For motion in depth, even if the target moves at a constant speed in the real-world , it would appear to be moving with acceleration on the retina. Therefore, the purpose of this study was to determine whether real-world and retinal motion affect speed perception in depth and to verify the influence of eye movements on both motion signals in judging speed in depth. We used a two-alternative forced-choice paradigm with two types of tasks. One stimulus moved at a constant speed in the real-world (world constant task) with three conditions: 80–60 cm (far), 60–40 cm (middle), and 40–20 cm (near) from the participant. The other stimulus moved at a constant speed on the retina  with three conditions: 4–8 deg (far), 8–12 deg (middle), and 12–16 deg (near) as the vergence angle. The results showed that stimulus speed was perceived faster in the near condition than in the middle and far conditions for the world constant task, regardless of whether it was during fixation or convergence eye movements. In contrast, stimulus speed was perceived faster in the order of the far, middle, and near conditions for the retinal constant task. Our results indicate that speed perception of a visual target approaching the observer depends on real-world motion when the target position is relatively far from the observer. In contrast, retinal motion may influence speed perception when the target position is close to the observer. Our results also indicate that the effects of real-world and retinal motion on speed perception for motion in depth are similar with or without convergence eye movements. Therefore, it is suggested that when the visual target moves from far to near, the effects of real-world and retinal motion on speed perception are different depending on the initial target position. It is crucial in our daily life to determine the speed of moving objects or people so that we do not collide when driving a car or walking on the street. It has been reported that speed perception involves signals from the retina, which are based on the vergence angle , 2. WhenIt has also been reported that speed perception is determined based on information from extra-retinal signals in 2D motion , 5 or 3DNine participants  volunteered to take part in this experiment. They reported having normal or corrected to normal vision and no known neurological or oculomotor disorder. This study was conducted in accordance with the Declaration of Helsinki, and all protocols were approved by the Research Ethics Committee at the Faculty of Health and Sport Sciences, University of Tsukuba. Written informed consent was obtained from all subjects before their participation.In this study, we measured speed perception using methods of psychophysics and eye movements. To attain the first purpose, we conducted a world constant task and a retinal constant task using a two-alternative forced-choice (2AFC) paradigm to measure psychometric functions for speed perception. For the world constant task, the target moved 80 to 60 cm (far condition), 60 to 60 cm (middle condition) and 40 to 20 cm (near condition), which was equivalent to 4.3 to 5.7 deg (far condition), 5.7 to 8.6 deg (middle condition) and 8.6 to 17.1 deg (near condition) as the vergence angle. The target velocities were 20.0 cm/s (1000 ms), 22.2 cm/s (900 ms), 25.0 cm/s (800 ms), 28.6 cm/s (700 ms) and 33.3 cm/s (600 ms) .For the retinal constant task, the target moved 4 to 8 deg (far condition), 8 to 12 deg (middle condition) and 12 to 16 deg (near condition) as the vergence angle, which was equivalent to 86 to 43 cm (far condition), 43 to 29 cm (middle condition) and 29 to 21 cm (near condition). The target velocities were 4.0 deg/s (1000 ms), 4.5 deg/s (889 ms), 5.0 deg/s (800 ms), 5.5 deg/s (727 ms) and 6.0 deg/s (667 ms) .p < 0.05 and the goodness of fit of the model was p ≥ 0.05, the point of subjective equality (PSE) was calculated based on the psychometric function. In this study, we defined PSE as the speed of the 50% point on the fitted psychometric function.The probability that a participant indicated “Faster” for each velocity in the 2AFC paradigm was calculated on three different tasks. Psychometric functions using a binomial logistic regression analysis were also derived from the probability in three different conditions. If the statistical significance level of the model obtained from the analysis was The position data of convergence eye movements, which are disconjugate eye movements evoked by a visual target moving from far to near, were derived by subtracting the right eye position from the left eye position. Convergence eye velocity was generated by digital differentiation of the position arrays using a central difference algorithm in MATLAB . The onset of smooth convergence eye movements was taken as the time that the average eye velocity reached >3 times standard deviation (SD) above the pretrial values during fixation.r. r was defined as small when r < 0.3, medium when 0.3 ≤ r < 0.5, and large when 0.5 ≤ r. The method of Holm was used to adjust the p values in multiple testing [p < 0.05.We conducted the paired t-test in order to compare the speed perception between three different conditions in each experimental task. The effect size was calculated as  testing . All stat(8) = 5.61, p < 0.05, r = 0.89 and t(8) = 7.20, p < 0.05, r = 0.93, respectively). These results imply that the near condition was perceived faster than the far and middle conditions.We hypothesized that if speed perception relies on real-world motion, there would be no difference in speed perception between conditions for the world constant task, and we would perceive a target faster in order of far, middle and near conditions for the retinal constant task. In contrast, we hypothesized that if speed perception relies on retinal motion, we would perceive a target faster in order of the near, middle and far conditions for the world constant task, and there would be no difference in speed perception between conditions for the retinal constant task. For the world constant task, a typical example of psychometric functions is shown in p’s < 0.05). The paired t-test also showed that the probability was significantly higher in order of the far, middle and near conditions at target velocities of 5.0, 5.5 and 6.0 deg/s . These results indicate that visual target speed is perceived faster in order of the far, middle and near conditions.For the retinal constant task, there were no significant models from the binomial logistic regression analysis for the far and near conditions because the velocities of all comparison stimuli were clearly different from the standard stimuli. Therefore, we compared the probability of responding that the comparison stimulus was faster than the standard stimulus, in which the target always moves at 4.5 deg/s in the middle condition, between each condition . These rThe above results suggest that speed perception for a visual target moving from far to near is influenced by real-world motion rather than by retinal motion. However, speed perception could be influenced by retinal motion or other factors when a target moves close to the observer.Eleven participants  volunteered to take part in this experiment. They reported having normal or corrected to normal vision and no known neurological or oculomotor disorder. This study was conducted in accordance with the Declaration of Helsinki, and all protocols were approved by the Research Ethics Committee at the Faculty of Health and Sport Sciences, University of Tsukuba. Written informed consent was obtained from all subjects before their participation.The experimental setup in experimental 2 was identical with that in experimental 1 . To attaMethods for calculating the PSE and convergence eye movements were identical with experimental 1. We also calculated the mean velocity of convergence eye movements from 100 to 400 ms after the motion onset, which allowed us to quantify the speed of the initial oculomotor response, based on potential differences in latency, acceleration, or peak velocity . FurtherWe analyzed the relationship between the probability, which a participant indicated “Faster” in the 2AFC paradigm, and the mean retinal slip velocity during convergence eye movements in three different conditions for eye movement task using the Pearson product-moment correlation coefficient. The other statistical analysis was identical with experimental 1.Typical examples of psychometric functions for the eye movements task are shown in t(10) = 5.13, p < 0.05, r = 0.85; near and middle condition in the fixation task: t(10) = 8.34, p < 0.05, r = 0.94; near and far condition in the eye movements task: t(9) = 4.05, p < 0.05, r = 0.80; near and middle condition in the eye movements task: t(9) = 7.36, p < 0.05, r = 0.93). The paired t-test also showed that the PSE values in the middle and near conditions for the fixation task were significantly lower than that in the eye movements task (middle condition: t(10) = -3.93, p < 0.05, r = 0.78; near condition: t(10) = -3.12, p < 0.05, r = 0.72). These results imply that the near condition was perceived faster than the far and middle conditions in both tasks and the fixation tasks in the middle and near conditions were perceived faster than the eye movements tasks. Therefore, these results suggest that the effects of real-world and retinal motion on speed perception for motion in depth are similar in tracking a visual target with convergence eye movements and fixation. However, these results also indicate that convergence eye movements alter the speed perception of a visual target moving from far to near.For the fixation task, the mean values (± SD) of the PSE in the far, middle and near conditions were 25.1 ± 2.2, 25.9 ± 1.5 and 19.7 ± 1.8, respectively . For ther = 0.34, p = 0.72; middle condition: r = -0.15, p = 0.69; near condition: r = 0.30, p = 0.37).The correlation analysis showed that there were no significant relationships between the probability that participants indicated “Faster” in the 2AFC paradigm and the mean eye velocity at 25 cm/s of target velocity in all conditions  have demonstrated that participants did not utilize the acceleration of the visual target to motion in depth when estimating the time to contact, which largely supports our results . HoweverThe first factor is retinal motion, which depends on the vergence angle. The visual target speed on the retina exponentially increases as the real-motion target approaches the peripersonal space, which is defined as the region of space immediately surrounding our bodies in which we can grasp and manipulate objects . This waThe second factor is the near-response cells, which are specific cells activated when animals view a near target , 14. ThuThe third factor is looming. The looming is one of the depth cues and represents that the visual target image on the retina exponentially increases as the target approaches the observer . In prevThe fourth factor is a threatening stimulus. Previous studies have shown that the observer’s brain activity changes when a threatening stimulus is presented to one. For example, it has been reported that the activity of the ventromedial prefrontal cortex is specifically modified when the observer is exposed to dangerous and emotionally negative stimuli –26. Low This study revealed differences in the effects of retinal and real-world motions on speed perception for motion in depth. Further study is necessary because we have not verified the effect when the target moves from near to far. We speculate that if we conduct the same experiment as this study when the target moves from near to far, speed perception would be different from the results obtained in the near condition of this study. That is because the effect of the threatening stimulus, which is one of the factors affecting speed perception in the near condition, is removed. Therefore, it is necessary to examine the effect of different depth motion directions on speed perception in retinal and real-world motion in the future.For the eye movement task, the target speed in the near condition was perceived faster than that in the far and middle conditions as well as the results of the world constant task. Thus, the speed of an approaching visual target could be perceived as the speed of the real-world under the middle and far conditions. It is also suggested that target speed in the near condition was perceived based on retinal motion, convergence eye movement or other factors described above, rather than real-world motion. Therefore, the effects of retinal and real-world motion on speed perception for motion in depth could be similar with or without convergence eye movements. However, a visual target in the middle and near conditions for the fixation task was perceived faster than that for the eye movements task. Our results also showed that there are significant positive relationships between the probabilities that participants indicated “Faster” in the 2AFC paradigm and the retinal slip velocities in the middle and near conditions for the eye movement task. Nefs and Harris (2007) have shown that a visual target moving in depth is perceived slower when observers pursue it with their eyes than when they keep their eyes stationary . Some prThe results of the present study indicate that there were no relationships between the probability that participants indicated “Faster” in the 2AFC paradigm, and the mean velocity of convergence eye movements. It rather seems to be the case that variations in eye velocity are not directly related to perceived speed, although pursuing a target may cause some perceptual bias compared with motion perception during fixation. The Bayesian model implemented by Freeman and colleagues (2010) suggests that a visual target motion is important for perceiving head-centered velocity . It has We aimed to reveal the effects of retinal and real-world motion on speed perception of a visual target moving from far to near, and to verify the influence of eye movements on both motion signals in judging speed in depth. Our results demonstrate that speed perception of a visual target approaching the observer is dependent on real-world motion when a target position is relatively far from the observer. In contrast, when a target position is close to the observer, speed perception is related to retinal motion or other factors rather than real-world motion. Thus, it is suggested that when the visual target moves from far to near, the effects of real-world and retinal motion on speed perception are different depending on the initial target position. Moreover, we found that stimulus speed was perceived faster in the near condition than in the middle and far conditions, regardless of whether the observer was fixating on a stationary spot or tracking a target with eye movements. Therefore, it is suggested that the effect of real-world and retinal motion on speed perception when the target moves toward the body could be similar when fixating on a stationary spot and tracking a target with convergence eye movements.


From 2013–2015, a CPAP quality improvement program (QIP) was implemented to introduce and monitor CPAP usage and outcomes in the neonatal wards at all government district and central hospitals in Malawi. In 2016 the CPAP QIP was extended into healthcare facilities operated by the Christian Health Association of Malawi. Although clinical outcomes improved, ward assessments indicated that many rural sites lacked other essential equipment and a suitable space to adequately treat sick neonates, which likely limited the impact of improved respiratory care. The aim of this study was to determine if a ward-strengthening program improved outcomes for neonates treated with CPAP. To address the needs identified from ward assessments, a ward-strengthening program was implemented from 2017–2018 at rural hospitals in Malawi to improve the care of sick neonates. The ward-strengthening program included the distribution of a bundle of equipment, supplemental training, and, in some cases, health facility renovations. Survival to discharge was compared for neonates treated with CPAP at 12 rural hospitals for one year before and for one year immediately after implementation of the ward-strengthening program. In the year prior to ward strengthening, 189 neonates were treated with CPAP; in the year after, 232 neonates received CPAP. The overall rate of survival for those treated with CPAP improved from 46.6% to 57.3% after ward strengthening (p = 0.03). For the subset of neonates with admission weights between 1.00–2.49 kg diagnosed with respiratory distress syndrome, survival increased from 39.4% to 60.3% after ward strengthening (p = 0.001). A ward-strengthening program including the distribution of a bundle of equipment, supplemental training, and some health facility renovations, further improved survival among neonates treated with CPAP at district-level hospitals in Malawi. Globally, 85% of neonatal deaths are attributed to complications from prematurity and low birthweight, intrapartum-related hypoxia, and infection . NeonateOver 91% of deliveries in Malawi occur in a health facility . HoweverTo address limitations in the capacity to treat neonates with respiratory distress, a quality improvement program (QIP) for neonatal care in Malawi was implemented in 2013–2015 to introduce the use of bubble Continuous Positive Airway Pressure (CPAP) at all government district and central hospitals in Malawi . In 2016In response to the challenges experienced during the CPAP QIP, in 2017 we coordinated detailed neonatal ward assessments at all government district hospitals and CHAM facilities to determine specific challenges and limitations at each site. For each hospital, members of their own administrative, clinical and technical/maintenance staff, together with MOH representatives, completed the ward assessments. Together, MOH representatives and research staff determined which parameters to evaluate; only parameters that directly impacted the quality of newborn care were prioritized to avoid burdening hospital staff and prevent disruption of work flow during the assessments. The assessments documented demand and capacity , staffing levels , and equipment inventory . Facilities lacking essential equipment and/or suitable space for a neonatal unit were selected for inclusion in a ward-strengthening program.We also assessed neonatal training programs at fifteen institutions that train nurses and clinicians to ensure sustainability in the transfer of knowledge and skills. The assessment showed that, despite the presence of neonatal care concepts in training curricula at all institutions, there were gaps in teaching these concepts, particularly due to poorly furnished skills labs. All fifteen training institutions were included in a program to strengthen skills labs for neonatal care.From 2017–2018, we implemented a ward-strengthening program at 12 government district hospitals and CHAM facilities to improve the care of sick neonates. Ward-strengthening activities included the distribution of a bundle of equipment , supplemTen of the 12 facilities lacked a separate or suitable neonatal unit. At these sites, the facility management team worked with the Ministry of Health (MOH) to identify a room or space for renovation, quantify material needs for the renovation, obtain quotations for the materials, and develop a renovation work plan. Renovations varied by facility, but generally included the expansion or partitioning of an existing space, plumbing installation and repairs, installation of lighting and electrical sockets, roofing and ceiling installation and repairs, and painting. For nine of the ten sites where renovations were completed, renovation costs ranged from $1,300 - $8,600 USD and were covered by project funds. At the remaining site, the cost of construction of a new ward ($38k USD) was supplemented by private donor funds.At each site, equipment was distributed according to the bed capacity of the unit ; for a uIn collaboration with the Paediatric and Child Health Association of Malawi (PACHA), members of the research team conducted training for the nurses and clinicians at the facilities using the Care of the Infant and Newborn (COIN) course. The COIN course, which is used nationally, focuses on diagnosis and triage of sick neonates, care at birth for all neonates, neonatal resuscitation, routine postnatal care of the neonate, emergency assessment and treatment of neonates, and identification of neonates in need of referral and safe transport .After the completion of the ward-strengthening program, we coordinated follow-up ward assessments at each healthcare facility. The follow-up ward assessments documented the same variables as the initial ward assessments . We compared the follow-up ward assessments to those conducted prior to the ward-strengthening program to evaluate changes in demand, capacity, and staffing levels.Fifteen health training institutions received support to establish skills labs for neonatal care, including equipment and teaching materials to facilitate hands-on teaching of essential skills in the care of neonates. Materials provided included training models, oxygen concentrators, glucometers, CPAP machines, suction machines, phototherapy lights, and teaching aids. Academic staff from these institutions also received training to encourage use of these resources within their institutions.We conducted an observational study to evaluate the impact of the ward-strengthening program at 12 district and CHAM hospitals located in rural areas  where CPDifferences in transfer rates and survival before and after ward strengthening were compared using a two-sided Fisher’s exact test. Differences between continuous variables  were assessed using a two-sided t-test for equality of means . Results were considered significant at the 5% level. Data were analyzed in Excel.The protocol was approved by the National Health Sciences Research Committee (NHSRC #1180) of Malawi and the Institutional Review Board of Rice University (13-102X). De-identified patient information was collected from standard Ministry of Health Acute Respiratory Illness forms, and the need for informed consent was waived by the ethics committees.Following implementation of the ward-strengthening program, there was a significant increase in survival for neonates treated with CPAP , and forThe admission weights, admission temperatures, and age on admission for neonates treated before and after ward strengthening are compared in We previously reported that implementation of a nurse-led CPAP QIP to treat neonates with respiratory illness in all government district and central hospitals in Malawi led to a significant increase in survival, particularly among neonates of low birth weight . FollowiDuring mentorship and supervisory visits to rural and district hospitals, many challenges were identified that likely adversely affected overall survival as well as limited the impact of improved respiratory care. Despite provision of supportive respiratory equipment, training, and peer mentorship along with the CPAP devices, additional equipment, training, and space were necessary to allow more comprehensive care of vulnerable neonates, who often have other complications in addition to respiratory distress. After the ward-strengthening program was introduced, survival rates for neonates weighing 1.00–2.49 kg with a diagnosis of RDS improved from 39.4% to 60.3%, approaching the 64.6% survival previously seen in a referral hospital .To identify facility-level barriers to improved neonatal care, the needs assessment were undertaken in each hospital by members of their own administrative, clinical and technical/maintenance staff, together with MOH representatives. Building modifications and equipment and staffing needs were thus directed by local requirements. Furthermore, MOH representatives and research staff collaborated to determine which parameters to evaluate, prioritizing those that directly impacted the quality of newborn care. Since hospital staff were familiar with the parameters being evaluated, assessments were completed as part of routine visits to the facilities, preventing the need for any intensive training and minimizing the disruption of clinical activities. Importantly, this collaborative effort gave ownership and investment to local hospital staff and in itself strengthened interdepartmental relationships.The most common requirement was that of space. Ten of the twelve facilities did not have a separate neonatal ward; if a separate area was available, it was too small or otherwise unsuitable. The absence of a neonatal ward is common in hospitals in many countries , 17. As The care of sick neonates also requires specialized training and skills. Neonatal nursing training programs are rare in low-income countries , and a hWe note that improved space, equipment, and training had important additional beneficial effects. Previously, sick and healthy neonates often remained with their mother in the postnatal ward, but the creation of a separate, well-equipped ward for sick neonates meant that staff were allocated specifically for their care; overall, nurse allocations to the neonatal wards increased and at least one clinician was assigned to the neonatal ward in each facility.As this observational study involved neonates undergoing routine care in a low resource setting, there were some unavoidable limitations. The primary outcome of this study was survival to discharge, as follow-up after discharge was not feasible. Few, if any, neonates had a chest x-ray to diagnose RDS, and gestational age as determined by a first trimester ultrasound was rarely available. Consequently, RDS was diagnosed clinically with the aid of an algorithm to help identify neonates who might benefit the most from CPAP. Although retinopathy of prematurity was addressed during trainings , we did When reflecting on our program, it is clear that efforts to improve the quality of neonatal care must be built on a solid foundation of good basic clinical care that is based on a holistic approach to the needs of a neonate. Improvement in one aspect of need (such as respiratory support) will falter without the provision of warmth, nutrition, infection control, and family-centered care given by trained staff in a dedicated space. A needs assessment is essential to identify gaps in overall care at the facility level so that these can be addressed with an appropriate bundle of interventions to improve care for all infants in the ward. Provision of materials is insufficient without training, supervision, and mentoring to ensure that equipment is used properly and protocols followed correctly.Some challenges are beyond the jurisdiction of hospital management to solve: an unreliable power system and irregular water supply are common and affect many of the facilities included in this program. While power and water outages were not systematically tracked during this audit, power outages were noted on the charts of 16.4% of neonates treated before, and 15.9% treated after ward strengthening. Furthermore, availability of essential commodities and consumables is a challenge if hospitals lack the resources or ability to procure them.It is encouraging to see that the physical changes made to the neonatal ward areas led to improved staff allocation to work in them. In initial discussions about possible space renovations, MOH representatives strongly advocated that each facility dedicate staff solely to planned new units to improve quality of care. Based on our observations, better staffing, more training and mentorship have improved morale, and by planning and creating the changes together, the hospital departments are more interactive. All of these factors have played a part in reducing neonatal mortality.To improve neonatal outcomes, healthcare facilities need the infrastructure, capacity, and resources to treat small and sick neonates. This means having a family-centered approach to care encompassing the provision of warmth, nutrition, and infection control given by trained staff in a dedicated space with functioning equipment. Providing CPAP within such a framework significantly improved survival for neonates with respiratory distress. These improvements in service availability lead to improved staff morale, increased the number of dedicated neonatal staff, and, most important, improved outcomes for admitted sick and small neonates treated with CPAP.


Mycobacterium marinum is a non-tuberculous mycobacterium that presents as a nodular granulomatous disease. The bacillus can infect humans when broken skin is exposed to a contaminated aquatic environment. M. marinum infections are usually isolated to the skin and soft tissues and can spread in a lymphatic distribution. A 26-year-old male cut his right ankle while spelunking in Tulum, Mexico. He presented to his primary care physician three months after he sustained the laceration with a nonhealing wound on the right lateral posterior ankle. Examination of the lesion demonstrated erythematous, violaceous, and hyperpigmented indurated plaques with satellite lesions noted at the right medial, posterior, and lateral ankle. The lesion characteristics raised initial suspicion for an invasive fungal infection. Biopsy of the lesion demonstrated epidermal ulceration covered by neutrophilic serum, marked underlying dermal acute inflammation, and granulation tissue. A mild perivascular, predominantly lymphocytic infiltrate was present in the deep dermis with no evidence of granuloma. Acid-fast bacilli culture plated onto chocolate agar confirmed the species M. marinum. Mycobacterium marinum is a non-tuberculous mycobacterium affecting 0.27 cases per 100,000 inhabitants that presents as a nodular granulomatous disease [M. marinum infections are usually isolated to the skin and soft tissue in immunocompetent individuals and spread in a sporotrichoid distribution [This article was previously presented as a poster abstract at the 2022 American Society of Dermatopathology Annual Scientific Meeting on October 22, 2022.A 26-year-old male with no significant past medical history sustained a small laceration on his right ankle while spelunking in Tulum, Mexico. Three months after the initial injury, he presented to his primary care physician with a nonhealing wound on his right ankle. Examination of the lesion demonstrated erythematous-to-violaceous indurated papules coalescing into plaques Figure . The lesM. marinum.Biopsy demonstrated skin with ulceration, neutrophilic serum, and adjacent reactive epidermal hyperplasia with an underlying neutrophil-rich superficial dermal infiltrate Figure . In the M. marinum is an endemic fish pathogen that is found in an assortment of aquatic environments such as natural water bodies, swimming pools, fish tanks, and aquariums [M. marinum infections are indolent with an intermediate incubation period of 16 days [M. marinum disease can take on a sporotrichoid pattern in about 20% of cases in which the local inoculation site is associated with nodular or ulcerating lesions that spread proximally up the lymphatics and to regional lymph nodes [quariums . M. mari 16 days . The inf 16 days . Over ti 16 days . The les 16 days . However 16 days . Though ph nodes .M. marinum infections may be confused with other differentials such as leishmaniasis, histoplasmosis, coccidioidomycosis, blastomycosis, and other fungal infections. Evaluation should include patient history, risk factors, duration of disease, site and morphology of lesions, as well as medical history [ M. marinum can be challenging due to the difficulty in culturing the bacteria and its nonspecific clinical polymorphism [ history . Diagnosmorphism . CultureDiagnosis is confirmed with bacterial growth in acid-fast bacilli culture at 25-30°C and biopsy. The histology shows suppurative inflammation in acute cases and mixed granulomatous inflammation with scattered multinucleated giant cells and neutrophils in long-standing cases [M. marinum infection is typically prolonged, requiring months of antibiotics to attain clearance. M. marinum can be particularly difficult to treat as an organism due to increasing multidrug resistance [Treatment of sistance . In casesistance . Clarithsistance . For sevsistance .This patient was empirically started on 100 mg of doxycycline twice daily. Two months into treatment, the erythema and tenderness of the lesions improved and began to heal without ulceration, necrosis, or discharge. Four months into treatment, the lesions continued to improve and the appearance of new lesions decreased significantly. The patient was counseled to stop doxycycline therapy after five months of treatment or follow-up if new lesions occurred. His treatment course lasted for five months. M. marinum is injury followed by exposure to contaminated marine environments. M. marinum primarily affects the superficial skin of extremities and classically appears as a nodular granulomatous disease. Though the infection may be self-limited in some individuals, the lesion has been reported to progress to a sporotrichoid pattern in others. Clearance of the infection can be achieved with monotherapy but may require polytherapy if it is severe and extends beyond a superficial cutaneous infection. This case demonstrates a classic history, clinical presentation, and histopathology of a rare infection in the United States.The primary form of inoculation by


YouTube plays an influential role in disseminating health-related information in the digital age. This study aimed to evaluate YouTube videos on transcutaneous electrical nerve stimulation (TENS) in terms of their information value and quality.In this descriptive study, we ranked the first 100 videos that met the inclusion criteria using the search term “transcutaneous electrical nerve stimulation” on October 30, 2022. These videos were classified according to the number of views, likes, dislikes, comments, duration, popularity and content categories. Reliability, quality, and accuracy of the videos were assessed using the Journal of American Medical Association (JAMA) Benchmark Criteria and Modified DISCERN Questionnaire and Global Quality Score (GQS). Video popularity were calculated by the Video Power Index (VPI).p = 0.226, p = 0.115, p = 0.812). Notably, there was a weak positive correlation between the JAMA scores and the number of views .Based on the GQS results, we found that 59, 27, and 14 videos had low, intermediate, and high quality, respectively. In addition, based on the JAMA results, 79 and 21 videos had poor and high reliability, respectively. No statistically significant difference was found between the JAMA, modified DISCERN and GQS scores in terms of the sources of videos (According to our study results, most YouTube videos on TENS were of low quality and reliability. Additionally, most videos were uploaded from sources created by doctors; the most frequently found content was about the TENS procedure, and content on complications of the procedure became less frequent as the videos became more recent. In particular, it was found that videos uploaded by academics have longer duration. It has been found that reliable videos with high JAMA scores also have high number of views. Accordingly, it can be concluded that videos with higher quality and more reliability that are created by healthcare providers will be more useful for patients seeking information about TENS. Pain is known to be a major global health problem, and it is estimated that one in five adults suffers from pain annually and one in 10 adults are diagnosed with chronic pain . In addivia electrodes placed on the skin surface . TENS exerts its effects by reducing pain perception through excitatory and inhibitory synapses in the dorsal horn of the spinal cord via the pain-gate mechanism, as proposed by Mellzack and Wall in the mid-1960s .https://www.youtube.com/) search engine. Further, a neutral term was used to create a broader pool of videos. In the event of disagreements between the two authors in the video evaluation, a third independent author (Y.E.) made the final decision. Notably, the Google Incognito window was used for the study to avoid bias due to search history and cookies. Videos with TENS-related content of acceptable audiovisual quality (without audio–video problems preventing accurate assessment) and those published in English were included in the present study. In contrast, non-English videos, videos without auditory or visual stimuli, and repetitive videos were excluded from the study  identified videos containing medical content using the search term “transcutaneous electrical nerve stimulation” on the YouTube , and it ranges from 0 to 5. Higher scores represent higher reliability  Table 1Table 1.The Journal of American Medical Association (JAMA) Benchmark is another reliability scale that examines online videos and resources based on four criteria: authorship, disclosure, currency, and attribution. It is used to assess the accuracy and reliability of the relevant videos. Each video was evaluated based on the four abovementioned criteria, and 1 point was awarded for each criterion found in the video. The final score ranges from 0 to 4. Higher scores represent higher reliability  Table 1Table 1. Global Quality Score (GQS) is a 5-point Likert scale used to assess the quality of videos. GQS scores range from 1 to 5, and higher scores represent higher quality. Videos with a score of 1 or 2, 3, and 4 or 5 are considered to have low, intermediate, and high quality, respectively  Table 1Table 1. For each video, the following five different engagement metrics were recorded: views, likes, dislikes, video duration (in seconds), and comments. The impact and popularity of the videos were determined using the Video Power Index (VPI). (VPI = like count/(like count + dislike count) × 100) .Video sources were categorized as academic sources, society/professional organizations, physicians, commercial and health-related websites. In addition, the country and continent of the upload were recorded. Further, the videos were evaluated based on whether they contained animation.n) and percentage (%), and continuous variables were expressed as mean ± standard deviation. Kolmogorov–Smirnov and Shapiro–Wilk tests were used to assess whether the data showed a normal distribution pattern. Mann–Whitney U and Kruskal–Wallis tests were used to analyze continuous variables between the study groups, whereas Pearson’s chi-square test was used to analyze categorical variables between the study groups. Spearman’s correlation test was used to analyze the correlation between variables. A p-value of <0.05 was considered to indicate a significant difference.Acquired data were analyzed using the Statistical Package for Social Sciences  24.0 software. Data indicating frequency were expressed as number (p = 0.469). Although there was no statistically significant difference in the number of videos with animations by continent (p = 0.303), a significant difference was found in the number of videos with animations by country (p < 0.001). Accordingly, 41.7% of the videos with animated content were from the US, whereas no animated content was found in videos from India , 21 non-English videos, 18 repetitive videos, and 17 videos with no sound or video content from the total 170 videos . If multom India .Regarding the evaluation of videos according to their sources, it was found that 40 (40%) were uploaded by physicians, 25 (25%) by health-related websites, 11 (11%) society/professional organizations, 13 (13%) by commercial websites and 10 (10%) videos by academic sources .p = 0.026). No significant difference between years was found between the other content categories , 31 (31%), 30 (30%), 81 (81%), and 44 (44%) contained information on indication, contraindications, complications, procedures, and TENS types, respectively. In particular, when analyzing the content categories by year, videos with information on complications were uploaded more frequently in 2018 or previous years .According to the results of GQS used for quality assessment, 59, 27, and 14 videos were determined as low, intermediate quality, and high quality, respectively. We found that the mean GQS score was 2.42 ± 0.95. In addition, based on the GQS, JAMA and DISCERN results, no significant difference was found between the video sources . Accordingly, videos with a long duration are mostly of academic origin. In addition, no statistically significant difference was found when video parameters were evaluated according to video quality scores (GQS) . When video parameters were evaluated according to the grouping based on JAMA scores , a statistically significant difference was found in the number of likes and comments . Accordingly, videos with high reliability received more likes and comments , 40 (40%), and 50 (50%) videos had completely sufficient, partially sufficient, and insufficient data. According to the JAMA results, 79 (79%) videos had low reliability, and the remaining 21 (21%) videos had high reliability. According to the DISCERN results, 23, 46, 14, 15, and 2 videos were of very poor, poor, fair, good, and excellent quality, respectively. The mean DISCERN score was 2.27 ± 1.04.r = 0.204, p = 0.041). A moderate positive correlation was found between the JAMA and modified DISCERN scores . A moderate to strong correlation was found between VPI scores and the number of views , likes , dislikes  and comments . No correlation was found between VPI scores and rating scales  (A weak positive correlation was found between the JAMA scores and views (= 0.658) .p = 0.001), likes (p < 0.001), dislikes (p = 0.001), comments (p = 0.001), and JAMA scores (p = 0.002), but there was no difference between the continents in terms of duration (p = 0.129). When the videos in the study were sorted by country, they were determined as follows; USA 68, India 11, United Kingdom 10 and other countries 11 . A statistically significant difference was found between the countries where the videos were uploaded in terms of the number of views (p = 0.008), likes (p = 0.005), dislikes (p = 0.016), comments (p = 0.033), VPI scores (p = 0.020) and JAMA scores (p = 0.024) as well as in terms of the animation content (p < 0.001)  . Videos In the present study, YouTube videos on TENS, which patients can use for many indications, especially pain symptoms, were evaluated according to content categories, quality, reliability, and user interaction criteria. For the study, we focused only on TENS, which is one of the noninvasive methods in pain management. A 2016 systematic review of 13 studies involving 267 patients reported that TENS led to significant improvements in pain lasting 2–24 weeks . TENS ise.g., 20 min per day, three times per day)  . The lacAlthough YouTube was originally created for entertainment purposes, medical information videos has been the subject of extensive research. Because YouTube has no inherent mechanism to verify the accuracy and reliability of uploaded videos, it is possible for patients to be exposed to misinformation. It is obvious that this misinformation can negatively affect healthcare consumers. For example, it has been reported that YouTube may have the potential to change patients’ beliefs about controversial issues such as vaccines . Lau et In the present study, when videos were evaluated based on their sources, videos uploaded by physicians (40%) and health-related websites (25%) accounted for a significantly higher number of videos. Similarly, Based on the analysis of video content categories, it was found that 81 (81%) of the videos evaluated in the present study provided information about the procedure, followed by 79 (79%) videos providing information about indications. Similar to the present study, Of the videos evaluated in the present study, 68%, 11%, and 10% were uploaded from the USA, India, and the UK, respectively. In terms of the continent, 71% of the videos were uploaded from America and only 29% were from non-American continents. Consistent with the results of the present study, via questionnaires may have impacted the results.This study has some limitations. First, only videos that were available on YouTube within a certain period were included in the evaluation. However, it is known that YouTube’s ever-changing dynamic content structure may lead to different results in a different study conducted in a different period. In addition, the exclusion criteria used during the selection of the first 100 videos also cause differences in the videos included in the study in different studies. Second, topic-related search terms can be vary and thus influence the results of the study. Therefore, we tried to maximize the quality of the results using a neutral term. Third, only videos in English were included in the study. Finally, although the two authors were blinded to each other, the subjective assessment of the videos The present study examining the analysis of TENS in YouTube, revealed that the videos were generally of low reliability and quality. It has been found that videos with high reliability scores have more views and academic videos have longer durations. It was determined that there was no difference between reliability and quality scores according to video sources. It has been determined that procedural issues are mentioned the most in the videos and complications are less mentioned as the videos became more recent. There is a need for videos with high quality and reliable content to be presented to patients and users. Future studies exploring the usefulness of this types of videos in patient health may produce significant positive improvements in public health.10.7717/peerj.15412/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.15412/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.15412/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.15412/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.15412/supp-5Supplemental Information 5Click here for additional data file.


Plasma Mg2+ homeostasis is maintained primarily through dietary intake and reabsorption in the kidneys. Inadequate dietary Mg2+ intake over several months results in slow depletion of plasma Mg2+ and chronic latent hypomagnesemia, a condition prevalent in women of reproductive age worldwide. Mg2+ is critical for normal embryonic development and neural tube closure. We hypothesized that DTG therapy might slowly deplete plasma Mg2+ and reduce the amount available to the embryo, and that mice with pre-existing hypomagnesemia due to genetic variation and/or dietary Mg2+ insufficiency at the time of conception and initiation of DTG treatment would be at increased risk for NTDs. We used two different approaches to test our hypothesis: 1) we selected mouse strains that had inherently different basal plasma Mg2+ levels and 2) placed mice on diets with different concentrations of Mg2+. Plasma and urine Mg2+ were determined prior to timed mating. Pregnant mice were treated daily with vehicle or DTG beginning on the day of conception and embryos examined for NTDs on gestational day 9.5. Plasma DTG was measured for pharmacokinetic analysis. Our results demonstrate that hypomagnesemia prior to conception, due to genetic variation and/or insufficient dietary Mg2+ intake, increases the risk for NTDs in mice exposed to DTG. We also analyzed whole-exome sequencing data from inbred mouse strains and identified 9 predicted deleterious missense variants in Fam111a that were unique to the LM/Bc strain. Human FAM111A variants are associated with hypomagnesemia and renal Mg2+ wasting. The LM/Bc strain exhibits this same phenotype and was the strain most susceptible to DTG-NTDs. Our results suggest that monitoring plasma Mg2+ levels in patients on ART regimens that include DTG, identifying other risk factors that impact Mg2+ homeostasis, and correcting deficiencies in this micronutrient might provide an effective strategy for mitigating NTD risk.In 2018, data from a surveillance study in Botswana evaluating adverse birth outcomes raised concerns that women on antiretroviral therapy (ART) containing dolutegravir (DTG) may be at increased risk for neural tube defects (NTDs). The mechanism of action for DTG involves chelation of Mg Neural tube defects (NTDs) are congenital malformations of the brain and/or spinal cord  that result from failure of the neural tube to close properly during early embryonic development. The etiology of NTDs is multifactorial, involving complex gene-nutrient-environment interactions. Numerous factors that contribute to NTDs are known, including maternal nutritional deficiencies, metabolic disorders , genetic variants, environmental toxins, and pharmaceutical exposures . It has In 2018, a surveillance study in Botswana evaluating adverse birth outcomes among women on different antiretroviral therapy (ART) regimens reported a 9-fold increase in the prevalence of NTDs among women who began treatment with dolutegravir (DTG) prior to conception . DTG is In response to the safety alert, the Botswana Harvard AIDS Partnership conducted a larger follow-up study, and found that the prevalence of DTG-NTDs  was lower than in the original report, although still elevated compared to other ART regimens (.10%) . NotablyThe preclinical embryo-fetal development (EFD) studies found no significant evidence of DTG-related maternal or developmental toxicity when pregnant animals were administered oral doses of DTG up to 1000 mg/kg/d . AnimalsAfter the report from the Tsepamo Study was released, several laboratories conducted investigations using animal or cell-based models to examine potential adverse effects of DTG, most of which focused on the folate pathway. Studies in Madin-Darby canine kidney II cells did not find a significant DTG or INSTI class effect on inhibition of folate transport . In contIn 2 different human embryonic stem cell (hESC) lines, DTG exposure resulted in cytotoxicity and a dose-dependent loss of pluripotency . In a 3-ex vivo and exposed to DTG during the window of neurulation (GD 9–11] (Rfc1 and downregulation of Folr1) were detected in GD 10.5 placentas ; however, when the mice were maintained on a folate-sufficient diet, a modest increase in Folr1 mRNA occurred only at the 2.5 mg/kg/d DTG dose, and no changes were observed at the 5x higher dose  used were calculated to approximate or slightly exceed human exposure based on established plasma levels in patients. However, the % of NTD-affected litters and the mean litter rate was small, which would make it difficult to detect with any certainty whether maternal folate supplementation in vivo has a protective effect.Collectively, key findings from human, cell-based, and animal studies demonstrate an important connection between very early (pre-gastrulation) exposure to DTG and potential for disruption of morphogenesis, axial patterning, gastrulation, and neural tube closure. Nonetheless, studies evaluating a possible connection between DTG and folic acid rescue or altered transport of folic acid in different models have yielded variable results, suggesting the relationship between DTG and folate may be indirect, rather than direct. In published 2+) ions in the active site of the HIV integrase, inhibiting its catalytic activity and preventing incorporation of viral DNA into the host genome  for pregnant women  Mgorldwide . Inadequgnesemia . Analysiinfected . In the nt women . Women ont women .2+ homeostasis in the body are the transient receptor potential melastatin (TRPM) divalent-selective ion channels TRPM6 and TRPM7  and the importance of Mg2+ in neural tube closure, we hypothesized that if DTG administration was initiated at conception, slow depletion of ionized Mg2+ in maternal plasma might occur over time, reducing the amount available for transport across the placenta as the pregnancy progressed. In dams unable to compensate and maintain Mg2+ homeostasis through increased dietary intake and/or increased renal reabsorption, the level of available Mg2+ might be inadequate to support normal neural tube closure. In addition, the unbound fraction of DTG that crosses the placenta might chelate Mg2+ present in coelomic or amniotic fluid and/or embryonic tissues. We hypothesized that in mice with pre-existing  hypomagnesemia due to genetic variation and/or dietary Mg2+ insufficiency at the time of conception/initiation of DTG treatment, Mg2+ levels would be more likely to drop below a critical threshold before the onset of neurulation. In other words, in mice with underlying nutritional and/or genetic risk factors that compromise Mg2+ homeostasis but still allow embryogenesis to proceed normally, the addition of DTG might ‘tip the balance’ to create the perfect storm for development of NTDs.Based on the known mechanism of action for DTG  to high. In the first set of experiments, these 3 mouse strains were maintained on the same diet and administered the same dose of DTG. Litter outcomes were compared across strains for susceptibility to DTG-NTDs based on genetic variation in plasma and urine Mg2+ levels. In the second set of experiments, we placed mice on diets with different concentrations of Mg2+  to examine the effect of reduced dietary Mg2+ intake on susceptibility to DTG-NTDs. In both experiments, plasma and urine Mg2+ levels were determined before mice were placed into timed mating. Dams were treated with vehicle or DTG by daily oral gavage, beginning on GD 0.5, and embryos examined for NTDs on GD 9.5. Plasma DTG levels were measured for pharmacokinetic (PK) analysis. In a third experiment, we analyzed whole-exome sequencing (WES) data from our inbred mouse strains to determine if the strain most susceptible to DTG-NTDs had variants in genes involved in Mg2+ homeostasis.To test our hypothesis concerning pre-existing perturbations in Mg2+ and the C57BL/6J strain for high (1.19 mmol/L) serum Mg2+. These 2 strains were purchased from Jackson Labs . The SWV and LM/Bc  strainsad libitum access to autoclaved water and pelleted irradiated rodent chow. For timed matings, nulliparous female mice were placed with a male overnight . The presence of a vaginal plug the next morning was considered GD 0.5. Plugged females were weighed, placed into a separate cage, and randomly assigned to a treatment group. All animal procedures were performed in accordance with the Public Health Service (PHS) policy on Humane Care and Use of Laboratory Animals and approved by the Creighton University Institutional Animal Care and Use Committee (IACUC).Breeding colonies and experimental mice were housed in Thoren micro-isolator cages with autoclaved corncob bedding and maintained on a 12-h light-dark cycle in climate-controlled AAALAC-accredited animal facilities at Creighton University. Mice were given ®: 50 mM N-methylglucamine in 3% mannitol )  was purchased from MedKoo Biosciences . Pregnant mice were dosed with vehicle (dimethylsulfoxide: Solutol v:w:v])  or DTG (According to the 2+ requirement in adult mice is 0.5 ppm of diet (0.05%). Magnesium in standard rodent diets ranges between 2–3 g mg/kg; the Envigo #8904 soy diet contains .3% Mg2+ by weight (5 times the NRC requirement) and was therefore considered a ‘high’ Mg2+ diet. Sources of Mg2+ in the soy diet include magnesium oxide (∼20%) and Mg2+ found naturally in grain ingredients (∼80%); it was therefore not possible to remove Mg2+ to create a soy-based version that was Mg2+ deficient. To test the hypothesis that low maternal plasma Mg2+ prior to conception would increase susceptibility to DTG-induced NTDs, it was necessary to switch to casein-based purified ingredient diets with defined levels of Mg2+ added back. The following casein-based purified ingredient diets were selected as experimental diets: TD.120,574 high Mg2+ diet (.3% Mg2+ oxide added to match the .3% high Mg2+ soy-based diet); TD.98341 control Mg2+ diet with 0.05% Mg oxide added back to match the NRC requirement; and TD.93106 Mg2+-deficient diet . The purified casein diets met or exceeded the NRC requirements for all other minerals and vitamins, including folic acid (2+ (.3%) soy diet; 2) high Mg2+ (3%) casein diet; 3) control Mg2+ (.05%) casein diet; and 4) deficient Mg2+ (<.003%) casein diet. C57BL/6J mice placed on severely Mg2+-deficient diets (<.003% Mg2+) demonstrate a significant decrease in plasma Mg2+ within 10–14 days, although they continue to appear healthy  for a minimum of 2 weeks. After this minimum period of acclimation to the experimental casein-based diets, blood and urine samples were collected for Inductively Coupled Plasma Mass Spectrometry (ICP-MS) determination of Mg2+ (and other cations).The SWV and LM/Bc breeding colonies have been maintained on Envigo Rodent Chow #8904 (a soy protein-based irradiated diet) for >20 years. C3H/HeJ and C57BL/6J mice (6–10 weeks old) were immediately placed on Envigo #8904 upon arrival at Creighton University and were maintained on this diet for a minimum of 1 month prior to analysis of basal plasma and urine cations. The National Research Council (NRC) estimate for the minimum Mg healthy . Our iniNon-pregnant female mice of each strain were placed into metabolic cages in groups of 3 mice per cage (n = 3 cages of each strain/diet) and allowed to acclimate for 24 h before urine collection began. Following adaptation, 24-h pooled urine samples were collected from each cage for 3 consecutive days to determine strain and/or diet-induced differences in urine cations. The volume of urine collected for each 24-h period was recorded, and samples were frozen at −80 °C for ICP-MS analysis.g for 8 min. Plasma samples were frozen at −80 °C for LCMS analysis of DTG or ICP-MS determination of cations.Baseline blood samples were collected via cheek bleed (submandibular vein) from all animals prior to timed matings, and again on GD 9.5. A Goldenrod sterile lancet  was used to lance the submandibular vein, and 3-4 drops (∼100 µL) of blood was collected into a Microvette CB 300 Lithium Heparin plasma separator tube  and then centrifuged at 2000 Pregnant dams were anesthetized with isoflurane and sacrificed by cervical dislocation on GD 9.5. The uterus was removed and examined under a dissecting microscope; individual implants were separated, assigned a number, and placed in ice-cold phosphate-buffered saline. Embryos were examined and the phenotype reported as normal , resorption, developmental delay , or NTD . Whole embryos were photographed under a Nikon SMZ 1500 stereomicroscope mounted with an AmScope™ 18MP USB3.0 digital camera. Placentas  and embryos were fixed in 10% formalin or frozen at −80°C.g for 5 min. Finally, 20 µL of supernatant was transferred into a vial containing 120 µL of 1 mg/mL EDTA (pH 8) with 0.1% formic acid. LC-MSMS separation was achieved using Agilent 1200 liquid chromatography devices and an AB Sciex 3200 Q-Trap mass spectrometer. The mobile phase consisted of an isocratic gradient of 0.1% formic acid in water:0.1% formic acid in acetonitrile (60:40) with a flow rate of 0.475 mL/min across an XBridge C18 column . The mass spectrometer identified DTG (m/z = 420.2 (277.2) and d5-dolutegravir (m/z 425.2 (277.2) in MRM scan mode with ESI positive polarity. All solvents used were HPLC grade and above. Linear best fit curves of calibrators allowed for quantification of control and unknown sample concentrations.DTG detection and quantification was performed at Creighton University using a previously published method  with minBlood was collected from separate groups of n = 3 non-pregnant LM/Bc female mice/time point for each of 10 different time points  following oral gavage administration of a single dose of DTG (750 mg/kg). Plasma DTG levels were determined by LC-MS/MS and PK Solver v2.0  was used2+ (and 17 other cations) were performed by Dr. Javier Seravalli at the Spectroscopy and Biophysics Core Facility, Redox Biology Center, University of Nebraska-Lincoln. These analyses used an Agilent Technologies ICP-MS 7500cs and an ESI SC-4 high-throughput autosampler, and the method was optimized for the analysis of eighteen elements (including 24Mg and 44Ca). Each biological sample was run in triplicate.ICP-MS analyses of plasma and urine Mgpost hoc analyses. Pairwise comparisons of the proportion of affected embryos in each litter between strains and treatments were evaluated by the Fisher least significant difference method. Index value pairwise comparisons were evaluated using the Kruskal–Wallis nonparametric test in Sigma XL (v9.0). Comparisons of cation levels in urine and plasma between strains and between diets as well as the effect of DTG on the incidence of NTDs in LM/Bc dams was performed by one-way ANOVA followed by Dunnett’s multiple comparisons test using GraphPad Prism version 9.5.0 for Windows. The level of significance p < 0.05 was used for all analyses. DTG pharmacokinetics were evaluated by noncompartmental analysis (NCA) using PKsolver . Exonic regions were enriched for one mouse of each strain using the SureSelect XT Mouse All Exon kit  according to the manufacturer’s recommendations. This kit targets 221,784 exons within 24,306 genes, with a total capture size of 49.6 MB. Prepared libraries were sequenced 2x100nt on an Illumina HiSeq at the David H. Murdock Research Institute (Duke University) in 2012. The resulting data was aligned against the NCBI37 mouse reference genome, the alignments cleaned, and the variants called, except without use of the contamination threshold parameter or allele phasing. To reduce artifacts and false positives, variants were required to have a read depth of at least 4, fewer than 10% of reads with mapping quality of 0, and a quality-by-depth of at least 7.2+ and susceptibility to DTG–NTDs between the different mouse strains, moderate (missense) and high  risk variants in known magnesium pathway homeostasis genes were examined. The set of magnesium homeostasis genes was assembled using a union of OMIM and Clinvar genes. A list of the 35 candidate genes analyzed is given in Variants were lifted over to the GRCm39 (C57BL/6J) reference genome using the Ensembl Assembly Converter tool and annotated using the Variant Effect Predictor (VEP) tool. To identify potential variants that might explain differences in basal plasma and urine MgPrimers for Sanger validations were designed using Primer3web (v 4.1.0). DNA was extracted from tail snips using the PureLink™ Genomic DNA Mini Kit (Invitrogen). Samples were amplified using the Phusion Flash High-Fidelity PCR Master Mix (Thermo Fisher) and 1 µM of primers. PCR was denatured at 98 °C for 10 s, followed by 35 cycles of 98 °C for 1 s, 48 °C for 5 s, and 72 °C for 15 s, and a final extension of 72 °C for 1 min. Products were cleaned using AMPureXP beads (Beckman Coulter) at 1.8X, eluted in water, and quantified using a Qubit BR dsDNA kit (Invitrogen). Sanger sequencing was performed at Genewiz/Azenta Life Sciences and consensus calling was manually checked from chromatograph traces using GeneSnap Viewer v5.3.2.2+ soy diet, average basal plasma Mg2+ levels were determined for each of the 4 mouse strains . The results are shown in 2+ values (average ± SEM) were lowest for C3H/HeJ  and LM/Bc (0.83 ± 0.03), and not significantly different from one another. Moreover, basal plasma Mg2+ for LM/Bc was significantly lower than SWV (1.05 ± 0.08) and C57BL6/J (1.29 ± 0.07). Based on these results, C3H/HeJ and LM/Bc were considered to have ‘low’, SWV ‘intermediate’, and C57BL6/J ‘high’ basal plasma Mg2+ levels.After a minimum of 4 weeks on the high Mg2+ across strains (2+ (average ± SEM) was not significantly different between C3H/HeJ (1.91 ± 0.09) and LM/Bc (1.91 ± 0.04). However, plasma Ca2+ was significantly lower in LM/Bc when compared to SWV (2.27 ± 0.14) with ‘intermediate’ and C57BL6/J (2.51 ± 0.03) with ‘high’ plasma Ca2+ levels.Similar results were obtained when comparing basal plasma Ca strains . Plasma 2+ soy diet, basal urinary excretion of Mg2+  in LM/Bc mice (41.07 ± 2.03) was significantly higher compared to that in C3H/HeJ  and SWV mice (22.82 ± 4.12). Meanwhile, in C57BL/6J mice, urinary Mg2+ was high (35.06 ± 4.21), and not significantly different from LM/Bc. However, unlike LM/Bc mice, C57BL/6J mice were able to maintain high basal plasma Mg2+ levels despite elevated urinary Mg2+ excretion (After a minimum of 4 weeks on the high Mg of Mg2+  and Ca2+Mg2+ (2+  were detxcretion .2+ excretion (µmol/24 h ± SEM) was also significantly higher in LM/Bc (4.42 ± 0.37) compared to SWV (3.23 ± 0.28) and C57BL/6J (1.73 ± 0.44)   but did gnesuria . Continu2+ levels, the LM/Bc strain was predicted to be susceptible to DTG-NTDs, whereas the SWV and C57BL/6J strains were predicted to be more resistant. The strain comparison with respect to NTD susceptibility on the high Mg2+ soy diet and DTG750 treatment is shown in p < .01). Unexpectedly, there were no significant differences between strains in the number of resorptions or developmental delays following exposure to DTG750.Based on basal plasma Mg2+ soy diet treated with vehicle or DTG750 are shown in Photos of GD 9.5 LM/Bc embryos harvested from dams on the high Mg2+ soy diet when pregnant dams were treated with DTG 500 mg/kg/d (DTG500). Of the n = 7 litters examined at this lower dose, all 62 viable embryos were developmentally normal, fully turned GD 9.5 embryos with a closed cranial neural tube . Additional DTG doses on this diet will need to be tested to definitively determine the NOAEL.No NTDs were observed in LM/Bc mice on the high Mg2+ soy diet are shown in The results of the LC-MS/MS pharmacokinetic analyses for plasma DTG in non-pregnant and pregnant LM/Bc mice on the high Mg2+ intake on DTG-NTD susceptibility, newly weaned female LM/Bc mice (from lactating dams maintained on the high Mg2+ soy diet) were placed on casein-based purified ingredient experimental diets with defined Mg2+ concentrations as previously described.To determine the role of reduced or deficient maternal dietary Mg2+ intake on plasma and urine Mg2+ levels prior to timed matings.Non-pregnant LM/Bc mice were placed on the 3 experimental casein-based diets to evaluate the impact of reduced dietary Mg2+ casein diet all died unexpectedly within 5–7 days. They appeared active and healthy prior to being found dead in their cage. Necropsy did not reveal any remarkable findings. Low blood levels of Mg2+ are known to cause cardiac arrhythmias and seizures. Dietary Mg2+ insufficiency coupled with renal Mg2+ wasting in the LM/Bc mice likely resulted in a severe drop in plasma Mg2+ and death due to a sudden cardiac event. Due to this unforeseen outcome, it was not possible to move forward with experiments examining the combined effect of DTG and Mg2+-deficient diet in LM/Bc mice. Instead, LM/Bc females were placed only on the high or control Mg2+ casein diets.The first group of female LM/Bc mice (n = 5) placed on the deficient Mg2+ concentrations were measured in female LM/Bc mice (n = 9) on the high Mg2+ soy diet at the time of weaning and then again in the same group of mice after 2 weeks (15 days) on the high Mg2+ casein diet. Another group of LM/Bc mice (n = 6) was placed on the control Mg2+ casein diet at the time of weaning and had plasma and urine Mg2+ measured after 2 weeks (15 days) and again after 8 weeks (60 days). LM/Bc mice maintained on the control Mg2+ casein diet for 8 weeks continued to appear healthy. It was determined that additional LM/Bc mice could safely be placed on this diet for up to 8 weeks and used for timed matings.Plasma and urine Mg2+ levels are shown in 2+ (mmol/L ± SEM) when mice on the high Mg2+ soy diet (0.83 ± .04) were placed on the high Mg2+ casein diet for 15 days (0.74 ± .04). LM/Bc mice on both high Mg2+ diets maintained similar basal plasma Mg2+ values. However, as predicted, when mice were placed on the control Mg2+ casein diet, average plasma Mg2+ levels dropped significantly after 15 days (0.56 ± .01) and slightly further after 60 days (0.49 ± .01), although the additional decrease was not itself statistically significant.The results for diet effect on plasma Mg2+ excretion on the experimental casein diets at time points corresponding to plasma Mg2+ quantifications are shown in 2+ casein diet, urinary Mg2+ excretion (µmol/24 h ± SEM) in LM/Bc mice was highly variable, and on average, significantly higher (87.90 ± 15.50) than on the high Mg2+ soy diet (41.07 ± 2.16). The .3% Mg oxide added to the purified casein diet is predicted to be more bioavailable and more readily absorbed from the gut than the .3% Mg in the soy natural ingredient diet due to chelation of Mg2+ by phytate in plant sources  and 60 days (14.26 ± 3.62).The results for urinary Mg sources . Monogas2+ intake on DTG-NTD susceptibility in the SWV strain, newly weaned female SWV mice  were placed on the Mg2+-deficient casein diet. Blood and urine samples were collected prior to the start of the experimental diet and again after 2 weeks (15 days) on the diet for ICP-MS determination of cations.To determine the role of deficient maternal dietary Mg2+ concentration) on SWV plasma Mg2+ levels are shown in 2+ levels (mmol/L ± SEM) in SWV mice on the high Mg2+ soy diet (1.05 ± 0.08), average plasma Mg2+ decreased significantly after 15 days on the high Mg2+ casein diet (0.77 ± 0.07), although the reason(s) for this are not clear and may involve compensatory mechanisms such as release of parathyroid hormone that shift the homeostatic ‘set point’ in response to excess dietary Mg2+.The results for diet effect  to levels comparable with those observed in LM/Bc mice maintained on control Mg2+ casein diet for 60 days (0.49 ± .01).2+ soy to high or deficient Mg2+ casein diets on urinary Mg2+ excretion in SWV mice is shown in 2+ casein diet, urinary Mg2+ excretion (µmol/24 h ± SEM) in SWV mice was highly variable, and significantly higher (138.52 ± 23.30) than on the high Mg2+ soy diet (22.82 ± 4.37), the same trend that was observed for the LM/Bc strain. Increased Mg2+ bio-accessibility and elevated urinary Mg2+ output after 15 days on the high Mg2+ casein diet might explain the corresponding decline in plasma Mg2+ in SWV mice.The impact of changing from high MgMg2+-Deficient Diet: After 15 days on the Mg2+-deficient casein diet, urinary Mg2+ excretion decreased significantly (3.38 ± 1.25) relative to the high Mg2+ casein diet, indicative of renal conservation and reabsorption of Mg2+ in the SWV strain to compensate for the severe restriction in dietary Mg2+ intake.2+ casein diet only. However, in LM/Bc dams on the high Mg2+ casein diet who were treated with DTG750 90 implants total), 3/16 litters  each had n = 1 exencephalic embryo. This was significantly fewer NTD-affected litters compared to LM/Bc dams treated with DTG750 that were maintained on the high Mg2+ soy diet   on the high Mg/litter) . The hig2+ casein diet for a minimum of 4 weeks continued to appear healthy and were placed into timed matings. Plugged females were randomly placed into one of 4 experimental groups: 1) diet-only control (no treatment); 2) vehicle; 3) DTG500; or 4) DTG750. No NTDs were observed in litters from LM/Bc mice on the control Mg2+ casein diet only (n = 14) or in litters from dams on the control Mg2+ casein diet who were treated with vehicle (n = 6). However, exencephalic embryos were observed when LM/Bc dams were administered either DTG750 (n = 13 litters) or DTG500 (n = 13 litters). In DTG750-treated dams, 11/13 litters (85%) had 1 or more exencephalic embryos , ranging from 1 to 6 per litter. In DTG500-treated dams, 8/13 litters (62%) had 1 or more exencephalic embryos , ranging from 1 to 5/litter. These results are depicted graphically in 2+ casein diet. The present results demonstrate that LM/Bc mice are susceptible to NTDs at a lower DTG dose when dietary Mg2+ intake is reduced and hence maternal plasma Mg2+ levels are lower at the time of conception and start of DTG treatment.LM/Bc mice placed on the control Mg2+-deficient casein diet, SWV females appeared in good health and were placed in timed matings. Plugged females received either no treatment (diet-only control) or the Mg2+-deficient casein diet + DTG750 to test whether severely reduced dietary intake of Mg2+ would increase susceptibility to DTG-NTDs . The results are shown in 2+-deficient casein diet only, demonstrating that maternal dietary Mg2+ deficiency alone compromises neural tube closure in SWV mice. In SWV dams on the Mg2+-deficient casein diet + DTG750, NTDs were observed in 6/8 (75%) litters . The results demonstrate a combined diet x drug effect and increased susceptibility to DTG- NTDs (in an otherwise relatively resistant strain) when dietary Mg2+ intake is reduced and hence maternal plasma Mg2+ levels are lower at the time of conception and start of DTG treatment.After a minimum of 4 weeks on the Mg2+ levels were lower prior to the onset of drug treatment due to reduced dietary Mg2+ intake. However, the LM/Bc strain was more susceptible to DTG-NTDs than the SWV strain when dietary Mg2+ levels were sufficient, suggesting an underlying genetic predisposition. To identify genetic variants that might contribute to perturbations in Mg2+ homeostasis and hence the increased susceptibility of the LM/Bc strain to DTG-NTDs, we reanalyzed a whole-exome sequencing data set previously generated from these 2 strains.A diet x drug effect and increased susceptibility to DTG-NTDs was observed in both LM/Bc and SWV mice when maternal plasma MgUgt1a1, Ugt1a3, Ugt1a9, or Cyp3a4 (genes involved in DTG metabolism) were found that were unique to LM/Bc . One high-risk variant in a known magnesium homeostasis gene was identified in LM/Bc but not in SWV–a heterozygous frameshifting variant in Hnf1b  shown in We also looked for variants unique to LM/Bc with a predicted deleterious effect (SIFT) that are intolerant to mutation in humans . This list included 17 SNVs in 14 genes  is a 611 amino acid serine protease that contains a conserved catalytic triad [reviewed in Fam111a in LM/Bc mice and corresponding amino acid changes in the protein are shown in FAM111A and the 9 predicted deleterious variants in Fam111a in the LM/Bc mouse strain are shown in The positions of the deleterious missense variants identified in de novo missense variants that have been identified result in ‘gain of function’ and hyperactivity of the protease , in which patients exhibit increased bone density, growth retardation, macrocephaly, facial dysmorphism, hypoparathyroidism, and electrolyte imbalances, including hypocalcemia and hypomagnesemia (602361), characterized by skeletal/craniofacial malformations, and perinatal lethality  proved to be the most resistant. Our WES study identified several unique variants in the Fam111a gene in LM/Bc mice. In humans, FAM111A variants are associated with hypomagnesemia and renal Mg2+ wasting, consistent with the phenotype observed in LM/Bc mice. Pre-existing perturbations in Mg2+ homeostasis appear to be an underlying risk factor for DTG-NTDs in LM/Bc mice. However, it will be important to corroborate these findings by testing other mouse models with mutations in Mg2+ homeostasis pathway genes.Numerous genetic variants predispose to hypomagnesemia. To test our hypothesis, we selected mouse strains on diverse genetic backgrounds with a range of inherently different basal plasma Mg2+ intake during pregnancy. Dietary intake of this micronutrient impacted both maternal plasma Mg2+ levels and susceptibility to DTG-NTDs. The food matrix also affected Mg2+ bio-accessibility, homeostasis, and susceptibility to DTG-NTDs. In LM/Bc dams maintained on a high Mg2+ (soy or casein) diet, none of the vehicle-treated litters were affected with an NTD. However, when exposed to DTG750, 63.2% of the LM/Bc litters on high Mg2+ soy diet had 1 or more NTD, while only 19% of litters were NTD-affected on the high Mg2+ casein diet. This was an unexpected difference, but can likely be attributed, at least in part, to phytate chelation of Mg2+ in the plant (soy)-based diet, which makes the 0.3% Mg2+ less bio-accessible , plasma Mg2+ levels dropped significantly. Despite this, mice appeared healthy, and embryos developed normally in vehicle-treated dams. However, when LM/Bc dams on this diet were treated with DTG750, 85% of litters had 1 or more pups with exencephaly, and when given a lower dose of DTG500, 62% of litters were NTD-affected. SWV female mice placed on a severely restricted Mg2+ deficient diet became hypomagnesemic prior to conception, and 25% of litters were NTD-affected in the diet only control group. The proportion of NTD-affected litters increased to 75% when SWV dams were treated with DTG750. In addition to the data presented, offspring from 2 additional SWV litters on Mg2+ deficient diet were examined at GD 12.5. In both, the entire litter was in the process of being resorbed, suggesting that severe dietary Mg2+ deficiency is incompatible with fetal development and term pregnancy. Moving forward, to reproduce a more likely scenario in humans, it would be preferable to test DTG-NTD susceptibility in mice on a ‘low’ Mg2+ diet that is compatible with a healthy pregnancy, rather than one so severely Mg2+ deficient that diet alone causes NTDs and fetal death.Insufficient dietary Mgountries . When LM2+ homeostasis and NTDs is exposure to the mycotoxin fumonisin B1 (FB1). FB1 is a common contaminant found in maize-based foods worldwide, including many countries in Africa where rollout of DTG has been widespread  in human and mouse blood   , and a Tyelocele .de novo missense variants in Fam111a in the LM/Bc strain. In LM/Bc mice, several of the predicted deleterious Fam111a variants are near the autocleavage site (FAM111A are associated with hypomagnesemia and hypermagnesuria (2+ transport is not clear. Fam111a knockout (KO) mice on a C57BL/6N genetic background maintained on standard rodent chow containing 0.22% Mg2+ are viable, and display no overt phenotype . It has been proposed that FAM111A is a potential modifier of CASR , or Bartter’s Syndrome, type V (both OMIM# 601198). ADH1 is characterized by low serum Ca2+ and hypercalciuria; Bartter’s Syndrome is more severe, and patients exhibit defective renal sodium chloride transport and increased urinary sodium excretion, which alters the electrochemical gradient necessary for Mg2+ and Ca2+ reabsorption. This leads to renal Mg2+ wasting, hypocalcemia, and hypomagnesemia (consistent with the phenotype observed in LM/Bc mice). In the thick ascending loop of Henley, CASR regulates paracellular transport of Ca2+ and Mg2+ through the cation-selective pore formed by claudin-16 (CLDN16) and claudin-19 (CLDN19) , and a missense variant in CLDN19 has been associated with NTDs  . Human Cith NTDs .Fam111a that in turn result in activation of CASR present a plausible link between altered Mg2+ homeostasis and mechanisms that contribute to failure of neural tube closure. In colon cancer cells, CASR activation stimulates secretion of WNT5A in the noncanonical Wnt signaling pathway (Wnt5a (in KO mice) results in axial shortening, limb truncations , a key enzyme involved in DNA synthesis  are illustrated in the STRING diagram in Another possible connection between GOF mutations in ynthesis . TYMS caynthesis . Inhibitynthesis  and humaynthesis . Collect2+ reabsorption, include proton pump inhibitors and diuretics, among others  , a prevag others . Of partgnesemia . Pentamipatients . Foscarnand Mg2+ . Monitor2+ homeostasis and hypomagnesemia can lead to various metabolic disorders, including altered glucose and lipid metabolism, insulin resistance, and T2DM  to appearance of clinical symptoms supports the hypothesis that DTG might result in a slow decline in plasma Mg2+ over time, although this remains to be verified in the clinical setting. The timing would also agree with the observation that NTDs were only observed in women who started DTG therapy 3 months prior to conception. Similarly, NTDs were only observed in animal models when DTG was started at the time of conception.Perturbations in Mgand T2DM , an estaand T2DM . Amniotiith T2DM , which cith T2DM . Patientith T2DM . If, in 2+ homeostasis as an underlying risk factor for DTG-NTDs, but it will also be critical to examine the impact of DTG on Mg2+ dynamics at the level of the placenta and embryo. The major Mg2+ transporters involved in intestinal uptake and renal reabsorption in the dam, TRPM6 and TRPM7, are also expressed in visceral yolk sac endoderm, extraembryonic chorion, trophoblast cells (2+availability to the developing embryo. Establishment of hemotrophic nutrition and exchange of nutrients (and drugs) between maternal and fetal circulation does not occur until ∼10–12 weeks gestation in humans, long after the neural tube is closed. Transplacental transfer of DTG has been demonstrated in women during the second and third trimester  and other medications which decreases their absorption from the gut and reduces the DTG Cmax and AUC  exposure to DTG and the potential for disruption of neural tube closure. In animal models, NTDs were not observed when DTG exposure started post-conception , a timef


Remarkably, the most highly phosphorylated messenger 1,5(PP)2-InsP4  readily adopts both conformations at physiological conditions. Environmental factors—such as pH, metal cation composition, and temperature—strongly influence the conformational equilibrium. Thermodynamic data revealed that the transition of InsP8 from the equatorial to the axial conformation is, in fact, an exothermic process. The speciation of InsPs and PP-InsPs also affects their interaction with protein binding partners; addition of Mg2+ decreased the binding constant Kd of InsP8 to an SPX protein domain. The results illustrate that PP-InsP speciation reacts very sensitively to solution conditions, suggesting it might act as an environment-responsive molecular switch.Inositol poly- and pyrophosphates (InsPs and PP-InsPs) are central eukaryotic messengers. These very highly phosphorylated molecules can exist in two distinct conformations, a canonical one with five phosphoryl groups in equatorial positions, and a “flipped” conformation with five axial substituents. Using  Myo-inositol polyphosphates (InsPs) are a family of messenger molecules that control a wide array of biological processes in eukaryotic cells a. In the2-InsP4, (from here on abbreviated as InsP8), which is generated via the phosphorylation of 5PP-InsP5 by PPIP5Ks (diphosphoinositol-pentakisphosphate kinases), or phosphorylation of 1PP-InsP5 by IP6Ks (inositol hexakisphosphate kinases). In recent years, InsP8 has emerged as a regulator of inorganic phosphate homeostasis in various organisms. The enzymatic activity of PPIP5Ks is directly regulated by phosphate concentrations, and cellular levels of InsP8 correlate with phosphate availability 8− complex is by far the most abundant species down to about pH 10, reflected by the almost unchanging chemical shift values. The third protonation event (from [K4(H2L)]8− to [K4(H3L)]7−) coincides with the conformational change, accompanied by a steep upfield shift of all monophosphate peaks around pH 8.5–9.0, consistent with our NMR data above ]7−, in which one K+ ion is coordinated to phosphates in position P6, P5α and P5β. The other K+ ion is coordinated to the pyrophosphate group at the 1-position, between P1α and P2 ]7−, the main K+-complex of InsP8 at physiological pH, is more than tenfold higher than that of the corresponding 5PCP-InsP5 complex ]6−, in which the Mg2+ ion is coordinated by the pyrophosphate group at 1-position, together with P2 (3Mg(H2L)]7− to [K3Mg(H3L)]6−). At pH 8.0 and 8.5, the P2 peak was too broad to detect, suggesting higher rates of exchange between conformations in the presence of one equivalent Mg2+, which move the system into the intermediate exchange range even with 31P-NMR detection.The speciation diagram b highlig with P2 c,d. As f8 and K+ or Mg2+ ions, and suggest an important role of the two pyrophosphate groups in Mg2+ binding. While in the axial conformation, InsP8 coordinates Mg2+ between its two pyrophosphate groups; once the conformation has changed to equatorial, the Mg2+ binding site is formed by the pyrophosphate group at position 1 and the phosphate group at position 2  from yeast VTC2 6−, in which Mg2+ is coordinated to P2, P1α and P1β. Another species present at physiological pH is the axial complex [MgK3(H2L)]7− in which Mg2+ is coordinated by P5α and P1α. Overall, the pyrophosphate groups in positions 1 and 5 play an essential role in Mg2+ binding, as the ion is always coordinated to either the two pyrophosphate groups  or P2 and PP1 . Notably, all metal complexes of InsP8 are far more stable than the equivalent ones formed by InsP6 or 5PP-InsP5.Phosphorous NMR studies revealed further details of the speciation of InsP2+ complexes of PP-InsPs were also found to be more stable than those of ATP and ADP, two other well-known cytosolic Mg2+ chelators. Formation constants of complexes between Mg2+ and ATP, previously reported at 150 mM NaCl and 37 °C [2+ + ATP4− → [MgATP]2−: log(K) = 4.34, Mg2+ + HATP3− → [Mg(HATP)]−: log(K) = 2.39. In contrast, we measured formation constants (log(K)) of 15.0 and 8.7 for [MgK3(H3L)] complexes of InsP8 and 5PCP-InsP5. It therefore appears feasible that the significant stability of these complexes influences their interactions with other biomolecules. Do metal ions bridge binding interactions between PP-InsPs and proteins? And if so, is this effect more pronounced for InsP8 than its less densely phosphorylated relatives? And how does this ultimately influence the strength and specificity of binding?The Mgnd 37 °C  are: Mg2One could envision signaling functions associated with the metal complexation and the conformational equilibrium of PP-InsPs. In light of their role as cellular ATP and phosphate sensors, additional sensing functions seem plausible ,48. Give8 binding to the VTC2 SPX domain were almost cut in half by addition of 1 mM Mg2+, and binding shifted towards a more enthalpically dominated interaction in the presence of Mg2+. It is tempting to speculate that the decrease in ΔH might reflect a more specific fit of an axial magnesium complex into the binding site, compared to the free, equatorial molecule, but structural evidence would be a prerequisite to support this hypothesis and is currently unavailable. Based on the current data, we cannot exclude the possibility that Mg2+ might lower the Kd independently of InsP8 conformation, for example, by altering the enthalpy and entropy of desolvation upon formation of the InsP8-Mg complex. It will be interesting to investigate the influence of Mg coordination more systematically for other proteins targeted by PP-InsPs, such as the mammalian SPX domain of XPR1, or the C2 domains of synaptotagmin isoforms.In the large majority of cases, it is unclear by which mechanisms proteins manage to recognize and distinguish the different PP-InsPs ,23. DiffOverall, our results highlight the immense complexity of PP-InsP speciation and how this speciation may influence their behavior. Solution conditions for PP-InsP-protein binding experiments in the literature differ widely regarding pH, salts, and other additives. For future biochemical studies, it will be important to consider carefully which molecular species are formed under the given assay conditions, how those conditions might affect the experimental outcome, and to what extent the conditions mirror cellular settings. An accurate understanding of this complexity will be indispensable in decoding the diverse roles of InsPs and PP-InsPs as cellular messengers.