pulse duration, so that the pulse duration ranged from 700 ms to 1000 ms. Slightly varying these pulse parameters produced highly variable effects on the resulting transient activity of neurons #1 and #2 Figs . We calc (0.003) . Thus, dhNaS (time constant of 100 ms) and hCaS (time constant of 485 ms) between the two neurons throughout the transient paw-shake-like response  between the two neurons. We concluded that the asymmetry in transient responses in the full model occurred due to the difference in how much the slow calcium currents in both neurons inactivate while they are depolarized during the pulse.Then for each value of ed model . This mehNaS plays any notable role in generating the asymmetric trends throughout paw-shake transience, we set hCaS constant in both neurons at the moment the pulse ended using the same example of transient activity that was used in Figs hCaS1 and hCaS2 were held constant at the values they had reached, and thereby, at different asymmetric values   . We foun 0.0242) . We appl 0.0242) . To quanhCaS constant at the same value for both neurons, namely at the average of the two values reached at the moment of pulse offset . The median BD slopes for these muscles, on the other hand, were not different from zero, except for VA . The DC slopes of the extensor bursts were significantly less than zero (except for VA), i.e. DC of these bursts decreased with consecutive cycles of paw-shaking ; but the VA DC was not different from zero .We calculated Pearson’s correlation coefficient (R) and performed linear regression analysis of BD, IBI, and DC for each paw-shake response within each muscle group. We used the one-sample Wilcoxon signed rank test to examine if the median linear regression slopes of BD, IBI and DC  were sig-4, n = 21 and for TA, p = 4.9x10-4, n = 12), while the IBI slopes were not significantly different from zero for IP and TA . The median DC slope for TA was positive but not significantly different from zero .In this study, we demonstrated that the model of a multistable half-center oscillator CPG, capable of producing steady-state slow locomotor-like and fast paw-shake-like rhythms ,18, coulWe also recorded EMG activity of selected hindlimb muscles while cats performed paw-shaking during walking. Measured paw-shake cycle periods increased in consecutive paw-shake cycles Figs  and 10 iIn the next sections, we discuss these findings in the light of the potential role of multistable CPGs and their possible operation during locomotion and paw-shaking in the cat.Central pattern generators are often modeled to produce stable steady-state rhythmic patterns, and each such pattern is an attractor state of the system. When a system exhibiting a steady-state pattern, e.g. a locomotor pattern, is perturbed, some transient activity may occur before the system returns to that attractor state and resumes its steady-state rhythmic pattern ,15,28. TStudies have shown in various neural circuits of different organisms that transient activity can have functional purposes sometimes more so than steady state activity. For example, olfactory responses are encoded by transient dynamics in many investigated species ,15,31,32We propose that a mechanism similar to the one we have modeled here could apply to other species where a brief behavior is required to respond to some environmental stimulus. For instance, the struggling behavior in Xenopus tadpoles and larval zebrafish ,11,12,34vasti (VA) and tibialis anterior (TA) muscles [Given that the recorded paw-shake cycle periods change in each successive cycle  and Na+ (INaS) currents would drive the kinetics of inactivation of the slow and fast regime, respectively. When transient activity is generated in our model, slow Na+ current is still the main driving current of the transient fast activity, but slow Ca++ current controls the way burst duration and interburst interval change over time and the way the transient activity terminates and returns to the slow rhythm. The transient rhythmic bursting is reliably controlled by the slowest variable of the system, hCaS, which drives the slow bursting rhythm, locomotor-like bursting.In the current study, we investigated the question of whether paw-shake responses can be produced as a transient response of the multifunctional locomotor CPG. Our HCO CPG model can exhibit transient paw-shake-like activity in two different types of parameter regimes, (1) when the model exhibits coexistence of paw-shake-like activity and locomotor-like activity, and (2) when the model exhibits only locomotor-like activity but is set nearby the stable paw-shake-like regime in parameter space. In (2), having CPG parameters set in the vicinity of the range supporting the fast paw-shake-like regime as an attractor relates to the concept of a conditional burster: a neuron producing endogenous bursting activity under certain physiological conditions, for example under certain neuromodulatory tone. If both slow and fast regimes are attractors as in case (1) then transient paw-shake-like activity could be induced due to a slow passage near basin of attraction of the fast regime as a result of perturbation which shifted trajectory of HCO activity close to the border between basins of attraction of the two regimes but did not make it cross the border. Our model uses a relatively novel mechanism for multistability of bursting regimes with largely distinct periods that involves the different kinetics of inactivation of two intrinsic slow currents . These thCaS2 was decaying towards 0. At the same time, the membrane potential of neuron #1 was hyperpolarized, and the slow calcium current was deinactivated, its inactivation state variable hCaS1 was growing towards 1. Thus, hCaS1 was larger than hCaS2 at the time the pulse was applied due to the asymmetric removal of inactivation of the slowest inward current. During the pulse, the slow calcium current inactivated in both neurons, i.e. the values of hCaS in both neurons were decreasing, but at the end of the pulse, hCaS was still greater for neuron #1 than for neuron #2  and tibialis anterior (TA) muscles [In this study, we investigated a simple half-center oscillator CPG generating two phases (flexor and extensor) of a steady-state rhythmic locomotor-like behavior and a paw-shake-like transient activity. One limitation of our model is that it does not consider sensory feedback despite that motion-dependent somatosensory feedback affects the centrally generated experimental paw-shake activity patterns. However, these effects appear to be limited to two muscle groups responsible for the atypical muscle synergy during paw-shaking–the  muscles . The pha muscles ,43–45 or muscles ,38,46,47 muscles ,38,46,47vasti and ankle flexor tibialis anterior, a.k.a. the paw-shake mixed synergy [vasti being stretched during the flexor phase. Thus, our previous modeling results, the reciprocal activity of hindlimb muscles with flexor- and extensor-related EMG bursts in this . First line of each file contains muscle name abbreviations (see (ZIP)Click here for additional data file.


Background: The existing risk of procedure-related miscarriage following invasive sampling for prenatal diagnosis is higher for twin pregnancies and some women are reluctant to test these typically difficultly obtained pregnancies invasively. Therefore, there is a need for noninvasive testing options that can test twin pregnancies at an early gestational age and ideally test the twins individually.Case presentation: A pregnant woman opted for cell-based NIPT at GA 10 + 5. As cell-based NIPT is not established for use in twins, the test was provided in a research setting only, when an ultrasound scan showed that she carried dichorionic twins.Materials and Methods: Fifty mL of peripheral blood was sampled, and circulating fetal cells were enriched and isolated. Individual cells were subject to whole-genome amplification and STR analysis. Three fetal cells were analyzed by chromosomal microarray (aCGH).Results: We identified 20 fetal cells all sharing the same genetic profile, which increased the likelihood of monozygotic twins. aCGH of three fetal cells showed the presence of two X chromosomes and a gain of chromosome Y. CVS from both placentae confirmed the sex chromosomal anomaly, 47,XXY and that both fetuses were affected.Conclusion: NIPT options can provide valuable genetic information to twin pregnancies that help the couples in their decision-making on prenatal testing. Little has been published about the use of cell-based NIPT in twin pregnancies, but the method may offer the possibility to obtain individual cell-based NIPT results in dizygotic twins. The risk of miscarriage following invasive testing is higher in twin pregnancies compared to singletons  . IncreasThe vast majority of NIPTs used in both public healthcare systems and by private providers are based on analysis of circulating cell-free fetal DNA (cffDNA) that sheds from the placenta to the maternal blood. We refer to these analyses as cell-free NIPT. Cell-free NIPT covers the common trisomies, sex chromosomal anomalies, and, for some types, specific targets associated with common microdeletions- and microduplications . While cIn contrast to cffDNA or cell-free NIPT, a newly launched test, Evita Test Complete, uses intact circulating fetal cells (extravillous trophoblasts) for detection of aneuploidies and genome-wide copy number variants down to 5 Mb . ExtraviAll current NIPTs  are screening tools and therefore, women who receive a positive NIPT result should be offered follow-up by invasive testing (CVS or AC).In the present case, we describe how cell-based NIPT (as wells as cell-free NIPT) detected Klinefelter syndrome in a twin pregnancy. Cell-based NIPT gave additional information indicating that this was a monozygotic twin pregnancy, as the genetic profiles of all the isolated cells were identical. This is strongly indicative of both fetuses being affected—a distinction not possible by the cell-free NIPT. This case report demonstrates how noninvasive testing options can provide valuable information to the pregnant couple using the traditional cell-free NIPT and cell-based NIPT, but also how cell-based NIPT may turn out to be particularly useful in twin pregnancies.A 37-year-old pregnant woman (GA 10 + 5) and her partner wanted genetic information about the pregnancy at an early gestational age. They contacted Aagaard Fertility Clinic Denmark, as they offer a commercial cell-based NIPT, EVITA TEST COMPLETE. The pregnancy was naturally conceived. At the clinic, ultrasound examination showed that the pregnancy was a dichorionic twin pregnancy by identification of a “lambda-sign” at the intertwin membrane. The couple was counselled about the limitation of the test, as it was not validated for multiple pregnancies. The couple still opted for cell-based NIPT. Therefore, the test was offered in a research setting.At gestational age 10 + 5, a 50 ml blood sample was collected in five Cell-Free DNA BCT tubes . The blood was processed as previously described with slight modifications . In brieTwenty mL of blood was sampled in Cell-Free DNA BCT tubes at GA 10 + 5 and the sample was analyzed retrospectively for research purposes. The cell-free NIPT analysis was conducted at the Department of Clinical Genetics, Aarhus University Hospital, as previously described .Chorionic villous sampling (CVS) and aCGH were carried out at GA 13 + 1 at the Department of Fetal Medicine and the Department of Clinical Genetics, Rigshospitalet, Copenhagen, Denmark, where the pregnant woman also had her first-trimester screening. Transabdominal approach using a needle guide with separate sampling from each placenta with a 18–20 G double-needle was performed. aCGH was performed using the Agilent SurePrint G3 180K ISCA oligo + SNP array . Labeling and hybridization were performed according to the protocol provided by Agilent.For cell-based NIPT, FACS single-cell sorting isolated 32 individual cells, which were subject to WGA and STR analysis. Nine tubes were, however, empty based on DNA concentration measurements following WGA. The STR analysis identified 20 fetal cells, two cells with inconclusive origin due to allele drop-out in the STR analysis and one maternal cell. All 20 fetal cells shared the same STR profile .A pool of single-cell WGA DNA from three individual fetal cells was analyzed by aCGH. The cell-based NIPT result showed presence of Y-chromosome and two X-chromosomes when compared to female control DNA, cell-based NIPT, The cell-based NIPT result was reported to the couple by a clinical geneticists (author IV) and invasive testing was recommended. The woman subsequently attended the first trimester screening at GA 12 + 5 where the nuchal translucencies were 1.2 and 1.3 mm, PAPP-A 0.514 MoM and fβ-hCG 0.34 MoM, which gave combined risks for trisomy 21 of 1:1,077 and 1:917, respectively. Due to the cell-based NIPT result, confirmatory prenatal diagnosis using invasive sampling from both placentae was carried out at GA 13 + 1. The invasive prenatal analysis indicated that the twins were monozygotic and demonstrated that both fetuses had the karyotype arr(X)x2,(Y)x1 (data not shown), in accordance with the findings of For research purposes, cell-free NIPT was conducted retrospectively, after invasive testing had confirmed the cell-based NIPT result. The fetal fraction was 8% and the result showed increased risk of 47,XXY in at least one of the fetuses and with two copies of chromosome 13, 18 and 21 .We report the detection of a sex chromosome anomaly (Klinefelter Syndrome) in a dichorionic twin pregnancy, using circulating fetal cells (extravillous trophoblasts) for NIPT. Twenty fetal cells were isolated from the maternal blood sample and STR analysis showed that they all shared the same genetic profile. This highly increased the likelihood of a pregnancy with monozygotic twins, although the presence of dizygotic twins could not be excluded. The finding of 47,XXY in circulating fetal cells indicated that at least one of the fetuses would develop Klinefelter Syndrome, but also that the pregnancy most likely was a monozygotic pregnancy with both fetuses affected. Invasive testing confirmed the sex chromosome anomaly and that both fetuses were affected.www.evitatest.com).The present case shows how noninvasive testing options, and in particular cell-based NIPT, can provide valuable information to prenatal diagnosis of multiple pregnancies. As always, the test limitations need to be communicated during genetic counseling and positive NIPT results must be confirmed invasively. The clinical applicability of cell-based NIPT is yet to be established in multiple pregnancies, however, the establishment of cell-based NIPT for detecting aneuploidies and CNVs in singleton pregnancies is already published  and has Specifically regarding multiple pregnancies, the utility of cell-free NIPT to screen for the common trisomies has been shown . HoweverFor cell-based NIPT, multiple pregnancies constitute some of the same challenges as has been described for cell-free NIPT but testing of circulating fetal cells has some strengths. In dizygotic twin pregnancies, it should be possible to isolate circulating fetal cells from both fetuses to obtain individual STR profiles and thus NIPT results. In monozygotic twin pregnancies, as in the present case, the identification and testing of multiple fetal cells with identical profiles will increase the probability that both fetuses have been tested. However, dizygotic twins can still not be excluded as the contribution of circulating fetal cells may solely originate from one twin. Invasive confirmatory testing is needed to obtain the prenatal diagnosis. More research is needed to validate the use of cell-based NIPT in twin pregnancies, but cell-based NIPT may contribute with valuable genetic information for couples who restrain or hesitate from invasive testing.The positive predictive value (PPV) for cell-free NIPT for SCAs is low  compared to the PPV for common trisomies and false positive results may lead to unnecessary invasive testing . KlinefeIn the study by Ronzoni et al., four cases presented with mosaic forms of SCAs in the pre- or postnatal follow-up after cell-free NIPT showed an increased risk of Klinefelter syndrome in the fetus . VariousIn Denmark, cell-free NIPT is a free-of-charge offer as an alternative to invasive testing if the first trimester risk assessment for Downs Syndrome is above 1:300. The pregnant woman in the present case received a low-risk result and was therefore not offered prenatal testing in the public setting. As the couple at an early gestational age were interested in reducing the risk of aneuploidy and CNVs, they had already opted for cell-based NIPT at a private clinic prior to the first trimester screening. This case shows how noninvasive testing options can provide significant information on the genetic status of fetus(es) for parents-to-be even before the first trimester screening. Increased use of NIPT early in pregnancy emphasize the need for new approaches for prenatal counseling before the first trimester risk assessment and optimally the available data should be integrated in the risk assessment algorithm if the result is normal. This would significantly reduce the residual risk of fetal aberrations and likely improve both prenatal care and parental anxiety both in case of negative and positive findings. In conclusion, this case report shows how cell-based NIPT demonstrated Klinefelter Syndrome at an early gestational age and thereby provided significant information for the decision-making to a couple expecting twins.


It is well-documented that autism can be reliably diagnosed by age two and that early signs emerge most often between 18 and 24 months. However, despite the increased awareness and focus on early diagnosis, the average age of diagnosis is over 4 years old; even later for Black children and those who are Medicaid-eligible. In this paper, we will propose a framework for accurate and accelerated autism diagnosis for children before age three. The proposed framework emphasizes a collaborative diagnostic process, which relies heavily on Birth to Three provider knowledge and expertise. Considerations for next steps are presented. This approach could increase access to diagnosis of young children soon after first signs of autism emerge. Autism Spectrum Disorder (ASD) is a neurodevelopmental disability that impacts social communication and repetitive behaviors that can be identified within the first 3 years of life , 2. In tMost children show clear signs of ASD  by 24 mohttps://sites.ed.gov/idea/The B-3 service delivery system, established by Part C of the Individuals with Disabilities Education Act (IDEA), is a federal program that supports states in providing individualized services for infants and toddlers with disabilities and their families . This syIncreased collaboration between B-3, medical providers, and diagnostic clinicians is an innovation that may improve outcomes for pediatric populations. For example, better integration of B-3 and newborn screening programs, programs that serve the same population but have different funding structures, recruitment approaches, and approaches to intervention, may improve services for children with Fragile X syndrome . Fragileearly intervention, but rather is considered on-time intervention.20 years ago, the National Research Council recommended that services for children with ASD should begin as soon as ASD is suspected . This goThe ‘On-Time Autism Intervention' project (OTAI) is a collaboration at the University of Washington (UW) in Seattle, Washington lead by the UW Autism Center and UW Haring Center for Inclusive Education. OTAI staff consists of researchers, clinicians, and educators with expertise in ASD diagnosis and intervention for young children. The goal of OTAI is to develop, with community partners, innovative strategies to increase equitable access to “on-time” diagnostic and ASD-specific intervention services for young children and their families.The OTAI identification and diagnosis practice framework consists of practice recommendations in five areas: (1) universal screening by B-3 staff, (2) supporting parental orientation toward diagnosis, (3) collaborative referral to diagnostic evaluation, (4) accelerated diagnostic evaluation, and (5) seamless transition to ASD navigation and intervention.To develop the framework, OTAI staff engaged community B-3 partners by visiting B-3 centers, meeting with and observing the work of B-3 providers. From there, a preliminary collaborative, accelerated diagnostic framework was developed and tested with a partner B-3 organization and revised based on observations and feedback from B-3 providers, parents, and OTAI staff. The framework employs reliable and valid clinical measures in the diagnostic process. The measures considered the ‘gold standard' include the Autism Diagnostic Observation Schedule (ADOS) , to direall roads should lead to B-3 programs as the first stop. This means that whoever raises concerns about ASD or developmental characteristics associated with ASD; pediatrician, parent, day care provider, or extended family member, the child should be referred to B-3 for evaluation for services. We propose that the following practices, implemented by B-3 centers, can reduce the gap between emerging ASD and accessing ASD services.The OTAI framework is informed by efforts over the past decade to help primary care physicians, B-3 early interventionists, and psychologists learn and implement universal screening for ASD and reduce health inequities . A fundaBased on existing recommendations for universal ASD-specific screening for primary care medical providers  and leveReview existing pediatric ASD screening records prior to B-3 evaluation, ORIf screening has not yet been conducted, or is not made available, B-3 centers should intentionally screen all children for ASD at their eligibility intake meeting.Standardized developmental testing, including parent reports, is conducted as part of the B-3 eligibility assessment. The OTAI model suggests universal screening, a novel addition to the standard B-3 assessments and questionnaires. B-3 staff should:Administer and score the Modified Checklist for Autism in Toddlers (M-CHAT-R/F), using the standardized follow-up questions  orReview results of any recent M-CHAT screening from a community provider see .- If the M-CHAT score indicates further ASD evaluation is warranted, the B-3 provider should assess the parent's orientation toward a diagnosis- If the M-CHAT does not indicate further evaluation is warranted, B-3 staff should:∗ Determine whether direct observation or reports from collateral contacts warrant further ASD evaluation.∗ If there is no need for further assessment, B-3 staff should make note of the administration date, continue surveillance for ASD signs, and repeat the M-CHAT every 6 months until 36 months of age , 36.∗ If at any point concerns about ASD are raised by parents or B-3 team, the B-3 provider should initiate the M-CHAT screening process as above.The OTAI framework proposes that services should be adapted to meet the needs of parents with different orientations toward receiving, understanding, and acting on diagnostic information. The OTAI framework is designed to help B-3 staff provide support consistent with parental orientation to a diagnosis to move the parent toward engagement with ASD-specific intervention when the time is right for them and in a way that is responsive to parental concern.Parental responses to early signs of ASD and referral for a diagnostic evaluation range from hesitancy due to fear of labels or stigma, to defensiveness when concerns are brought up by providers, to acceptance or feelings of validation , 38. WheParents respond to screening results differently depending upon their orientation toward diagnosis.Action: Some parents have been thinking and learning about ASD and are waiting for a provider to suggest they seek out a diagnostic appointment.Contemplation: Some parents may be open to the possibility of ASD but are weighing the pros and cons and are still not quite ready to act and schedule a diagnostic evaluation.Precontemplation: Some parents may be adjusting to the developmental concerns that led them to B-3 services and have not considered the idea that their child may have ASD. These parents are unlikely to schedule a diagnostic evaluation and if they do, they may not be comfortable with the diagnosis or with seeking ASD-specific services.If concerns about ASD are indicated on the M-CHAT, but parents are contemplating or not yet contemplating a diagnosis, clinicians should support parental orientation toward a diagnosis and intervention by preparing for difficult conversations and engaging families in conversation.Prepare for difficult conversations: B-3 providers should prepare to engage parents using MI strategies to collaboratively explore the ASD characteristics that need further evaluation and to highlight a child's strengths in addition to behavioral and developmental concerns .wait and see.” B-3 providers are experts at supporting families. Sharing additional information about ASD and engaging families in these conversations should aim to support parental orientation toward diagnostic evaluation.Engage in conversations: B-3 providers can engage in ongoing conversations with families about child developmental concerns and positive ASD screens thereby avoiding the pitfall of implicitly or explicitly encouraging families to “Parents in the action stage may benefit from:- Information about local diagnosing providers- Ongoing discussion and information specific to their child- Help scheduling and completing the diagnostic evaluation- Post-diagnosis navigation support.Parents in the contemplation and precontemplation stages may benefit from:- Discussing the behaviors on the M-CHAT that suggest ASD- Opportunities to ask questions about the assessment, their child's behavior, and other concerns.- Conversations about ASD using MI strategies to support orientation towards diagnostic evaluation over the first few months in B-3 services.- Child skills and needs in relevant developmental domains Services the child is currently receiving through B-3 agencyB-3 team impressions of likelihood of ASDB-3 team impressions of parental concern regarding ASD see Child strengthsFamily support needsIn collaboration with B-3 partners, OTAI developed a tool to measure parental orientation towards diagnosis and collect other information relevant to the diagnostic process. This B-3 provider-rated form (available upon request) is designed to support the B-3 provider in tailoring discussions to the parental level of concern and, once a parent decides to pursue a diagnosis, to facilitate efficient and relevant conversations between the B-3 team and the diagnosing clinician. Utilizing the knowledge and expertise of the B-3 provider specific to each family and child is a novel contribution that could support both parents and diagnosing providers when it comes to accelerating access to timely diagnosis. Information for the diagnostic clinician includes:Once a parent decides to pursue a diagnostic evaluation, an innovative collaborative diagnostic model is recommended. The OTAI model suggests the formation of partnerships between B-3 agencies and local diagnosing clinician(s); psychologists, pediatricians, neurologists, or other professionals, consistent with state law. The collaborative diagnostic model delineates roles for the B-3 provider and diagnostic clinicians and weaves these roles together in a meaningful way to best support the family and accelerate the diagnostic process. B-3 referral for a collaborative diagnosis includes two steps:Parental release of information to share B-3 records with diagnostic clinicianConversation between B-3 and diagnostic clinician to share relevant information about the child and familyThe accelerated diagnostic evaluation utilizes recommended practices  for accu- Contact family and schedule first diagnostic visit. Diagnosing clinicians should set a goal to complete the first visit as soon as possible and track how long this takes.- Contact B-3 provider to discuss case with child's team.- Review records and assessment results provided by B-3 team; medical and developmental history, standardized developmental assessment, M-CHAT screening results, and other relevant records.Diagnostic clinician - Pre-meeting data review:Following the pre-meeting data review, the diagnostic clinician will conduct a single, 3-h diagnostic appointment consisting of a parent interview, child-parent play observation, ADOS administration, and diagnostic disclosure. The child, caregivers, and B-3 provider should be in attendance. Involving the B-3 provider in the diagnostic evaluation and disclosure as standard practice is novel and facilitates a coordinated transition between diagnosis and post-diagnosis services. The diagnostic disclosure should include examples of observed child behaviors across the core ASD diagnostic categories, intervention recommendations, and individualized family support information.Following the diagnostic appointment in accordance with existing clinical practice recommendations, the diagnostic clinician will write a diagnostic report to be shared with the family and B-3 intervention team. The report should include behavioral observations, diagnostic information and scores, intervention recommendations (including a prescription for ABA), and family support resources.Once a family has received an ASD diagnosis, they should immediately be offered ASD navigation support as part of their ongoing B-3 services. Navigators can be B-3 service providers of any discipline who have additional training in helping families find ASD-specific services and resources. The family should be assigned an ASD navigator and meet quickly  to review the evaluation process, psychologist feedback, and recommendations. The primary role of the navigator is to provide parents with follow-up support, information, and resources after diagnosis. Navigators help parents understand their child's ASD characteristics, seek out resources, pursue and evaluate interventions for the child, and process emotions related to the diagnosis.The OTAI project suggests that, with adequate funding and support, B-3 intervention service systems can serve a critical and novel role in the effort to increase equitable access to timely diagnosis of ASD through community partnerships with diagnosing clinicians. Novel practices are proposed that are in line with accepted clinical ractice and use valid and reliable measures. Telephone surveys conducted with B-3 providers in the state of California found that although 85% of agencies conduct screening for ASD, only 39% offer diagnostic evaluations prior to age three . The OTAStudies examining the parent perspective of diagnosis suggest a need for improvement and innovation. When given opportunities to share about their experiences of the diagnostic process, parents highlighted a need for more information and follow-up after diagnosis , 44. SimThere are several limitations and considerations for future work. Our project staff engaged closely with community providers to increase acceptability and feasibility of the referral and diagnostic process. Despite our efforts to ensure a good fit in our region, this may not be applicable to other communities. Finding diagnostic providers to engage in novel, collaborative diagnosis may be challenging due to structural barriers like low insurance reimbursement or lack of providers with expertise in diagnosis of children under age three. Furthermore, there are questions related to costs of training B-3 providers and logistics, such as who will provide the training, that could impede the implementation of this framework. Finally, the OTAI project was small and focused locally. Scaling up the project to meet the complex and different needs of other regions has not yet been tested. Future efforts are needed to engage community partners to make adaptations to the model that will increase acceptability and feasibility. Additionally, well-designed implementation research is needed to better understand the impact of these practices.The OTAI Project to date has focused on conducting development field work with partner B-3 agencies. OTAI staff have conducted all diagnoses. The next phase of research will implement the OTAI framework with community diagnostic clinicians and new B-3 agencies. In this next phase, OTAI will provide training but will not provide direct services within B-3 agencies. This will achieve two major purposes, (1) larger scale implementation and testing of the framework, and (2) feedback from community partners to inform novel design and implementation considerations. Further refinement of the OTAI framework will help ensure acceptability and feasibility in a wide range of communities.For decades large-scale studies have documented delays between first concerns and ASD diagnosis, with greater delays for children of color and those with fewer resources. This research and practice gap suggests the need for innovative advances toward sustainable implementation . The OTAThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.AP, JG, IS, and AE devised the project, main conceptual ideas, and framework outline. AP wrote the manuscript with input from all authors. JG implemented and field tested the framework. IS and AE were in charge of overall direction and planning of the project. All authors discussed the results and commented on the manuscript.This project was supported by funding from the Seattle Foundation. Use of facilities and resources supported by: Intellectual and Developmental Disabilities Research Centers (P50HD103524-02).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.


Here, we introduced a machine learning-based algorithm as an alternative for a more accurate search of new pharmacological candidates, which takes advantage of Recurrent Neural Networks (RNN) for active molecule prediction within large databases. Our approach, termed PharmaNet was implemented here to search for ligands against specific cell receptors within 102 targets of the DUD-E database, which contains 22886 active molecules. PharmaNet comprises three main phases. First, a SMILES representation of the molecule is converted into a raw molecular image. Second, a convolutional encoder processes the data to obtain a fingerprint molecular image that is finally analyzed by a Recurrent Neural Network (RNN). This approach enables precise predictions of the molecules’ target on the basis of the feature extraction, the sequence analysis and the relevant information filtered out throughout the process. Molecule Target prediction is a highly unbalanced detection problem and therefore, we propose that an adequate evaluation metric of performance is the area under the Normalized Average Precision (NAP) curve. PharmaNet largely surpasses the previous state-of-the-art method with 97.7% in the Receiver Operating Characteristic curve (ROC-AUC) and 65.5% in the NAP curve. We obtained a perfect performance for human farnesyl pyrophosphate synthase (FPPS), which is a potential target for antimicrobial and anticancer treatments. We decided to test PharmaNet for activity prediction against FPPS by searching in the CHEMBL data set. We obtained three (3) potential inhibitors that were further validated through both molecular docking and in silico toxicity prediction. Most importantly, one of this candidates, CHEMBL2007613, was predicted as a potential antiviral due to its involvement on the PCDH17 pathway, which has been reported to be related to viral infections.The discovery and development of novel pharmaceuticals is an area of active research mainly due to the large investments required and long payback times. As of 2016, the development of a novel drug candidate required up to $ USD 2.6 billion in investment for only 10% rate of approval by the FDA. To help decreasing the costs associated with the process, a number of  Al. Al67]. The obtained binding energy for Zolonadrate agrees well with that reported elsewhere . In thisCHEMBL250434 -6,6-dimethyl-2-bicyclo[3.1.1]hept-2-enyl]methyl]piperidin-4-yl]-1,3-benzothiazol-2-amine)  has two After corroborating the interaction through molecular docking, the top-3 candidates were analyzed with the aid of the online servers GUSAR, for cytotoxicity, and DIGEP-Pred for genotoxicity. PCDH17 gene, which encodes for a protein that contains six extracellular cadherin domains, a transmembrane domain, and a cytoplasmic tail, which makes it different from classical cadherins sulfonyl-2-chloro-4- mercaptophenyl acetate (PubChem CID:380934) was established as non-toxic for IV administration routes and upregulates the in vitro and in vivo, given that only the most promising candidates are further analyze. We implemented this approach here and put forward PharmaNet, a deep learning architecture for predicting binding of a molecule to possible protein target receptors. PharmaNet´s algorithm represents the 2D structural information of a molecule as a molecular image and process it with modern computer vision recognition techniques. Our architecture is trained end-to-end and consists of a convolutional encoder processing phase followed by an RNN. This approach allows multiclass classification with a single model. The conventional metric for this type of task has been the Receiver Operating Characteristic curve (ROC-AUC); however, we propose that a more accurate metric is the area under the Normalized Average Precision (NAP) curve. Under this framework, PharmaNet outperforms the state-of-the-art algorithm by one order of magnitude (from 1% to 65.5%) in the AD dataset, and has a perfect performance in identifying the active molecules for 3 receptors of the 102 targets: CXCR4, FPPS and KITH. We selected FPPS as target to apply our model in the search of active molecules within the large database of pharmacological molecules, ChEMBL. We chose the 10 best candidate molecules to investigate into detail interactions with FPSS via molecular docking. We also conducted an in silico evaluation of toxicity with the aid of online servers. We found that the compound identified with the ID CHEMBL2007613, i.e., amino]sulfonyl-2-chloro-4-mercaptophenyl acetate) exhibits potential antiviral activity, which needs to be corroborated in vitro. We expect that our algorithm opens new opportunities for the rediscovery and repurpose of pharmacological compounds that otherwise might be disregarded in importance by the pharmaceutical industry. Moreover, we are currently exploring its potential in the reverse problem, i.e., searching for multiple receptor targets for molecules with certain physicochemical properties.One important challenge in modern drug discovery is to accelerate the search for new and more potent therapeutic molecules but in a more assertive manner as thus far, conventional approaches are extremely costly, time consuming and largely ineffective. Pharmaceutical companies have spent billions of dollars in development but only a small percentage of candidates have made it to the market. Novel artificial intelligence algorithms provide an alternative route for a more comprehensive search of candidates in already available and large databases of pharmaceutical compounds. Also, those algorithms reduce the number of experiments needed


Covid-19 pandemic, continues all over the world with the increasing number of confirmed cases and performed tests day by day. It has been shown that collecting nasopharyngeal samples, as the most commonly prefered method to perform RT-PCR, has disadvantages like causing discomfort and bleeding in patients. Sample collecting procedure also renders healthcare professionals by exposing them to the risk of transmission of the virus related to the direct contact with patients. These disadvantages make this procedure undesirable for the researchers and forces them to search for an alternative technique. At this point, saliva appears as an opportunity, regarding its high viral load. On the other hand, this high viral load poses a threat, especially for professions such as dental practitioners, with too much aerosol exposure. Since dentistry is a branch of health that constantly needs direct operations, it is necessary to be protected from the virus as much as possible while caring for the patient. A literature review was done using electronic databases “PubMed,” “Google Scholar,” and “Cochrane Database,” on January 2021. Studies have proposed many different preventive measures in this regard. Therefore, the purpose of this review is to draw attention to the saliva by bringing together the recent research and also to provide information and a perspective to dental clinicians about both prevention and a potential diagnostic technique. New coronavirus disease COVID-19) has spread to more than 200 countries globally. Worldwide, a total of 105,658,476 confirmed cases of COVID-19, of which 2,309,370 deaths were reported to WHO till 08 February 2021.9 has spr6Coronaviruses are enveloped RNA viruses which are presenting the classic structure with “spike protein” in the membrane envelope. The interplay between this spike protein and angiotensin converting enzyme 2 (ACE 2) receptors are in charge of the virus entrance into the cells. The dispersion of ACE-2 receptors in various parts of the body may show us the possible routes of infection.11Currently, direct contact and/or spreading of air droplets are the most likely routes of transmission. This is reinforced by findings that SARS-CoV-2 can be isolated in aerosol (<5μm) for at least three hours.A literature search was done using electronic databases “PubMed,” “Google Scholar,” and “Cochrane Database,” on January 2021 and these were used to identify the articles in English language that cover the relevant objectives. Multiple search keywords were used, such as saliva and covid-19, covid-19 diagnosis, and dentistry and covid-19.Saliva is a special body liquid produced by the major and minor salivary glands. This special liquid is formed mostly of water 94-99%), with 0.5% organic substances and 0.2% inorganic substances. Saliva has a crucial role in cleaning, digesting food, protecting the oral cavity, lubricating the oral mucosa and fulfillment of the balance in the oral cavity. A healthy adult generally produces about 600 ml of saliva each day. Apart from secretion from salivary glands, saliva also carries white blood cells, exfoliated epithelial cells, serum elements and oral microorganisms with their metabolites. More than 700 microbial species which are associated with several diseases have been identified in saliva. It offers a suitable place for the colonization and growth of oral microorganisms. Additionally, it prevents the overgrowth of specific pathogens to protect the natural balance of the oral cavity. Saliva can also act as a keeper and block the passage of pathogens to the respiratory tract and digestive system.4-99%, wiThere are at least three separate ways for SARS-CoV-2 to occur in saliva: (1) Infection of the salivary glands release particles from the salivary ducts into saliva; (2) SARS-CoV-2 in the blood can reach the oral cavity through gingival fluid; and (3) Saliva reaches the mouth together with the droplets of the virus in the upper and lower respiratory tract.14The most widely adopted way of SARS-CoV-2 spread is direct contact with respiratory droplets. The use of dental instruments such as high speed and low speed turbines or ultrasonic cleaners in daily dental practice causes splashes of saliva and blood droplets.16After ultrasonic cleaning, contamination of the inner surfaces of clinical clothes and masks with saliva has been reported. Even though surgical masks defend the mucous membrane in the oral cavity and nose from splashing, they can not ensure full protection against infections.15Our knowledge about the constancy of the SARS-CoV-2 in saliva droplets or aerosols is very limited. A study showed that the SARS-CoV-2 genome was discoverable in 66.7% of air samples taken from corridors outside patient rooms in addition to air samples taken from the rooms of the patients.All these observations demonstrate an elevated risk of airborne transmission of the SARS-CoV-2 to dental practitioners. Apart from direct spread there is also an indirect way too. Liu et al.18Researchers suggest different protection measurements to avoid Covid-19 in dental practice. These suggestions mentioned the importance of personal protective equipment (PPE) for dental professionals. Also includes using mouth rinses before dental treatments and using rubber-dam during dental approaches.The major way of spread is the airborne droplet in dental practices. Therefore, during the COVID-19 pandemic, all the protection barrier equipment is a priority for all dental professionals. Medical hand gloves, medical face mask, protective eyewear, medical cap, face shield, and special protective suits are examples for PPE. Medical Goggles provide the most trustworthy eye protection from aerosols, but also the face shields are stated as an alternative as an eye contamination protection measurement. Studies report that N95 respirator masks or FFP3 respirator are recommended in addition to room ventilation to avoid airborne transmission.19In their recent review, Vergara-Buenaventura and Castro-Ruiz25The dental rubber dam is an ideal equipment that is used for isolation in the dental practice, especially during endodontic and adhesive procedures. Rubber-dam helps to reach a proper cross-infection control for both the dental professionals and the patients. The dam provides an excellent isolation from the oral cavity, thus aerosol is not mixed with the saliva of the patient. Which has a high viral load as mentioned before in this review. Several studies report that using the rubber dam is an excellent barrier to the potential spread of the infectious disease in the dental practice.The real-time polymerase chain reaction (RT-PCR) with samples taken from nasopharyngeal swabs is accepted as a gold standard currently. A nasal mid-turbinate swab by a healthcare team or self-collection with a conical swab is also accepted as an alternative. However, various studies stated that the samples taken by this method have low sensitivity for detecting SARS-CoV-2.28Generally, samples taken from the upper respiratory tract as nasopharyngeal or oropharyngeal swabs. Additionally, for patients presenting with pneumonia, they can also be collected from the lower respiratory tract as sputum or endo-tracheal aspirate.30Apart from physical discomforts, pychological effect of repetitive diagnostic and treatment procedures  should be considered too. Zaidi and Jawaid were reported that moral trauma caused by the COVID-19 has left indelible scars on the human psyche.Vaz et al.Azzi et al.Saliva collection procedure or nasopharyngeal swabs applied to 44 hospitalized COVID-19 patients by healthcare professionals and 121 samples obtained in the research by Wylli et al.However, in another study, SARS-CoV-2 was discovered in only four saliva samples of 31 individuals whose oropharyngeal swabs were positive.Studies have shown that saliva specimens have higher viral load or lower cycle threshold against nasopharyngeal swab specimens.Under the limitations of this review, most of the researchers stated no significant difference in viral load between nasopharyngeal and saliva samples. These studies concluded that saliva is a non-invasive sample type for the diagnosis of COVID-19 virus and viral load monitoring.38Several groups have also studied the potential of saliva as a clinical sample for COVID-19 virus detection. In this regard, a study of 12 patients confirmed by PCR detection of viral RNA using nasopharyngeal or sputum samples found that coughing saliva from 11 patients was positive for SARS-CoV-2. Importantly, no viral RNA was detected in saliva samples collected from 33 patients whose nasopharyngeal samples tested negative for SARS-CoV-2. Consistent results were obtained by the same group examining a different set of patients, showing that SARS-CoV-2 was detectable in self-collected saliva of 20 out of 23 confirmed patients.29Another approach to collecting saliva samples is through oral swabs that are easily administered even for non-professionals. In two studies examining saliva samples collected with oral swabs, 15 (50%) of 39 patients and 25 (100%) of 25 patients, respectively, tested positive for SARS-CoV-2.The approved saliva-based COVID-19 test kit is built on the current TaqPath SARS-CoV Test developed by the Rutgers Clinical Genomics Laboratory to qualitatively identify RNA from the virus. This test uses primers and probes for respiratory, nasopharyngeal and oropharyngeal samples, validated by the Emergency Use Authority (EUA). Collection protocols and nucleic acid extraction buffers are changed to ensure that the saliva sample is tested. Saliva samples can be transported and stored at ambient temperature but must be processed within 48 hours of collection. The recommended system for RNA extraction is Chemagic Viral DNA / RNA 300 Kit H96 and PerkinElmer ChemagicTM 360. The RT-PCR can be performed using Applied Biosystems TaqPath Combo Kit on the ThermoFisher Applied Biosystems QuantStudio 5 Real-Time PCR System or the Applied Biosystems ViiA7 Real-Time PCR System.39The saliva collection method and types of collection devices are critical issues in this detection technique. There are three main types of saliva which includes whole saliva, parotid gland and minor gland samples and the collection method of each type varies accordingly. When the aim of sampling is to detect respiratory viruses by molecular analysis, it is beneficial to collect all types of saliva from suspected patients.In daily dental practice, high-speed water-cooled turbines are used frequently. Therefore a high amount of aerosol is created. The virus in this aerosol remained stable for three hours on air and 72 hours on stainless steel or plastic surfaces. Dentist should always protect himself with protective clothing. Additionally, the risk of contamination can be reduced by protective measures such as gargling with various antiseptic solutions before the procedure and performing dental procedures under rubber dam isolation. The high viral load in saliva is both a threat and an opportunity for humanity. Under the limitation to the results of the studies reported in this review, the use of saliva in the diagnosis of Covid-19 is a cost-effective, reliable and easy-to-apply technique compared to other disturbing detection forms. Further salivary studies should be encouraged to reduce the research gap in this area.ISS: conceived, designed and write the manuscript MGC: did data collection review, editing of manuscript and final approval of manuscript


During the COVID-19 pandemic, most countries implemented lockdowns that motivated changes in the dietary patterns, physical activity, and body mass index (BMI) of consumers worldwide, as well as the emergence of new food marketing strategies in social media. We sought to design and validate a methodology for monitoring and evaluating the Facebook marketing strategies of multinational fast-food chains in response to the COVID-19 pandemic.We developed three datasets. First, a dataset with the Uniform Resource Locators (URLs) of 1015 Facebook posts of five fast-food chains present in Argentina, Bolivia, Guatemala, and Peru. Second, a dataset of 106 content-analyzed posts we used in a pilot to determine intercoder reliability using statistical tests. Third, a dataset of a final sample of the 1015 content-analyzed posts that we used to determine the variables most frequently used. Following a mixed-methods approach, we developed 29 variables that recorded general information, as well as the marketing strategies we identified in the posts, including 14 COVID-19 specific variables. These data should help to monitor the social media marketing strategies that fast-food chains have introduced during the COVID-19 lockdowns, thus providing initial evidence about how they could be contributing to an unhealthy food environment. The objective was to design and validate a methodology for monitoring and evaluating the COVID-19 related Facebook marketing strategies of five multinational fast-food chains  present in Argentina, Bolivia, Guatemala, and Peru. The pandemic context led governments to implement lockdowns that influenced changes in the Body Mass Index (BMI), dietary patterns, and physical activity of children and adolescents , as wellPrevious studies analyzed the online marketing strategies food and beverage brands have used, including those adapted to social media platforms like Facebook –20. OtheThese data are unique since they contain a sample of 1015 Facebook posts collected from the country-specific fan pages of the previously mentioned fast-food brands during COVID-19 lockdowns. We also include information about the validation of our methodological tools. This work can help researchers analyze and compare the Facebook marketing strategies fast-food brands have introduced during this period, thus providing initial evidence about how they could have been contributing to an unhealthy food environment. The tools from this study can also empower public officials to monitor the social media environment over time in order to identify the marketing strategies of fast food companies that appear to be detrimental among vulnerable populations.We stored three datasets and the complementary documentation in the open data repository of the Inter-University Consortium for Political and Social Research (openICPSR) . The firWe restricted the period of data collection to one month before the start of the lockdown measures and three months after in each country: (1) Argentina: February 20–June 20th (lockdown started March 20); (2) Bolivia: February 22–June 22nd (lockdown started March 22nd); (3) Guatemala: February 16–June 16th (lockdown started March 16th); (4) Peru: February 16–June 16th (lockdown started March 16th).We used a mixed-methods approach to develop the methodological tools that would allow us to examine COVID-19 related content. This entailed two phases. During phase one, we manually extracted the screenshots and URLs of a random sample of 180 Facebook posts published within the specified months (around nine per brand for each country) and we assigned them unique identification codes. We included posts in every format allowed by the social media site: video, graphics interchange format (GIF) image, static image (one or more), slider, and text. Then, two researchers analyzed simultaneously these posts following a qualitative content analysis method, which led to consolidating a qualitative analysis codebook , based on the qualitative tool designed during phase one and on previous research . The codThe total sample of Facebook posts we collected for this study only includes those published by the selected brands during the four-month periods we specified before. Therefore, it does not provide a full picture of the evolution of the marketing techniques on Facebook or other social media platforms such as Instagram or Twitter for the duration of the COVID-19 pandemic, as they needed to adapt to a rapidly evolving context in which lockdown measures relaxed and tightened. This may also impact the comprehensiveness of our methodological tools, which could be subject to minor changes. Furthermore, given that Facebook posts include branded content under intellectual property rights, we were unable to include screenshots of the posts in our data deposit, as it is impossible to obtain the appropriate permissions from the fast-food companies. Instead, we have only included the URLs of each post we analyzed, which could produce data loss in the long term.


The deposition of a classic turbidite by a surge-type turbidity current, as envisaged by conceptual models, is widely considered a discrete event of continuous sediment accumulation at a falling rate by the gradually waning density flow. Here, we demonstrate, on the basis of a high-resolution advanced numerical CFD  simulation and rock-record examples, that the depositional event in reality involves many brief episodes of nondeposition. The reason is inherent hydraulic fluctuations of turbidity current energy driven by interfacial Kelvin-Helmholtz waves. The experimental turbidity current, with realistic grain-size composition of a natural turbidite, used only 26 to 33% of its in-place flow time for deposition, while the remaining time went to the numerous episodes of sediment bypass and transient erosion. The general stratigraphic notion of a gross incompleteness of sedimentary record may then extend down to the deposition time scale of a single turbidite. Sediment deposition from continuous turbidity current is highly discontinuous due to autogenic flow energy fluctuations. Thus, at the upper end of this range, a single turbidity current is occasionally capable of transporting more sediment than the annual global output of all rivers combined  flows, generated by an abrupt release of a fixed flow volume, and the sustained (longer-duration) flows, possibly with multiple pulses, which can stream for weeks to months and are generally attributed to a multisource release or river hyperpycnal effluent  simulation of a natural-scale, average surge-type turbidity current. Deep-sea turbidity currents are difficult to observe and monitor in nature  waves , with a −1 . The K-H−1 , and theAn important direct hydraulic consequence of the K-H waves and energy fluctuations were the corresponding instantaneous changes of the flow bottom shear stress, which fluctuated between 0.19 and 22 Pa. The plots of sediment accumulation and bed shear stress show alternating brief episodes of sediment accretion, nondepositional bypass, and transient erosion , insets.For the episodes of accretion, we have predicted the mode of sediment deposition , and a velocity of 1.8 m s−1. The mode of flow release imitated an average surge-type turbidity current generated by localized mid-slope slumping or issued from the outlet of a modest slope channel  from the inlet gate lasted 12 min. The flow monitoring along its axis was over a downslope distance of 800 m, spanning a bathymetric range of 80 to 150 m. With its tangential gradient and bathymetric range, the seafloor topography in the experiment resembled the slope of a large Gilbert-type delta or morphology of a large slump scar at shelf margin.In the experiment, a nonchannelized turbidity current was released from an inlet gate 12 m wide onto a submarine slope of 20° that tangentially flattened out to horizontal over a distance of 135 m. A similar tangential seafloor topography characterizes more than 70% reported cases of a submarine slope to basin-floor transition in natural settings . The assumption seemed reasonable because nonchannelized turbidity currents have little propensity for substrate erosion (


With many countries experiencing a resurgence in COVID-19 cases, it is important to forecast disease trends to enable effective planning and implementation of control measures. This study aims to develop Seasonal Autoregressive Integrated Moving Average (SARIMA) models using 593 data points and smoothened case and covariate time-series data to generate a 28-day forecast of COVID-19 case trends during the third wave in Malaysia. SARIMA models were developed using COVID-19 case data sourced from the Ministry of Health Malaysia’s official website. Model training and validation was conducted from 22 January 2020 to 5 September 2021 using daily COVID-19 case data. The SARIMA model with the lowest root mean square error (RMSE), mean absolute percentage error (MAE) and Bayesian information criterion (BIC) was selected to generate forecasts from 6 September to 3 October 2021. The best SARIMA model with a RMSE = 73.374, MAE = 39.716 and BIC = 8.656 showed a downward trend of COVID-19 cases during the forecast period, wherein the observed daily cases were within the forecast range. The majority (89%) of the difference between the forecasted and observed values was well within a deviation range of 25%. Based on this work, we conclude that SARIMA models developed in this paper using 593 data points and smoothened data and sensitive covariates can generate accurate forecast of COVID-19 case trends. The novel coronavirus, also known as COVID-19, was first detected in Wuhan, China on 31 December 2019. Within a short period, this virus has spread globally affecting many countries worldwide ,5,6.Similarly, Malaysia reported its first wave of COVID-19 infection from 25 January to 16 February 2020 involving 22 cases, of which the majority were imported cases from China. Following this, the second wave occurred from 27 February to 19 September 2020 involving 10,167 cases. Currently, Malaysia is experiencing a resurgence with the third wave, which began on 20 September 2020 . As of 3Several studies have modeled COVID-19 case trends to guide decision making on the management and control of the pandemic . In addiTo address these challenges, where disease parameters are not available, especially for novel diseases like COVID-19, time-series models such as the Auto-Regressive Integrated Moving Average (ARIMA) or Seasonal ARIMA (SARIMA) models can be used as suitable modeling methods that are relatively simple to develop and are able to produce forecasts with a single time-series dataset . WhereasIn Malaysia, ARIMA models were developed to forecast daily COVID-19 case trends during the second wave from 18 April to 1 May 2020 . Being aWhile time-series models are simple to develop and can generate accurate forecasts, few studies to date have forecasted COVID-19 case trends using these models in Malaysia ,30. Therhttp://covid-19.moh.gov.my/ (accessed on 1 November 2021)) [National- and state-level COVID-19 daily cases from 22 January 2020 to 5 September 2021 (593 data points) were sourced from the Ministry of Health (MOH) Malaysia official website (r 2021)) .p values < 0.05 indicate that the data are stationary [Statistical Package for the Social Sciences (SPSS) version 26.0  was used to develop, train and validate the time-series (SARIMA and ARIMA) models. Time-series models with a Ljung Box test (18) with a non-significant p-value, the lowest Bayesian information criterion (BIC), the lowest root mean square error (RMSE) and lowest mean absolute error (MAE) were selected to generate forecasts of COVID-19 cases for a duration of 28 days (6 September 2021 to 3 October 2021). The Augmented Dicky Fuller (ADF) test was performed in R programming software version 4.0.3 to confirm data stationarity, wherein ationary . In addiThe model development of SARIMA consists of two stages, namely, parameters estimation and model identification. The first stage was to estimate the non-seasonal  and seasonal  parameters, where p refers to the order of autoregressive (AR) component, d refers to the degree of differencing of the original time series and q refers to the order of the MA component. These were determined by the autocorrelation function (ACF) and partial autocorrelation function (PACF). In the parameter estimation stage, the unknown coefficients corresponding to the AR and MA components of the ARIMA model were estimated by implementing a multivariate regression analysis . All datp-value, the lowest RMSE, lowest BIC and lowest MAE.In the model identification stage, independent and dependent variables were used in several combinations in order to identify the best SARIMA model. The independent variable (covariate) used in the model identification was the state-level COVID-19 daily case data. We used case data from the state of Selangor as the covariate. The covariate was found to have no evidence of multicollinearity by using the variance inflation factor (VIF) test . The depA total of 415 data points were used for model training (22 January 2020 to 11 March 2021) and 178 data points were used for validation (12 March 2021 to 5 September 2021). This corresponds to a 70–30% partition of total data points for model training and validation, which is suggested as the optimal ratio in the literature . During p value > 0.05), a two-degree differencing was applied to transform the series to stationarity as shown in p-value < 0.05, therefore confirming stationarity. In addition, the model was validated using the Ljung-Box Q test, which suggested that the ACF for the residuals at different lag times was not statistically different from zero (the Ljung-Box Q test statistic (18) value was 52.628). The majority of the ACF and PACF residuals were within the 95% CI as shown in This study developed three different models arising from combinations of   with the inclusion or exclusion of seasonal component and the independent covariate. The three models used are shown in The SARIMA model   forecasts showed a downward trend of COVID-19 cases during the forecast period from 6 September 2021 to 3 October 2021 as shown in Being a novel disease, a detailed understanding of COVID-19 disease dynamics is limited resulting in difficulties to predict disease outbreaks. However, to ensure a balance between lives and livelihood, forecasting of disease outbreaks has become an important approach in assisting the management and control of the COVID-19 pandemic. It is evidence in the literature that forecasting using ARIMA models was able to provide reliable forecast results during the second wave of COVID-19 pandemic in Malaysia .Despite having limited and stochastic data, a 7-day cyclical trend was observed in the time-series data, whereby a reduction of cases was usually observed during the weekends, which could be due to variation in data collection and surveillance during weekends. Hence, analysis using a seasonal component was carried out to determine the most appropriate time-series model. This is the first study to develop SARIMA models to forecast COVID-19 cases in Malaysia during the third wave (6 September 2021 to 3 October 2021), wherein this study found that SARIMA model   was able to provide an accurate forecast despite the presence of limited stochastic data. To improve forecasts using SARIMA models, several measures were taken. Among them, these include using 593 data points for model development, smoothing time-series data of both the dependent and independent variables, which improved the identification of signals, and using appropriate sensitive covariates.n = 593), SARIMA models could be developed to generate forecasts of COVID-19 case trends up to a duration of 28 days. This is evident by the accurate forecasts of the COVID-19 case trends generated by the SARIMA model   which were similar to the observed downward case trends during the forecast period. In addition, the observed cases (7-day MA) were within the 95% CI of the model forecasts. Furthermore, 89% of the differences between the forecasted and observed values were well within a deviation range of 25%. Therefore, these findings indicate that the SARIMA model   generated an accurate forecast. Similar findings have been reported in the literature, wherein 593 data points generally improved the SARIMA model accuracy [The study found that with the available number of data points  was used so that the model may transmute the true data trends into signals. This, in turn, improved the overall trend of the dependent variable and provided better signals for data training and validation, which subsequently resulted in a more accurate forecast. Similar approaches using moving averages have been utilized in the past to obtain true signals from time-series data when developing ARIMA models .In addition, to improve the SARIMA model forecast, appropriate covariates such as the daily subnational Selangor state case report was used, as it accounts for the majority of the COVID-19 cases and has been consistently reporting high numbers of COVID-19 cases since the beginning of the pandemic in Malaysia. Furthermore, the case trends in Selangor state closely mirrored the observed national daily COVID-19 case trends. To further improve the model, as with the national’s time-series data, the COVID-19 daily cases in Selangor were also smoothened using a 7-day moving average in order to reduce the noise and improve signal detection. The generated goodness of fit showed very minimal difference between the smoothened and non-smoothened SARIMA model, but in terms of the model forecast, the model forecast with smoothened covariates showed a better resemblance to the actual COVID-19 daily cases. This study found that using a single appropriate covariate (Selangor state COVID-19 incidence with smoothening) is sufficient to develop accurate SARIMA models. This could be due to the selection of covariates that are truly representative of the COVID-19 case trends as well as having the ability to reflect the national-level trends to further improve the model forecast.Daily data were used in this study, and the weekly variation on reporting resulted in the seasonal model pattern observed in the study. Previous papers published by Hernandex et al. (2020) and Alraj et al. (2021) report ARIMA  and ARIMA  for forecasting COVID-19 cases using time-series model, respectively, which are different from the SARIMA model reported in this study. This variation could be attributed to methodological variations such the use of data smothering, covariates and seasonal effects as applied in this study, which were not reported in the work done by Hernandex et al. (2020) and Alraj et al. (2021) ,35. In aThere are several strengths of this study. First, a total of 593 data points were used for model training and validation. Second, data were smoothened to remove noise to obtain better signals. Additionally, we included a smoothened covariate that was able to generate good signals for the model development. Overall, these measures resulted in the development of an appropriate SARIMA model, which was able to produce reasonably accurate forecasts of COVID-19 cases. The duration of the data used for model development remain the main limitation in this study. However, this limitation was addressed by using covariates to develop the model. Future studies should look into developing time-series models with the availability of more data points as time progresses as well as using more covariates that are reflective of the transmission dynamics.n = 593) and appropriate covariates would provide more signals to train and validate the models, hence resulting in more accurate forecasts. This is especially important as the third wave has been by far the most challenging one faced by the health authorities in Malaysia. In addition, this study uses smoothened data to obtain more appropriate signals during the model development in order to improve the model accuracy.Since COVID-19 is a novel disease with continuously changing disease dynamics, using time-series data (This study showed that SARIMA models developed using 593 data points with sensitive covariates and smoothened data can generate a reliable and accurate forecast of COVID-19 case trends up to 28 days in Malaysia. The SARIMA models that were developed were used to advise the extension of the national MCO from phase 1 to 2 during the early stages of outbreak in Malaysia [