BackgroundsDuring the COVID-19 pandemic, visitor restrictions in healthcare settings adversely affected patients. Video calls have emerged as an essential digital alternative that can decrease patients’ anxiety and improve satisfaction. This study investigated whether family-initiated video calls could mitigate delirium symptoms and risky behaviors and enhance patients’ comprehension of instructions.MethodsThis observational study used medical chart data and the Diem Payment System from a single acute care hospital in Fukuoka, Japan. The study involved patients hospitalized between May 2020 and August 2021 who used video chat systems. Patients or their relatives used video calls through Skype. The frequency of video chat use served as the primary exposure. Changes in the patients' risky behaviors and instruction comprehension upon discharge were the primary outcomes.ResultsA total of 532 patients were included in the study, with an average age of over 70 years. After implementing the inverse probability of treatment weighting adjustment, an improved balance across age, sex, BMI categories, and other variables was observed. The effects of video calls on risky behaviors and instruction comprehension varied. Patients with three or more video calls showed distinct effects compared with those with fewer calls. When hospitalization was limited to three weeks, video calls noticeably influenced risky behaviors , but not instruction comprehension .ConclusionsThe use of video calls as a visitation method in acute care hospitals during a pandemic suggests that video calls reduce risky behaviors in patients with a three-week stay. This alternative to physical visitations contributes positively to patient safety and supports ongoing efforts to prevent the spread of COVID-19. In response to the coronavirus disease 2019 (COVID-19) outbreak, medical facilities implemented visitation restrictions to mitigate the spread of the virus . This diExisting research has shown a strong correlation between pandemic-related visitation restrictions and a high incidence of delirium among patients with acute cerebrovascular disease in stroke units . This coAmong these are increased patient risk behaviors such as self-extraction of intravenous used in treatment, falls, and self-injurious behavior, as well as poor understanding of instructions, which hinder recovery from illness and injury. These are serious problems that increase the nursing burden on nurses who have the most contact with patients during hospitalization.Video call visits provide tangible benefits to patients and their families, including increased satisfaction, increased motivation, reduced anxiety, decreased depressive symptoms, and reduced distress ,8. EspecThis study aimed to ascertain whether the frequency of video calls, acting as a substitute for in-person visits due to visitation restrictions in acute care hospitals, is associated with an improvement in patients' risk behaviors related to delirium symptoms and instruction comprehension during the COVID-19 pandemic.Study designThis observational study was a retrospective study using Diagnosis Procedure Combination (DPC) and medical record data of patients at a single hospital in Japan.Data sourceDiagnosis Procedure Combination Per-Diem Payment System data and medical chart data from a high acute care hospital in Fukuoka, Japan, were used. The Diagnosis Procedure Combination Per-Diem Payment System is a national acute-care inpatient database in Japan that provides administrative claims data for a fixed payment system for medical expenses . The datPatient selectionThis study analyzed the data of patients hospitalized at Shinkomonji Hospital between May 2020 and August 2021 who used a video chat system.OutcomesThe primary outcomes were risky behavior and understanding of instructions, each evaluated as a binary value of presence or absence. Risky behavior was defined as self-removal of intravenous infusions during treatment, falls, self-injurious behavior, and any other behavior that nurses or others judged to be potentially risky. If there has been no hazardous behavior within the past week or there is no record of such behavior, the patient is rated as "none". If the patient has engaged in risky behavior within the past week and there is a record of such behavior, it is rated as "Yes". The understanding of instructions is evaluated based on whether the patient is able to understand and carry out medical and treatment instructions, regardless of the disease in question. The evaluation criterion is "none" if the patient always acts appropriately in response to medical and treatment instructions, or if there is no record of actions not taken in accordance with instructions. The evaluation criteria are as follows: "Yes" if the patient has at least one instance of behavior that is not in accordance with medical or medical treatment instructions, and if there is a record of the behavior that is not in accordance with the instructions . These oVideo chatVideo calls for visitations were made upon request from patients or their families. The patient was accompanied by a nurse in his room, and the family used the visiting room in the ward. Each visitation session lasted for 20 min. Skype was used for video calls; Skype uses Voice over Internet Protocol technology to communicate using voice and video over a real-time connection via the Internet . Visits Other variables2) and created five categories. BMI category was assigned based on the modified World Health Organization classifications of <18.5 kg/m2, 18.5-24.9 kg/m2, 25.0-29.9 kg/m2, 30.0-34.9 kg/m2, ≥35 kg/m2 [We extracted information on age, sex, length of stay, destination of discharge, use of general anesthesia, and ward admitted to . We calculated body mass index (BMI) as weight (kg) divided by height squared  was used to adjust for differences in baseline characteristics and to estimate the average treatment effect using probability weights assigned to video chat frequencies of one, two, three, and ≥four times .The weights used in the IPTW were determined using the propensity scores (PSs). PSs were created using a multivariate logistic regression model to predict the probability of video chat frequencies of one, two, three, and ≥four times. IPTW values were calculated using the baseline characteristics of each patient to estimate the average treatment effect . The varAfter weighing the baseline, baseline covariates were compared using absolute mean differences (AMDs) . To compMissing values were handled by multiple imputations using chained equations  generatiSensitivity analysisWe conducted sensitivity analyses. The length of hospital stay was limited to the number of days within three weeks . Maybe, the longer the length of hospital stay, the more effective the inference from the video chats. Moreover, the longer the length of hospital stay, the greater the effect observed in the patient’s status on whether risky behavior is present and whether instructions are understood at discharge.This study was approved by the Clinical Research Ethics Committee of Shinkomonji Hospital . The retrospective and anonymized nature of the data exempted the study from requiring explicit participant consent [A total of 756 patients who were admitted to Shinkomonji Hospital from May 2020 to August 2021 used Skype. Of these, 532 patients with information on the number of times they used Skype were included in the study.Tables Table The findings from the peripheral structural model in Table As shown in Table However, instructional comprehension improved significantly after the third video call, as suggested by the value of Exp(Coef)=0.61 . This improvement was not observed in the second or subsequent video calls, suggesting that comprehension had no significant effect.When the duration of hospitalization was limited to three weeks or less (see Table When the duration of hospitalization was limited to three weeks or less, the change in instructional comprehension due to the frequency of video calls was not significant.During the COVID-19 pandemic, our study revealed that, under visitation restrictions in acute care hospitals, video calls proved to be an effective alternative to in-person visits. These video calls contributed to a reduction in the patients' risk behaviors and improved their understanding of instructions. Notably, hospital stays were within three weeks, and patients who engaged in video calls three or more times exhibited a statistically significant improvement in their risk behaviors.Previous research has validated the efficacy of video calls in acute-care settings. Specifically, the use of recorded family video messages as a non-pharmacological nursing intervention for patients with delirium has been shown to significantly lower the incidence of delirium . FurtherAccording to the current study, no statistically significant difference in comprehension of instructions was observed when the hospitalization period was limited to three weeks which could be due to multiple factors. A longer hospitalization period may have contributed to the stabilization of the patient's condition, adaptation to the hospital environment, and improvement in activities of daily living as a result of rehabilitation, which may have contributed to improvement in the patient’s comprehension of the instruction. However, the benefits of three or more video calls within a three-week hospital stay underscore the significance of this study. These results provide a strong rationale for introducing video calls into hospitals’ daily operations. Video calls not only support patient and family communication but also contribute to increased patient satisfaction . The impThis study has several limitations. First, the study was conducted in a single acute care hospital in Fukuoka, which limits its applicability when taking the results to other healthcare settings and different cultural contexts. Further, the focus on the impact of Skype video call-based visitation did not provide insight into other communication methods and platforms. Factors such as the quality, length, and emotional content of video calls, which could significantly influence the results, have not been fully explored. The facilities studied prohibited the use of cell phones in the hospital, with the exception of some compartments, because of their potential to affect the operation of medical equipment. However, it is not known whether they personally used cell phones to conduct video calls with family members.Second, the selection of patients for the study did not involve a randomized controlled trial, which could over-reflect certain demographic attributes and introduce selection bias. In addition, limitations of the tools used to assess the effectiveness of the intervention may have resulted in a certain bias in the results. It is also possible that confounding factors not identified during the research process may have influenced the results and their interpretation. In the future, it will be important to take these factors into account and explore optimal visitation methods using video calls, especially after a pandemic.This study determined the impact of the frequency of video calls with family members as an alternative to restricted visitation on patients' risk behaviors and understanding of instructions in a single acute care hospital during a COVID-19 pandemic. Results showed that increasing the frequency of video calls to three or more times significantly reduced patient risk behaviors when the length of hospital stay was limited to three weeks or less. These findings have important implications for clinical practice and provide a compelling case for incorporating video calls into regular visiting practices to prepare for new pandemic hospital visitation restrictions.


Pc-Sw fracture response to changes in boundary conditions. Results show that aperture is Gaussian-distributed and the coefficient of variation is scale-independent. The aperture contraction during normal stress increments causes higher capillary pressures and steeper Pc-Sw curves, while shear displacement results in invasion anisotropy. The three-parameter van Genutchen model adequately fits the fracture capillary response in all cases; the capillary entry value decreases with fracture size, yet the fracture Pc-Sw curve normalized by the entry value is size-independent. Finally, we combine the fracture and matrix response to infer the rock mass response. Fracture spacing, aperture statistics and matrix porosity determine the rock mass capillarity-saturation Pc-Sw curve. Fractures without gouge control the entry pressure whereas the matrix regulates the residual saturation at high capillary pressure Pc.The fractal topography of fracture surfaces challenges the upscaling of laboratory test results to the field scale, therefore the study of rock masses often requires numerical experimentation. We generate digital fracture analogues and model invasion percolation to investigate the capillarity-saturation  Examples include energy resource extraction such as hydrocarbons and geothermal4, the vadose zone and environmental remediation6, geological storage of CO2 and nuclear waste8, and hydrothermal alteration and ore deposition9.Mixed fluid conditions are common in fractured rocks and affect both engineering applications and natural processes2 injection due to viscous fingering, and aggravated by fractures and heterogeneity). On the other hand, long-term analyses must be based on capillarity-saturation curves at thermodynamic equilibrium.In the absence of gouge, fractures provide preferential flow pathways that dominate fluid flow. Then, invasion can readily bypass wetting (and even non-wetting) fluids in the matrix and fast injection studies tend to conclude low displacement efficiency .This study explores the relationship between capillary pressure and saturation in fractured rocks, with emphasis on fracture-matrix interaction First, we investigate the effects of normal stress, shear displacement and fracture surface topography on the fracture capillary response. We generate digital fracture analogues that resemble natural fractures and bring the two rough surfaces together to create the fracture pore space. Then, we use network model simulations to investigate the effects of surface roughness and matedness on the fracture capillarity-saturation curve, and its evolution during normal loading and shear displacement. Fracture generation, response to normal stress and shear displacement, and fluid invasion modeling are described next.G(λ) [m3] follows a power law with respect to wavelength λ [m]40  [m] for a certain wavelength λ is related to the spectral density as G(λ) = C |X(λ)|2, where the scaling factor C = N∆x/4 [m] depends on the selected Fourier pair and transform definition . We use the Inverse Fast Fourier Transform to synthesize the surface roughness profile in space by assuming a uniformly distributed random phase (see similar surface generation models in Refs.41). The fractal topography implies lack of a characteristic scale or representative equivalent size. However, fractal geometries have limits in natural systems; in our case, the longest wavelength is the fracture size under consideration.The power spectral density of rock fracture surfaces ]40 Fig. a:1\documα-factors .σ [MPa]. The rock reaches its yield stress σy [MPa] at the true fracture contact area Ac [m2]. Then, equilibrium implies,Af [m2] is the total fracture area. The two fracture surfaces interpenetrate a vertical distance δv to satisfy the computed contact area Ac . The height havg of each cell is the average aperture in the original fracture within the corresponding area Δl × Δl, so that After shear displacement and normal loading, we map the resulting l × Δl defines the aperture resolution. While the pressure-dependent saturation of a given pore depends on the pore geometry, the global trends exhibit limited sensitivity to local details: results from a focused numerical study reveal that aperture averaging in Δl × Δl affects only the lower end of the capillary-saturation Pc-Sw curve, i.e., at high capillary pressures and low degrees of saturation. Furthermore, apertures h are much smaller than wavelengths in natural fractures (h/λ <  < 1—Ref.27); therefore, the in-plane radius of curvature is negligible and capillarity-saturation results are unaffected by aperture averaging when Δl < 10Δx. Previous studies show that the in-plane curvature can influence invasion patterns48; however, our fracture generation model ensures h/λ <  < 1 and the capillary pressure is only a function of the aperture.The selected cell size Δ31, fluid transport into the matrix50, water evaporation and vapor pressure equilibration51. While these processes have characteristic time scales, the equilibrium assumption disregards any transient trapping of the wetting phase.The invasion percolation algorithm assumes equilibrium at any given pressure , and non-trapping of the wetting phase. Various pore-scale phenomena justify the non-trapping assumption, including Fig. : corner Pc = Ts/h . The largest aperture connected to the inlet is invaded first and defines the entry value. All apertures connected to the non-wetting phase throughout the medium are candidates for invasion. Invasion proceeds by monotonically increasing the capillary pressure to define the capillary pressure versus saturation Pc-Sw curve.We implement the Young–Laplace equation in terms of the aperture-induced curvature, Lx × Ly = 1 × 1 m, 0.1 × 0.1 m and 0.01 × 0.01 m subjected to zero normal stress . The selected fracture variables represent natural conditions and highlight size effects. Each curve represents an average of 100 numerical realizations in order to obtain statistically representative results. The mean aperture size distribution is Gaussian-distributed in the three cases, with almost the same coefficient of variation sh/μh defined in terms of the aperture standard deviation sh and the mean aperture μh  − Sw curves are scale-independent.The capillary pressure mentclass2pt{minimAs the normal stress increases, the aperture size distribution shifts towards smaller values and a cutoff at zero aperture emerges as contact yield results in zero aperture contact points Fig. a. The coPc-Sw curve increases .The shearing of unmated, uncorrelated fracture surfaces results in statistically identical aperture fields and the capillarity-saturation 27. Figure Pc-Sw curves for initially mated fractures: as the shear displacement increases, the mean aperture increases and the capillary pressure decreases for a given degree of saturation.Shear displacement causes aperture changes only when there is some initial degree of matedness between surfacesPc-Sw curves -particularly at low Pc values- when the capillary pressure is controlled at a boundary that is normal or parallel to ridges.Shear displacement induces anisotropy in the aperture field of initially mated fractures as aperture ridges emerge transverse to the shear direction. Results in Fig. Pc-Sw curves combine to define the capillary response of the fractured rock mass. Consider three orthogonal fracture sets with the same spacing d in the three directions so that the repetitive unit is of size n, the mean aperture size μh and the fracture spacing d determine the volume of voids in the matrix μh/d and high matrix porosity n:The fracture and matrix nηF = 1 − ηM. The fracture and the matrix share the same capillary pressure at equilibrium, therefore, the resultant Pc − Sw curve is a void-volume average of the saturation contributed by the fracture and the matrix SwRM = SwF(1 − ηM) + SwMηM.Conversely, the storativity in fractures gains relevance in rock masses with low matrix porosity Pc-Sw curves and combine them for various matrix void fractions ηM to estimate the rock mass capillary response. We compute the fracture Pc-Sw curve using the algorithm described above, and a similar algorithm for the matrix where pores are represented as connected tubes with capillary pressure Pc = 2Ts/r (see model details in Ref.53). Results in Fig. ηM fraction increases, the rock mass capillary-saturation curves approach the matrix Pc-Sw curve.Let’s consider the matrix and the fracture Pc-Sw. This will determine the original oil saturation profile in the reservoir and the residual oil after production, the long-term CO2 and H2 storage capacity, the distribution of LNAPLs and DNAPLs contaminants and environmental remediation strategies, the residual distribution of hydraulic fracturing fluids and the relative permeability of the resulting fractured rock mass.The long-term saturation of a fractured rock mass will depend on the capillary pressure and the degree of saturation at equilibrium sh/μh independent of fracture size? . Then, the fracture surface topography z satisfies a Gaussian distribution, z ~ N .The numerical study revealed surprising emergent properties. In particular, why is the aperture coefficient of variation ze? Fig. . The fraw in Eq.  and randzt and bottom zb surface topographies, h = zt − zb,. Therefore, the fracture aperture values also exhibit a Gaussian distribution when the two surfaces are uncorrelated h ~ N . The scale invariant features in our numerically generated fractures result from the fractal nature of the surface roughness56.The fracture geometric aperture is the subtraction of the top fh allows us to obtain the lower bound of the Pc-Sw curve: the pore volume distribution is fv = (h × Δl2)fh, then, the cumulative distribution function of fv  normalized by the integral of (h × Δl2) corresponds to the wetting phase saturation Sw with capillary pressure Pc = Ts/h.The aperture Gaussian distribution The capillarity pressure vs. saturation response of fractured rock masses is needed for long-term analyses. However, the time needed to reach thermodynamic equilibrium and size-dependent fracture topology limit our ability to experimentally gather relevant capillarity-saturation curves.Pc-Sw curve for a fractured rock mass is determined by pore-scale characteristics in the fractures and the matrix, including aperture and pore size statistics, spatial variability and connectivity.Numerical experiments show that the capillary pressure versus saturation sh/μh is scale independent.The fracture surface roughness can be synthesized as a sum of independent sinusoids. A power law relates the sinusoidal amplitudes to their wave wavelength. The central limit theory emerges and the aperture size follows a Gaussian distribution. The mean aperture increases with fracture-size, yet, the coefficient of variation While the wetting phase could remain occluded in the matrix during non-wetting fluid invasion, trapping in fractures is limited. Wetting fluid connectivity allows for the wetting fluid to escape through a network of connected corners formed by the surface roughness or via the permeable porous matrix. Long-term equilibrium in liquid–vapor systems further promotes non-trapping conditions in fractures.Pc-Sw curves. Capillary invasion reflects a slight anisotropy during shear and affects saturation at low capillary pressures. The three-parameter van Genutchen model captures the fracture Pc-Sw response and its evolution with normal stress and shear displacement. While the capillary entry value is inversely proportional to the fracture size, the normalized capillarity-saturation curves (Pc/P0) − Sw follow similar trends and are scale independent.The increase in normal stress contracts the aperture and results in higher capillary pressure, increased aperture variability, increased true contact area and steeper Pc-Sw curve controls the entry pressure, whereas the matrix regulates the rock mass residual saturation at high capillary pressures.At equilibrium, fracture spacing and aperture statistics combine with the matrix porosity to determine the capillary pressure versus saturation curve for a rock mass and its storativity. In the absence of gouge, the fracture


This study aimed to investigate the effect of the pathological staging of acute histological chorioamnionitis (HCA) on laboratory indicators and to conduct further studies to reassess the threshold values used by clinicians to identify acute HCA in febrile parturients undergoing epidural analgesia.A retrospective study of febrile mothers receiving epidural analgesia at Nanjing Maternal and Child Health Care Hospital from January 1, 2018 to December 31, 2018. The participants were grouped by the progression of acute HCA, and the laboratory parameters were compared between groups. The ability of C-reactive protein (CRP), neutrophil-lymphocyte ratio (NLR), monocyte-lymphocyte ratio (MLR), and monocyte-leukocyte ratio (M%), alone or in combination, to identify acute HCA in febrile parturients undergoing epidural analgesia was assessed using logistic regression and ROC curves.p < 0.0001). Based on the ROC curves, the following thresholds were selected to define increased laboratory indicators for identifying acute HCA: CRP ≥ 6.90 mg/L, NLR ≥ 11.93, and MLR ≥ 0.57. In addition, the AUC of the best logistic regression model predicting HCA ≥ stage 2 was 0.710, so these inflammatory markers were more precise in predicting HCA ≥ stage 2.The area under the curve (AUC) of the best logistic regression model predicting HCA climbed to 0.706 (CRP + MLR). Maternal CRP, NLR, and MLR significantly and progressively increased with the progression of acute HCA (Increased CRP (≥ 6.90 mg/L), NLR (≥ 11.93), and MLR (≥ 0.57) may help clinicians to identify early potential acute HCA in febrile parturients receiving epidural analgesia and to monitor progression to optimize clinical treatment options.http://www.chictr.org.cn, ChiCTR2100053554).The study was registered in the Chinese Clinical Trial Registry on November 24, 2021 (The online version contains supplementary material available at 10.1186/s12884-023-06026-1. The term “chorioamnionitis” literally refers to inflammation of the chorionic and amniotic layers of the fetal membranes, and its use conveys the etiology of microbial infection when this may not always be the case . The defThe most important clinical symptom of chorioamnionitis is maternal fever , and theAlthough non-invasive laboratory markers have been thoroughly explored as a prenatal diagnosis of febrile maternal HCA, the link with pathological staging has received little attention. Therefore, this study aimed to investigate the effect of the pathological staging of acute HCA on laboratory indicators and to conduct further studies to reassess the threshold values used by clinicians to identify acute HCA in febrile parturients undergoing epidural analgesia.This was a population-based diagnostic accuracy study including febrile parturients who received epidural analgesia at Nanjing Maternity and Child Health Care Hospital between January 1, 2018, and December 31, 2018. Because the retrospective clinical investigation caused no damage to the patients involved, the Medical Ethics Committee of Nanjing Maternity and Child Health Care Hospital agreed to waive informed consent . The flow chart of the study procedure is presented in Fig. Patient demographics and laboratory indicators were extracted from electronic medical record system. Demographic data include maternal age, gestational week, gravidity, parity, meconium-stained amniotic fluid (MSAF) , oxytociHistologic examination of the placenta is performed in febrile pregnant women as part of routine clinical care. Collected placentas were fixed in 10% neutral buffered formalin, paraffin embedded, sectioned, and stained with hematoxylin and eosin (H&E) for histological examination. Acute HCA is defined as placental tissue  with mild to severe acute inflammatory alterations. The progression of acute HCA according to the Blanc’s classification , 19, basAny elevated maternal temperature during labor is referred to as intrapartum hyperthermia . Because20% of febrile parturients may have both epidural hyperthermia and intrapartum infection, suggesting that the two conditions are not entirely independent , 18, 19.P < 0.05.Data were analyzed using SPSS software , and plotted using GraphPad Prism 9.0.0 software , Origin Pro 2021 , and Adobe Illustrator . All continuous variables were tested for normality using the Shapiro-Wilk Test. Continuous data are expressed as mean ± standard deviation, and categorical data are expressed as number (percentages). Clinical characteristics of different groups were compared by the chi-square test for categorical variables and ANOVA for continuous variables, and those variables that were statistically different were screened for the next step of the analysis. A multivariate logistic regression was used to develop a predictive model to analyze the predictive factors associated with HCA. The predictive values of single laboratory indicators and the multivariate model were evaluated using receiver operating characteristic (ROC) analysis and the area under the curve (AUC). The variables needed to make up the best regression model were screened based on the AUC of the logistic regression model. ROC curves were created to identify the optimal dichotomized cutoff scores of independent predictors to distinguish between patients with and without HCA. Utilizing the cutoff scores, we compared the frequency of increased predictors according to the progression of acute-HCA with Pearson’s chi-square test on the one hand, and assessed the predictive performances by calculating sensitivity, specificity, positive and negative predictive values, and positive and negative likelihood ratios on the other hand. All statistical tests were two-sided, and statistical significance was defined as A total of 1775 patients were included in this study, of which 187 were epidural hyperthermia (control group) and 1588 were HCA. Of the 1588 patients with acute HCA, 231 had stage 1, 175 had stage 2, and 1182 had stage 3. There were no significant differences in the maternal age, gestational week, gravidity, parity, turbidity of amniotic fluid, oxytocic manner, sex and weight of the newborn, degree of perineal laceration, and amount of bleeding between the different stages of acute HCA and the control group. Only labor duration was substantially extended with the progression of acute HCA among the clinical features (Supplementary Table P)1 = 0.108 + 0.058×CRP + 2.219×MLR , and Logit (P)2 = -0.980 + 0.059×CRP + 0.094×NLR + 0.278×M% . Maternal MLR, CRP, NLR, and M% are important risk factors for acute HCA, and ROC curves were constructed to assess their ability to identify acute HCA. Compared to independent factors  = -0.096 + 0.059×CRP + 2.269×MLR . The best logistic regression model had an AUC of 0.710 , so these inflammatory markers were more precise in predicting HCA ≥ stage 2.In addition, stage 2 HCA had higher pathological and clinical significance than stage 1 HCA, so we reclassified two groups (control group vs. stage 2 and 3 HCA) to perform binary logistic regression analysis. The best logistic regression models for predicting HCA ≥ stage 2 with laboratory parameters were as follows: Logit , NLR ≥ 11.93 , and MLR ≥ 0.57 .The logistic regression model for predicting acute HCA with multiple laboratory indicators (CRP + MLR or CRP + NLR + M%) has a higher AUC than any single laboratory indicator. Based on the staging of placental pathology, this study found that CRP, NLR, and MLR increased gradually with the progression of acute HCA. These inflammatory markers were more accurate in predicting HCA ≥ stage 2 than in predicting HCA.Bacterial staining and biochemical analysis of amniotic fluid and histopathological examination of the placenta are important methods for the diagnosis of placental inflammation . The mosAlthough epidural analgesia does not raise the risk of intrapartum infection , it is dIn the early stages of chorioamnionitis, infection and inflammation are limited to the chorion and amnion membranes, and IL-6 is released into the maternal blood, leading the mother to produce CRP . As a re9/L), and foul-smelling amniotic fluid, in addition to maternal fever [The main clinical signs associated with infected chorioamnionitis include uterine tenderness, maternal tachycardia (≥ 100 beats/min), fetal tachycardia (≥ 160 beats/min), maternal leukocytosis  with the progression of acute HCA. Second, unlike previous studies, this study proposes that maternal MLR is a biomarker for the diagnosis of acute HCA. Third, this study recommends maternal CRP, NLR, and MLR as combined maternal biomarkers that can be widely used in basic medical institutions to distinguish epidural hyperthermia from acute HCA. The diagnostic criteria for chorioamnionitis varied depending on the type of classification used. Therefore, hospitals using the Blanc’s classification may be able to use the results of this study as a reference, while hospitals using other classifications cannot . The incHowever, since previous placental histopathology was limited to febrile parturients, only febrile parturients were included in this study. Further diagnostic accuracy studies are needed to screen for subclinical HCA in parturients without fever to verify the applicability of the results in this study. In addition, this study only included women with singleton term pregnancies, and further studies are needed to determine whether there are similar biomarkers for febrile women with early and late labor. Since we use individual blood samples during fever to calculate inflammatory factors, we were unable to reflect long-term inflammatory status. The positive and negative likelihood ratios for the three prenatal biomarker cutoffs used to identify acute HCA in this study remain low.Maternal CRP, NLR, and MLR increased significantly and progressively with the progression of acute HCA, and increased CRP (≥ 6.90 mg/L), NLR (≥ 11.93), and MLR (≥ 0.57) may be considered as biomarkers to identify the development of acute HCA in febrile parturients receiving epidural analgesia. These indicators may help clinicians identify potential acute HCA patients early and monitor disease progression to optimize clinical treatment options.Below is the link to the electronic supplementary material.Supplementary Material 1


Each category was scored on a 3-point scale from 0 (absent) to 2 (proficient). A video scoring 20–24 points induced low cognitive load, 13–19 points induced moderate cognitive load, and less than 13 points induced high cognitive load. Three reviewers independently evaluated the videos and then agreed on scores and feedback. Results: All 132 videos were evaluated. Mean video total score was 20.1 ± 0.7 points out of 24. Eighty-five percent of videos were rated low cognitive load, 15% were rated moderate cognitive load, and 0% were rated high cognitive load. The following components scored the highest: audio quality and matching modality. The following components scored the lowest: signaling and call-to-action. Conclusions: Understanding the use of a pragmatic tool is a first step in the evaluation of InPACT at Home exercise videos. Our preliminary findings suggest that the InPACT at Home videos had low cognitive load. If future research confirms our findings, using a more rigorous study design, then developing a collection of instructor-led exercise videos that induce low cognitive load may help to enhance youth physical activity participation in the home environment.Background: Exercise videos that work to minimize cognitive load (the amount of information that working memory can hold at one time) are hypothesized to be more engaging, leading to increased PA participation. Purpose: To use a theory-based pragmatic tool to evaluate the cognitive load of instructor-led exercise videos associated with the Interrupting Prolonged Sitting with ACTivity (InPACT) program. Methods: Exercise videos were created by physical education teachers and fitness professionals. An evaluation rubric was created to identify elements each video must contain to reduce cognitive load, which included three domains with four components each [ The current generation of children are the first to have their entire childhood influenced by the internet and mobile devices such as smartphones and tablets . This trCurrently, there are several opportunities for children to view and participate in online physical activity videos. Exergames  have shown promise to enhance physical health by using technology to encourage physical activity participation. For example, a study by Silva et al. found that exergaming provided the same acute physiological benefits as conventional exercise in a group of young adults . Exergamhttp://inpactathome.umich.edu, accessed on 7 August 2023) and broadcast on public television . However, the quality of these online resources has not been established as there is no validated tool to evaluate the quality of exercise videos designed to increase youth physical activity engagement.Another online resource, the Interrupting Prolonged Sitting with Activity (InPACT) at Home program is an evidence-informed physical activity program that utilizes instructor-led exercise videos . The InPOne way to determine the quality of exercise videos is to consider the amount of cognitive load they induce, which is a key determinant of viewer engagement. The cognitive load theory has been extensively used to guide the creation and evaluation of educational videos. This theory builds on the model of human information processing to reduce the demands on the viewers working memory to help them engage effectively ,10,11. TFurther, there are three main types of cognitive load that an instructor needs to consider when creating online exercise videos to reduce the demands on working memory to improve engagement: (1) the level of difficulty of instructions presented (intrinsic load), (2) the mental energy needed to link current knowledge to the new information (germane load), and (3) the distractions that prevent processing new information (extraneous load) ,16. WhenThe cognitive load theory has not been used to assess the quality of structured exercise videos; however, it has been used to examine motor skill acquisition. For example, Rekik et al. assessed gender differences in learning baseball skills from video modeling vs. static pictures . AuthorsPrevious research in the education literature has also demonstrated that instructional videos that induce a low cognitive load  are associated with increased participant learning and performance. In a study that used cognitive load theory principles to create video-based and live instructor-led online training sessions and their effects on performance outcomes, authors found that participants performed significantly better on easy tasks compared to more difficult ones independently of the type of instruction used . FurtherCognitive load has primarily been taken into consideration in previous studies that utilize video-based methods to improve youth learning outcomes. However, there are no pragmatic tools available that enable fitness professionals and physical education teachers to evaluate the quality of exercise videos. This is important, as many general and physical education teachers are using exercise videos to promote youth physical activity in their classrooms and gyms .The overarching objective of this study was to use a theory-based pragmatic tool to evaluate the cognitive load of the InPACT at Home instructor-led exercise videos. More specifically, we examined differences in cognitive load by video type . This study is a first step toward the construction and validation of a publicly available tool that can be used to determine the cognitive load of instructor-led exercise videos. Data generated from this study will provide a systematic process to evaluate instructor-led exercise videos. From this evaluation, future researchers can use a more rigorous study design to assess video quality. Our long-term goal is to be able to develop and disseminate a pragmatic tool that enables us to generate video improvement recommendations to enhance existing videos and provide guidance for developing new instructor-led exercise videos that promote youth physical activity engagement.There were a total of 132, 8-min exercise videos created by 14 instructors. The InPACT at Home exercise videos are organized into the following categories: (1) cardio-based  (2) strength-based , (3) sports skills , and (4) mindfulness .http://inpactathome.umich.edu, accessed on 7 August 2023). The current study was observational and did not evaluate the opinions of children. However, youth mood, attitudes, and recommendations for improvement after engaging in InPACT at Home exercise videos were previously evaluated in a pilot study [Examples of equipment used were basketballs, a dining room table chair, weights, or heavy objects , a tennis ball, steps on a staircase, tape, a jump rope, a towel, a shirt, a wall, and a soccer ball. The exercise videos can be found on the InPACT at Home website  and two additional evaluators from iEval services) was previously contracted to develop the video evaluation rubric. These evaluators each have over 15 years of extensive experience using observational rubrics in early childhood, K-12, after-school programs, and university instructional settings. This rubric was created to help with the development of the InPACT at Home program. technical domain. The technical domain is defined as the aspects of the video that relate to its visual and audio communication to the viewer. Within the technical domain the components are visual quality , audio quality , matching modality , and signaling . For example, in a video leading to low cognitive load for the technical domain, the viewer can clearly see and hear what the instructor is saying and doing. The instructor demonstrates the movements as they are being explained verbally, and on-screen text such as what the sets and reps for the movements are included.The rubric consisted of three domains which included four components each. The first domain is the content domain. The content domain is defined as the instructor’s stated objectives of the video and the instructor’s ability to meet those objectives. Within the content domain, the components are instructional objective , met objective , call-to-action , and bias . An example of a video leading to low cognitive load for the content domain would be a video in which the instructor clearly states the goals and how the participant will reach those goals, works to meet those goals throughout the video, ends the video with encouraging next steps to continue being active, and is free of bias.The second domain is the instructional domain. The instructional domain is defined as how the tasks and directions given by the instructor in the video are organized. Within the instructional domain, the components are learner engagement , content organization , segmenting , and weeding . An example of a video leading to low cognitive load in the instructional domain is one in which the instructor continuously encourages the learner, presents the information in organized short segments and deletes any information that does not support the goal.The third domain is the The internal evaluation team consisted of three research staff  members who were trained by a member of the external evaluation team (W.T.) during a 2-h virtual training session to understand the rubric elements and use the rubric to evaluate the exercise videos. Prior to the training session, the research staff reviewed the rubric. During the training session, the external evaluator verbally described the domains and components in the rubric and gave relevant examples of how each component would appear in the video. Once the rubric was understood, the internal evaluation team watched two videos and independently evaluated them for practice. After each video was evaluated, scores were discussed as a team with the external evaluator, and discrepancies were identified. Where discrepancies were present the internal evaluation team discussed why they chose their scores and ultimately came to a consensus. This allowed the internal evaluation team to become familiar with the rubric in practice and to have the opportunity to ask the external evaluator clarifying questions before starting data analysis.Data analysis of the InPACT at Home videos occurred from September 2021 to March 2022. The three video evaluators independently scored each of the videos. The evaluator assessed all videos from one instructor to better identify common areas of improvement based on instructor style. For example, if one instructor created 10 videos, all 10 videos were evaluated before evaluating another instructor. After the evaluators completed an independent review of approximately 20–30 videos, the team met to discuss their independent evaluations for each video. If there were differences for any of the components between the three evaluators, then the team discussed that score and came to a consensus. When clarifying questions occurred during the evaluation process, the video evaluation team reached out to consult the program evaluator. This process occurred until each of the three individuals evaluated all 132 videos. The symmetry of the evaluator’s performance was measured using an intra-class correlation coefficient and the interrater reliability after the independent evaluation and prior to consensus was moderate at 0.72.For each component within the three domains, the evaluation team used the rubric to determine the points that should be awarded based on whether the key component was absent, developing, or proficient (see detailed descriptions of scoring criteria in p < 0.01). We subsequently used a non-parametric Kruskal–Wallis H test to assess differences in overall video scores by video type. Data were analyzed using SPSS Version 27.A sum score of all 12 components within all three domains was the total score for each video. Videos could score up to a total of 24 points. Videos with total scores below 13 points were classified as having high cognitive load, scores between 13–19 points were classified as moderate cognitive load, and scores between 20–24 points were classified as low cognitive load. The mean total overall video score was calculated by adding the individual composite scores of all videos and dividing by the total number of videos. The mode for each individual category was also calculated and the components in least and most need of improvement were highlighted to inform video improvement. A Shapiro–Wilk test was performed to test the normality of the data and showed that the distribution of total cognitive load scores departed from normality (p > 0.05). A total of 132 exercise videos were evaluated from September 2021–March 2022. The mean total score for the videos was 20.1 ± 0.7 points out of 24. The mean total score for the cardio videos was 20.0 ± 0.7. The mean total score for the mindfulness videos was 20.2 ± 0.9. The mean total score for the sports skills videos was 20.2 ± 0.5. The mean total score for the strength videos was 20.3 ± 0.6. There were no significant differences in overall video score by video type  classified as low (scoring between 20–24 points), 20 videos (15%) classified as moderate (scoring between 13–19 points), and 0 videos (0%) classified as high cognitive load (scoring less than 13 points). http://inpactathome.umich.edu, accessed on 7 August 2023). Given the widespread dissemination of these exercise videos during the COVID-19 pandemic, there was a need to evaluate the exercise videos for quality; however, no validated assessments were readily available to do so [signaling  and call-to-action (content domain). This study is the first step in developing a systematic process by which to evaluate exercise video content.The overarching objective of the current study was to use a theory-based pragmatic tool to evaluate the cognitive load of instructor-led exercise videos associated with the InPACT at Home program  in the videos . HoweverWhile the InPACT at Home videos were developed with components to decrease cognitive load, instructors were given creative agency for what to include within the given duration of the videos. In the current study, within the three domains, the videos scored the lowest in the technical domain (mode: 6.0 out of 8). The technical domain is important because it assesses the ability to clearly see, hear, and understand what is being asked of the learner throughout the video and when performed well can help increase retention and transfer of information as well as increase engagement ,28.In the current study, the component in the technical domain that was most in need of improvement was signaling—as it was absent from all videos. Signaling is defined as on-screen text or symbols that are used to highlight important information throughout the video. Signaling has been shown to improve a student’s ability to retain and transfer new knowledge ,29. In aThe InPACT at Home videos scored the second highest in the content domain (mode: 7 out of 8). The content domain is important because it assesses if there is a clear objective and material in the video as well as ensures that the video works to meet the stated objective throughout. Assessing content helps to analyze if the video is accurate, useful, and free from bias. In the content domain, the component most in need of improvement for the InPACT at Home videos was call-to-action. Call-to-action in this study is defined as the instructor making a clear and direct statement encouraging the viewer to continue to be physically active each day. A reason that call-to-action scored low could be because each of the videos used the same script for the ending and only reminded the child to fill out a daily exercise log to keep track of their activity without including a specific encouraging call to stay active after using the video. A recommendation to improve the call-to-action in the videos would be to explicitly tell the viewer to keep using the videos each day to help them reach the recommended 60 min of physical activity needed to maintain health. Another recommendation to improve the content domain score of videos is to ensure that every video is self-contained and does not refer to a previous or next video since students may not be watching them in that order. Referring to the other videos would distract the learner from the current video objectives leading to a higher cognitive load.Finally, InPACT at Home videos scored high across all four components in the instructional domain (mode: 8 out of 8). The instructional domain is important because it assesses how the information is included and organized in the video. The lowest component score in the instructional domain for the InPACT at Home videos was weeding. The weeding component refers to eliminating information that does not contribute to the learning goal or help build relationships between the instructor and viewer. Weeding, or the deletion of extraneous information, has been shown to improve retention and transfer of knowledge and lower learning difficulties . Brame rA few strengths of this study should be noted. First, the rubric was developed by an experienced program evaluator and is grounded in theory. Second, the videos were analyzed by three reviewers which minimized potential data analysis errors and allowed for higher reliability and trustworthiness of the analysis. Third, the videos were professionally filmed in a controlled studio environment. Fourth, the videos were designed and performed by certified physical education teachers and fitness professionals. The present study also had limitations. First, the video review rubric has not yet been validated. Future studies should assess the psychometric properties of the rubric to determine its validity. Second, the videos were professionally developed and recorded in a public broadcasting studio, thereby limiting their generalizability to other recording environments. This is important to mention, as many physical education teachers and fitness professionals are recording amateur exercise videos using tablets and cellphones in their homes and gyms . Future The current study used a pragmatic tool informed by theory to evaluate the cognitive load of the InPACT at Home instructor-led exercise videos. Understanding the cognitive load induced by instructor-led exercise videos is a novel area of research and has important implications for the utility of such a resource to promote sustained physical activity participation in young people. This was the first step in creating a systematic process to evaluate the quality of exercise videos. An important next step is to employ a more robust experimental design to assess the psychometric properties of our video evaluation rubric to determine its validity. Once established, future video developers  can utilize the evaluation rubric to improve existing videos and guide future video development.Development and dissemination of instructor-led exercise videos aligns with the Comprehensive School Physical Activity Program (CSPAP) framework to promote youth physical activity outside of school hours, and within the home and community environments . While t


Underage drinking is a public health concern. However, few studies have examined the association between alcoholic beverage advertising and underage drinking, particularly in countries with low underage drinking rates, such as Japan. Therefore, we aimed to investigate the relationship between exposure to advertising in various media and alcohol drinking among Japanese adolescents.We conducted a cross-sectional study involving 15,683 adolescents (51% girls) using data from a nationwide lifestyle survey in 2021 among junior and senior high schools across Japan. Media types were websites, stores, and public transportation. We defined current drinking as alcohol consumption of ≥1 day in the 30 days preceding the survey. Multivariable logistic regression was used to examine the association between exposure to alcohol advertisements and current drinking, adjusting for sex, grades, school area, lifestyle (bedtime and having fun at school), and addictive behaviors .p for sex interaction >0.1), except for that of exposure to web advertisements; its association with current drinking was more pronounced in girls (p for sex interaction = 0.046). Exposure to a larger cumulative number of different alcohol advertising media was independently associated with a higher prevalence of current drinking among all students, boys, and girls .The prevalence of current drinking was 2.2% (2.3% of boys and 2.0% of girls). Students who were exposed to any alcohol advertising media had higher odds of current drinking compared with those who were not . Students who were exposed to web, in-store, and public transportation advertisements had odds ratios of 1.44 , 1.62 (1.28–2.05), and 1.45 (1.06–1.98) of current drinking, respectively, compared with those who were not. The association of exposure to alcohol advertising media with the prevalence of current drinking was similar among boys and girls (all We found an association with a dose-response relationship between exposure to alcohol advertisements and current drinking among adolescents in junior and senior high schools across Japan. Our findings highlight the need for further advertising regulations to prevent underage drinking.https://doi.org/10.1265/ehpm.23-00127.The online version contains supplementary material available at  Underage drinking is a public health issue because of its negative effects  –4. The gAlcohol advertising is perceived as attractive by some students . PreviouUsing data from a nationwide survey conducted in 2021 among junior and senior high school students across Japan, we aimed to clarify the association of exposure to advertising through various media  and the cumulative number of different advertising media with drinking behavior. Many countries have declining underage drinking rates, and reports from countries with quite low underage drinking rates, such as present-day Japan, are useful for developing future global strategies and effective advertising control policies to prevent underage drinking .Drinking behaviors of Japanese junior and senior high school students have been reported in our national survey , 16, 17.The survey population consisted of seventh to twelfth graders in junior and senior high schools from all over Japan using the National School Directory. All students enrolled in the sample schools comprised the target population. The distribution of school characteristics  was selected as representative of the survey population .In 2021, a paper- or web-based survey was conducted using a questionnaire. The questions were the same for both methods. The schools were randomly assigned to participate in either the paper- or web-based survey. Two-thirds and one-third of the schools were assigned to web- or paper-based surveys, respectively. For the paper-based survey, letters were sent to the principals of the selected schools requesting their cooperation along with questionnaires and envelopes for each enrolled student. Classroom teachers in the participating schools explained the survey to their students and assured them of privacy protection. The questionnaire included a statement requesting honest answers and assuring participants that their answers would not be revealed to teachers or parents. Written informed consent was obtained from all participants. In accordance with the Ethical Guidelines for Medical and Health Research Involving Human Subjects, informed consent was obtained from each high school student and the parent or guardian of each junior high school student because this was an anonymous, non-invasive questionnaire survey. The nature of confidentiality and voluntary participation was explained to all students by the teachers, and the completed questionnaires were returned to our working group in sealed envelopes . In the Using a questionnaire about the exposure to the types of media on alcohol advertising in the past 30 days, we tabulated the exposure to each type of advertising media , as well as the cumulative number of different advertising media to which participants were exposed. The question about advertising media was formed by discussing it in the context of Japan based on previous research , 20. TheFor the question about current drinking , participants were considered current drinkers if they answered that they consumed alcohol for one or more days. The participants provided data on demographic and school characteristics, including sex, grade, school type (junior or senior high school), and school area. School area was categorized into eastern Japan (Hokkaido-Tohoku and Kanto districts), central Japan , and western Japan . Participants responded to lifestyle behavior questions, including smoking status and bedtime. For the smoking-related question (“How many days did you smoke during the past 30 days?”), participants were considered current smokers if they answered that they had smoked for one or more days. Bedtime was divided into two categories: before and after 12:00 a.m. Participants were asked if they felt happy at school with the following choices: “Yes, I do,” “Neutral,” and “No, I don’t” . Parentap-value of 0.05 or less. We handled missing data using pairwise deletion. Multicollinearity was assessed using the variance inflation factor (VIF) for all models. All analyses were performed using the STATA statistical software .Participants’ demographic characteristics were presented as numbers and percentages and were listed by current drinking status and sex; differences in characteristics were evaluated with a chi-square test. We also tabulated the characteristics according to the two survey methods (paper-based or web-based) and examined the difference between them using a chi-square test. We used multivariable logistic regression to calculate the ORs and 95% confidence intervals (CIs) for the presence of current drinking according to the type of media advertising alcohol. The following adjusted models were constructed: Model 1 was adjusted for invariant factors such as sex, grades, and school area , and Model 2 was additionally adjusted for students’ lifestyles (bedtime and having fun at school) and addictive behaviors . Covariates considered relevant to underage drinking or advertising exposure were included in the multivariable models; however, school type was not included due to multicollinearity with grades. We repeated the analysis stratified by sex (boys vs. girls) and examined the multiplicative interactions between sex and alcohol advertising for current drinking. To test for systematic errors by survey method (paper-based or web-based), we conducted multivariable analyses stratified by survey method using the same logistic regression models. In addition, we performed a multivariable analysis in which all advertisements  were simultaneously adjusted for all advertising media in the multivariable model (other covariates were the same as in Model 2). Furthermore, multivariable analysis by school type (junior or senior high school) using the same logistic regression models was conducted to confirm the differences. Since many current drinkers were smokers  and 17 (27.4%), respectively, participated in this study. Of 95,548 eligible students , 15,832  responded to the questionnaire survey . In the present analysis, we excluded 149 students with missing data ; therefore, data for 15,683 students  were analyzed. Data with the following missing values  were included: bedtime (36), having fun at school (88), smoking (98), parents’ alcohol consumption (18), frequency of alcohol consumption (23), and amount of alcohol consumption (12). Missing values for covariates were duplicated within each participant; the multivariable analyses included data from 15,683 participants for Model 1 and 15,483 participants for Model 2. Table p values for sex interaction >0.1), except for that of exposure to web advertisements which had a more pronounced association in girls (p for sex interaction = 0.046). Exposure to a larger cumulative number of different alcohol advertising media was independently associated with a higher prevalence of current drinking among all students, boys, and girls  . These results suggest that there are no obvious systematic errors affecting multivariable analysis due to survey methods  among Japanese adolescents in the present study. A cross-sectional study in 2016 of 3,291 high school students in two prefectures in Japan reported that 9.6% were current drinkers, and our previous cross-sectional study in 2017 of 64,152 junior and senior high school students in a nationwide study reported that 5.6% were current drinkers, indicating a lower drinking rate in the present study in 2021 . AccordiOur study found a positive dose-response relationship between the cumulative number of different alcohol advertising media exposure and current drinking among Japanese adolescents. A dose-response relationship has been reported between past-year exposure to television advertisements and current alcohol consumption in a previous study . Our finThe strengths of our study are its large sample size, its contribution to addressing the scarcity of reports on East Asian populations with low drinking prevalence compared to the West, its representation of Japanese junior and senior high school students (a nationwide survey), being the first to identify a positive relationship between the cumulative number of different alcohol advertising media exposure and current drinking, and exploring sex differences in exposure to advertisements. However, this study has several limitations. First, because this was a cross-sectional study, causal relationships could not be identified. Current drinkers may be more likely to have a lasting impression of alcoholic beverage advertisements than non-drinkers; therefore, current drinkers may have a higher awareness of alcohol advertisements . ConsequExposure to alcohol advertisements is positively associated with current drinking among junior and senior high school adolescents in Japan. The association between advertising and current drinking has been confirmed in Japan where underage drinking rates are low. Furthermore, we found that the odds ratios of current drinking were higher with exposure to a larger cumulative number of different alcohol advertising media for both sexes (dose-response relationship). Our results revealed sex differences in the strength of the association between exposure to alcohol advertisements on websites and current drinking. Although further studies are required to determine the relevant factors, and there are possible biases , our results reveal an association between alcohol advertising and underage drinking among low underage drinking rate population and highlight the direction for further advertising regulations to prevent underage drinking.


Herein,we introduce a new quinoline-based fluorescent sensor, MagZet1, thatdisplays a shift in its excitation and emission wavelengths, affordingratiometric detection of cellular Mg2+ by both fluorescencemicroscopy and flow cytometry. The new sensor binds the target metalwith a submillimolar dissociation constant—well suited fordetection of changes in free Mg2+ in cells—and displaysa 10-fold selectivity against Ca2+. Furthermore, the fluorescenceratio is insensitive to changes in pH in the physiological range,providing an overall superior performance over existing indicators.We provide insights into the metal selectivity profile of the newsensor based on computational modeling, and we apply it to shed lighton a decrease in cytosolic free Mg2+ and altered expressionof metal transporters in cellular models of drug-induced liver injurycaused by acetaminophen overdose.Magnesium(II) plays catalytic, structural, regulatory,and signalingroles in living organisms. Abnormal levels of this metal have beenassociated with numerous pathologies, including cardiovascular disease,diabetes, metabolic syndrome, immunodeficiency, cancer, and, mostrecently, liver pathologies affecting humans. The role of Mg A thorough understandingof the mechanism connecting abnormal magnesium levels to these andother liver pathologies, however, is still lacking. The inflammatoryresponse associated with liver damage and disease is thought to involvethe activation of several Ca2+-dependent pathways and theelevation of Ca2+ levels in affected tissues. In fact,it has been proposed that Mg2+ deficiency promotes inflammationthrough the disruption of its natural Ca2+ antagonism.18 Unfortunately, the study of cellular Mg2+ in this andother systems in which Ca2+ may play a role has been hamperedby the scarcity of tools and methods for the dynamic detection ofthe former cation without interference from the latter. To meet thisneed, we set out to develop a new fluorescent indicator that couldbe used for the selective detection of Mg2+ over Ca2+ in live cell imaging applications.Recently, our team identified the upregulationof cyclin M4 (CNNM4),a protein involved in regulating Mg2+ in cells.Our thermodynamic analysis of metal binding to APTRA,19 a pentadentate metal-binding motif commonly used in Mg2+ indicators that exhibits limited selectivity,21 suggested that decreasing the denticity of carboxylate-based sensorscould decrease the affinity for competing divalent cations and improveselectivity for the target Mg2+.22 The metal binding profile of sensors of the KMG family, based onbidentate metal-binding motifs, illustrates this effect.24 Bidentate sensors, however, allow the formation of ternary sensor-Mg2+-biomolecule complexes that obscure the response to the “free”metal.25 Considering this shortcoming andbased on the design principles that arose from our thermodynamic analysis,the group of Kikuchi developed a tridentate quinoline-2,8-dicarboxylate(QDC) chelator with excellent Mg2+/Ca2+ selectivityand low tendency to form ternary complexes.27 Unfortunately, all sensors built thus far using this chelator displayfluorescence quenching, i.e., turn-off response, upon Mg2+ coordination as well as strong pH dependence, which limits theirutility. The selective detection of free Mg2+ in the presenceof Ca2+ thus remains challenging. Addressing this gap,we report herein a new wavelength ratiometric sensor,MagZet1, that affords the selective detection of Mg2+ freeof Ca2+ and pH interference under physiological conditions.We demonstrate the application of the new indicator in both fluorescencemicroscopy and flow cytometry, shedding light on a decrease in thelevel of cytosolic free Mg2+ correlated with the alteredlevel of expression of CNNM4 in a cellular model of DILI.Cationselectivity has been a long-standing challenge in the developmentof fluorescent indicators for the study of Mg28 To capitalize on the properties of the QDC moietyin the design of a wavelength ratiometric sensor for Mg2+, we sought to functionalize the electron-deficient quinoline withan electron-donating amino group to complete a push–pull electrondonor–acceptor system. We surmised that coordination of Mg2+ to the acceptor moiety would stabilize an internal chargetransfer (ICT) excited state, resulting in a red shift in the electronicspectra.30Intensiometric, turn-on, and turn-offsensors are the most common types of fluorescent indicators used tovisualize metal cations in cellular imaging. The intensity of theirfluorescence emission, however, depends on their cellular concentrationas much as on the concentration of the target metal itself. Ratiometricindicators showing a wavelength shift are of greater analytical value,as they offer a response that is independent of sensor concentrationand minimizes the effect of light source fluctuations and other variables.Supporting Information). Treatmentof the sensor with increasing concentrations of Mg2+ inaqueous buffer  leads to a hypsochromicshift in absorption from 488 to 430 nm31 and a bathochromic shift in emission from 500 to 530 nm . Nonlinear fit of the fluorescenceemission data as a function of metal concentration was used to estimatethe apparent dissociation constant for Mg2+, Kd,Mg = 0.22 ± 0.01 mM. This apparent affinity iswell suited for the detection of physiological concentrations of freeMg2+ (0.5–1.2 mM),32 whilethe lower affinity for Ca2+  suggests that bindingof this cation would not be a source of interference in the cellularmilieu. Overall, the spectral shift and favorable preliminary bindingdata of MagDMA supported the feasibility of the ratiometric detectionof biological Mg2+ with our design. The low emission quantumyield of the compound  states, leading to nonradiative relaxation. MagZet1 1, an ana2+-free and -saturated forms, respectively  also resembles that of MagDMA and is well-tuned for maximumsensitivity in the physiological range of intracellular free Mg2+ concentrations.The effect of azetidine substitution on thephotophysical propertiesof the resulting sensor was readily apparent. In aqueous buffer atpH 7, MagZet1 shows quantum yields of 40 and 76% in the Mgectively 1, which dination1. This s2+ or equimolar concentrations of first-row d-blockmetals, with the exception of Zn2+. An increase in ratiowas observed with the latter, but upon closer inspection, we noticedthat the Zn2+ complex is significantly less emissive thanthe Mg2+ complex and the emission profiles of the two areclearly distinct, which could help discriminate between the metals. Competition experiments conductedwith one equivalent of other metals in the presence of 1 mM Mg2+ showed no significant interference by the other divalentcations in the detection of physiological levels of Mg2+. Some fluorescence quenching was observed in the presence of Co2+ and Ni2+, suggesting metal binding. The fluorescenceis restored, however, in the presence of Mg2+.The optical response of MagZet1 to other biologicallyrelevantdivalent cations was also investigated 2. No sig2+ and Zn2+ to MagZet1 were further investigated . The dissociation constant for the Ca2+ complex was determined to be Kd,Ca =1.7 mM; thus, typical intracellular concentrations of Ca2+ are not expected to interfere with Mg2+ detection. Theaffinity for Zn2+, on the other hand, is higher than foreither main group metal but still rests outside the range of typical basal concentrationsof Zn2+ in cells (0.01–0.1 nM).37 Accordingly, MagZet1 should not suffer from significantinterference from Zn2+ in the detection of Mg2+ in typical samples. Care should be taken, however, when the sensoris to be employed in samples that are particularly rich in Zn2+, such as neurons.39The thermodynamics of binding of Ca2+ and Ca2+ complexes wereoptimized by DFT calculations using the M062X hybrid functional andthe 6-311+G basis set, as shown to be well suited for push–pullsystems and noncovalent interactions.40 Input structures were generated from crystal structures of Mg2+ complexes of 2-quinoline carboxylic acids.41 The coordination spheres of the metals were completed withaqua ligands to achieve typical coordination numbers for Mg2+ (six) and Ca2+ (eight).Computational studies were used togain insight into the cation selectivity of MagZet1. Ground stategeometries of the Mg2+ complex, the metal is located approximatelyin the plane of the sensor 42 show dihedral angles around 180°, with an averageCa–O distance of 2.3(1) Å (44 This out-of-plane twistof the carboxylate in [MagZet1·Ca(H2O)5], not observed in the Mg2+ complex, likely weakens theCa2+–carboxylate interaction and overall bindingof the metal.In the optimized geometryfor the Mge sensor 3. In the2.3(1) Å 3.43,44 T45 was investigated by bothabsorption and fluorescence spectroscopy (S7). Theabsorption maximum shows a shift from 480 nm in the acidic form to380 nm in the basic form, with an isosbestic point at 409 nm. Theemission spectrum also shows a large blue shift with increasing pH,and the intensity of the emission increases significantly as the sensoris deprotonated. From single-wavelength readings, a pKa of 7.1 was determined. The ratio of fluorescence emissionintensities at 530 and 500 nm, however, shows almost no pH dependencein the physiologically relevant range of pH= 5.5 to 8 in the presenceor absence of metal (em = 630 nm) comparedto both deprotonated and metal-bound forms . The overall fluorescence intensity, however, is lowerat lower pH values; thus, studies that involve such conditions mayrequire higher concentrations of the sensor, and studies below pH5 may be hampered by a low signal-to-noise ratio. The isosbestic pointfor the deprotonated and metal-bound forms of the sensor is 390 nm.This wavelength was used for fluorescence excitation in all photophysicalcharacterization studies conducted in vitro.The response of MagZet1 to changes in pH in the biological range(pH 5–8)troscopy 4 and S7.of metal 4. This snd forms 4C. As su2+·sensor ternary complexes,25 which may hamper the study of free Mg2+ in biologicalmatrices. Therefore, the ability of MagZet1 to form ternary complexeswith MgATP was tested. The addition of high concentrations of MgATP,prepared in situ from a one-to-one mixture of Mg2+ andATP, led to a gradual increase in wavelength and intensity of thefluorescence emission (2+] (from competition with ATP)and ternary [MagZet1·Mg2+·ATP] complexes (S9). Using this model and a dissociation constantof 50 μM for MgATP,46 we determineda dissociation constant of 100 mM for the ternary complex. This valuefar exceeds the total concentration of Mg2+ in the cell;7 thus, MagZet1 could be used to monitor changesin intracellular free Mg2+ with little interference fromthe bound forms.An established shortcoming of low-denticitysensors and chelators is their propensity to form biomolecule·Mgemission 5. The flomplexes 5 and S9.2+ .We also investigated the effect of various aminoacids and metabolitesthat may bind the metal or interact with the fluorescent indicator,including histidine, glutamate, aspartate, cysteine, glutathione,citrate, pyruvate, and malate. The presence of these species at physiologicallevels neither altered the fluorescence output of the sensors norits response to Mg2+ in live cells was investigated. The sensor was converted into anacetoxymethyl ester form, MagZet1-AM , a rapid and low-toxicity Zn2+ chelator,51 did not result in significant changes in thefluorescence ratio, thus ruling out the detection of either of thesemetals by MagZet1 .Given the excellent propertiesshown in the cuvette, the ability of MagZet1 to detect MggZet1-AM 1, to enae sensor6. To ver content 6. Experi2+ by flow cytometry, taking advantageof the shift in theemission wavelength elicited by metal binding to MagZet1. Flow cytometryis a powerful technique that enables the rapid analysis of large populationsof cells, providing robust measurements and statistics. This technique,however, has not been widely used for the study of Mg2+ due to the lack of suitable probes. Mag-Indo-1, an analogue of theCa2+ sensor Indo-1 suitable for such applications,52 suffers from significant interference from competingdivalent cations and interactions with proteins,54 photobleaching, and is no longer available from most commercialsources. As such, there is a need for new emission ratiometric sensorsfor free Mg2+ compatible with the technique.We then sought to investigate the detection ofintracellular MgFigure S12), typicallyassociated with cell toxicity. Suspensions of MagZet1-stained HeLacells in the absence and presence of excess extracellular Mg2+ and metal ionophore 4-Br-A-23817, or coloaded with EDTA-AM, werethen analyzed. Histograms showed significantly different fluorescenceratios arising from the sample treated with excess Mg2+  and 530/30 bandpass  filters.Treatment with the sensor did not have a negative impact on cell shapeor granularity S12, typi55 APAP hepatoxicity occurs through the formation of a reactivemetabolite, N-acetyl-p-benzoquinoneimine(NAPQI), which depletes glutathione and leads to Ca2+ redistribution,mitochondrial dysfunction, and ER stress in hepatocytes.57 CNNM4, a protein involved in regulating Mg2+ transport,14 was found to be markedly upregulated in hepatocytes from patientssuffering DILI generated by APAP overdose, whereas its silencing resultedin restoration of Mg2+ serum levels and amelioration ofvarious hallmarks of the disease in animal models.16 With a sensor capable of the selective detection of Mg2+ against Ca2+, we set out to investigate changesin Mg2+ in a cellular model of DILI by flow cytometry.Acetaminophen(APAP) is a common drug used to treat mild to moderate pain. Thisdrug, however, is responsible for more than 50% of overdose-relatedacute liver failure cases in the United States.59 and displaydistinct characteristics of DILI within several hours of treatmentwith sublethal concentrations (5–10 mM) of APAP.16 Fluorescence microscopy revealed diffuse cytosolicstaining in THLE-2 cells treated with 5 μM MagZet1-AM for 30min at room temperature, followed by an additional 30 min de-esterificationperiod at room temperature .We thus used similar sensor loading conditions in the preparationof samples for flow cytometry. THLE-2 cells treated with 10 mM APAPdisplayed a decrease in the fluorescence ratio after just 3 h of exposureto the drug (S14–S15). Analysis of theexpression of various proteins involved in magnesium transport revealedan upregulation of CNNM4, consistent with prior observationsin primary hepatocytes from DILI patients and animal models.16 An upregulation of TRPM6 wasalso detected by RT-qPCR  cells express the levelsof phase I and phase II drug-metabolizing enzymes and transporterscomparable to primary hepatocytesperature S13.We t2+ in Ca2+-rich environmentsby fluorescence techniques requires tools that selectively detectMg2+. While much effort has been put into achieving thisgoal, current sensors still lack on multiple fronts.64 Addressing this gap, we developedMagZet1, a ratiometric sensor based on a 2,8-quinoline dicarboxylatechelator, which displays a 10-fold selectivity for Mg2+ over Ca2+ and a dissociation constant well suited forthe detection of typical cellular concentrations of the metal. Uponmetal binding, MagZet1 displays a red shift in fluorescence emission,making it one of the few emission ratiometric sensors available forstudying Mg2+. Significantly, the fluorescence emissionratio is solely dependent on the concentration of free Mg2+ and does not suffer from significant pH interference in the biologicalrange. Moreover, the sensor has a low propensity to form ternary complexeswith polyphosphates at typical biological concentrations, making MagZet1an excellent tool for the study of cellular free Mg2+.The study of Mg2+ homeostasis. Flow cytometry allowsthe robust analysis of large numbers of cells, providing greater insightinto population heterogeneity and the detection of small but significantchanges. With a suitable sensor in hand, we employed flow cytometricanalysis to shed light on a decrease in the level of cytosolic Mg2+ in a cellular model of APAP-induced liver injury. The changesin metal levels correlate with upregulation of CNNM4 in the cells,previously observed in liver biopsies from DILI patients,16 and are consistent with CNNM4 playing a rolein promoting metal extrusion from the liver. The selectivity for Mg2+ over Ca2+ displayed by the sensor is imperativefor the study of this system, in which changes in ER Ca2+-releasing capacity and ER stress reveal alterations in Ca2+ homeostasis at play. We anticipate that the new sensor and methodologyreported herein will likewise open the door to the study of Mg2+ in other systems, thus far largely inaccessible, in whichCa2+ levels are high or may be altered. As such, it mayhelp to shed light on the intricate interplay between the two cationsthat, for the lack of better tools, had only been studied from a Ca2+-centric perspective.The emission ratiometric response of MagZet1 expands the possibilitiesbeyond conventional microscopy techniques to applications in flowcytometry, a powerful technique that has thus far been underutilizedin the study of Mg


Work hour restrictions imposed on orthopedic surgery residents since the early 2000s have reduced educational opportunities at the workplace and encouraged alternative strategies for teaching outside the clinical setting. Preoperative templating is essential for safe and effective total hip arthroplasty (THA) and is accurate in predicting final implants. We sought to determine the effectiveness of a video tool for teaching orthopedic residents basic THA templating skills.We developed a video-based teaching tool with instructions on proper THA templating techniques. Ten cases were selected for testing, after excluding patients with severe hip deformities and poor-quality radiographs and only retaining those with concordance between templating by the senior authors and implanted components. The study subjects included three postgraduate year 1 (PGY-1), three PGY-2, and three PGY-5 residents, and three adult reconstruction fellows (PGY-6). Templating skills were assessed before and after watching the instructional video. The evaluation included the size and positioning of femoral and acetabular components, as well as the restoration of leg length. Each templating session was repeated twice. Variance was measured to evaluate consistency in measurements. A linear mixed model and F-test were used for statistical analyses.The number of years in training significantly affected performance prior to exposure to the instructional video. Post-exposure, there was a significant improvement in the accuracy of sizing and positioning of acetabular and femoral components for PGY-1, PGY-2, and PGY-5 residents. The results achieved were comparable to PGY-6 examiners, who did not gain substantial performance benefits from the instructional video. Limb length restoration was less affected by experience or exposure to the video. Component positioning and sizing, as well as leg length discrepancy (LLD), showed a significant decrease in variance after the intervention in all study groups.Video learning is reliable in teaching invaluable skills to orthopedic surgery residents without encroaching on work hours. We conceived a concise video to train orthopedic residents to perform THA templating with proper technique and demonstrated its efficiency and reproducibility. In 2003, the Accreditation Council for Graduate Medical Education (ACGME) set an 80-hour restriction on weekly resident work hours in an effort to minimize fatigue-related medical errors. While the success of this policy in improving patient safety has been a topic of debate, it inherently leads to a reduction in the number of clinical encounters and opportunities for resident education -4. As a This prospective study was approved by the Thomas Jefferson University hospital institutional review board IRB # 11E.296). The senior corresponding author, who is a board-certified, fellowship-trained adult reconstruction surgeon, developed two video-based teaching tools for THA templating. The first tool involved a four-minute and 27-second video demonstrating the use of the computerized digital templating system . The video provided instruction for the use of the digital templating software, including program access, selection of relevant cases and radiographs, selection of appropriate digital implants, manipulation of the size, offset, positioning, and orientation of the digital implants, and use of length and angle tools. The second teaching tool . Preoperative and six-week postoperative pelvic radiographic films were reviewed for 29 consecutive cases previously performed by the senior author. Fourteen cases were excluded because they demonstrated inadequate or poor-quality radiographs, severe deformity of the hip, a contralateral THA, or moderate to severe contralateral hip arthritis. Preoperative pelvic radiographs of the remaining 15 cases were templated by the two senior authors using the principles demonstrated in Video Orthopedic surgery trainees who took part in the study included three postgraduate year 1 (PGY-1) residents, three pos-graduate year 2 (PGY-2) residents, three postgraduate year 5 (PGY-5) residents, and three postgraduate year 6 (PGY-6) adult reconstruction fellows. Trainees were chosen among their peers based on their availability during the timeframe of the study. It is important to note that formal didactic training in THA templating was not a part of the resident or fellow teaching curriculum at our institution. All trainees watched the first training video and then placed digital acetabular and femoral component templates for the 10 cases, based on their preexisting skills. The trainees were also instructed to mark the femoral head length of choice on the femoral component. All trainees repeated this task 2-4 weeks later. After another two-week holiday, all trainees watched Video After each templating session, the results were measured and recorded. These included the implant sizes, the acetabular component inclination (measured as the angle between the inter-teardrop line and the face of the acetabular component), and the varus/valgus angle of the femoral component . In addition, the vertical distance between the center of the axis of the acetabular component and the chosen neck length of the femoral head was measured. This value was compared with the calculated preoperative leg length discrepancy (LLD).For each measurement, the error was calculated as the absolute value of the difference between the subjects’ measurements and the correct value. For stem and cup sizes, the correct value was defined as the component sizes used at the time of surgery. For acetabular component inclination, the correct values were defined as a range between 40° and 45°. For femoral component positioning, the correct value was defined as 0°, i.e., the axis of the stem is colinear with the axis of the femur.A linear mixed model is a statistical model that accounts for fixed and random effects. The model design may be used in cases in which there are repeated measurements on the same statistical unit. A linear mixed model predicting absolute error, treating the 12 subjects (residents/fellows) and the 10 pelvic radiographs as independent random effects, was used to look at the interaction of intervention and year of training while controlling for repeated observations with the same subjects and radiographs. In addition, for each group of subjects within the same PGY level, the standard deviation for the net error (as opposed to the absolute error) was used to determine the variance of pre- and post-intervention measurements. This standard deviation indicates how much variation exists among residents of a given year. The F-test was then used to determine changes in variance after watching the second video. All statistical analyses were conducted using the R statistical platform .Acetabular component sizing Figure  and inclAnother improvement was in the variability of estimates. Figure The advent of ACGME-mandated restriction of resident work hours has translated into difficulties in effectively training orthopedic surgery residents in a timely manner ,3. As a Video learning is becoming increasingly widespread and has been shown to significantly improve medical student and resident physician knowledge and skills ,16. TechTemplating in THA helps establish an adequate preoperative plan, identify individual osseous morphology, and prevent complications such as LLD, instability from component malpositioning, implant loosening from undersized cementless implants, and periprosthetic fracture from oversized components . DigitalThe results of our investigation demonstrated that prior to learning from our video tool, residents of increasing PGY levels showed a corresponding increase in accuracy for the selection of acetabular and femoral component sizes, femoral varus/valgus positioning, and acetabular component inclination. This goes along with prior literature showing increasing templating accuracy with experience ,6. MittaAfter watching the instructional video, our study subjects demonstrated improvement in both performance, as represented by means of component positioning and size, as well as consistency, as demonstrated by decreased variance. Junior and senior residents were able to perform at the same level as arthroplasty fellows after watching the video. Adult reconstruction fellows showed to be proficient at properly sizing and positioning components, even without watching the instructional video. In regard to accurate templating of LLD, although post-intervention performance witnessed more consistency, it was still suboptimal.Our study did present several limitations. We only included simple cases of patients with hip osteoarthritis undergoing THA. Performance would probably have been different if taking into consideration more complicated cases such as patients with proximal femoral deformities, etiologies other than osteoarthritis, and revision THA. Our study is limited to the residents and fellows training at a single institution with its own background in education on THA templating. A single implant was used for homogeneity of the study; however, in clinical practice, a multitude of implants are used, and most surgeons will adapt the implant to the patient’s osseous anatomy, especially in regard to the proximal femur. We did only include 12 subjects in our study; nonetheless, this is higher than prior studies addressing this topic in the literature ,29. It iVideo-based learning is a proficient tool to teach skills that need to be acquired during orthopedic residency, including THA templating. We designed a concise video to teach orthopedic surgery residents how to perform adequate templating for THA and demonstrated its effectiveness and reliability. Making this tool universally available will help orthopedic surgery residents learn proper THA templating in a time-efficient manner, without encroaching on their work hours and clinical duties. In the future, we plan to include advanced instructions addressing complex cases such as severe acetabular dysplasia, proximal femoral deformity, and revision THA.