Studying language evolution brings a crucial perspective to bear on questions of human prehistory. As the most linguistically diverse region on earth, East and Southeast Asia have witnessed extensive sociocultural and ethnic contacts among different language communities. Especially, the Kra-Dai language family exhibits tremendous socio-cultural importance in these regions. Due to limited historical accounts, however, there are several controversies on their linguistic relatedness, ambiguities regarding the divergence time, and uncertainties on the dispersal patterns. To address these issues, here we apply Bayesian phylogenetic methods to analyze the largest lexical dataset containing 646 cognate sets compiled for 100 Kra-Dai languages. Our dated phylogenetic tree showed their initial divergence occurring approximately 4000 years BP. Phylogeographic results supported the early Kra-Dai language dispersal from the Guangxi-Guangdong area of South China towards Mainland Southeast Asia. Coupled with genetic, archaeological, paleoecologic, and paleoclimatic data, we demonstrated that the Kra-Dai language diversification could have coincided with their demic diffusion and agricultural spread shaped by the global climate change in the late Holocene. The interdisciplinary alignments shed light on reconstructing the prehistory of Kra-Dai languages and provide an indispensable piece of the puzzle for further studying prehistoric human activities in East and Southeast Asia. Kra-Dai language family exhibits great linguistic diversity and tremendous socio-cultural importance in East Asia. In this study, the authors found that Kra-Dai languages initially diverged ~4,000 years ago in Southern China coinciding with prehistoric demic and agricultural diffusions likely driven by climate change. Language is the carrier of socio-cultural activities and is often shaped by complex demographic dynamics5. Therefore, studying language evolution brings a crucial perspective to bear on questions of human prehistory. However, ethnolinguistic prehistory remains poorly understood in East and Southeast Asia.East and Southeast Asia host several great ancient civilizations and the world’s most populous countries, exhibiting great genetic and socio-cultural diversities7. Their geographic distributions are surrounded by or intermingled with the settlements of the four other language families: Austronesian, Austroasiatic, Sino-Tibetan, and Hmong-Mien  is spoken by nearly 100 million people and geographically distributed in a vast region encompassing South China, Mainland Southeast Asia (MSEA), and Northeast Indiaien Fig. 6. The ac9. Similarly, the placement of Hlai is also an ongoing debate whether Hlai is a primary branch or a lower position as a sister of Ong-Be and Tai10. Moreover, it remains no consensus on the explicit relations of Ong-Be with the Tai and Kam-Sui branches, respectively11. In addition, there are ongoing debates on the low-level branches of Tai languages such as the division between the Central and Southwestern branches on a par with the Northern branch15. Different proposals on Kra-Dai classification have different implications for the topological structure of Kra-Dai phylogeny ranging from more rake-like to more hierarchically nested16.Three fundamental issues remain dubious and then hamper reconstructing the history of the Kra-Dai languages. The first issue is linguistic relatedness. The Kra-Dai languages primarily comprise five well-described branches: Kra, Hlai, Ong-be, Tai, and Kam-Sui. However, their relationships are still controversial. The foremost one is the position of Kra. Some scholars advocate the Kra languages as a primary branch in the Kra-Dai language family while others suppose that Kra languages should be below the Kam-Sui group8. However, the initial divergence time of the Kra-Dai languages is ambiguous due to lacking available prehistoric records. In historical linguistics, some scholars regard the Kra-Dai language family as a very old phylum and speculate their initial divergence to have occurred 5000–6000 years ago18. In contrast, other scholars consider that the first split of Kra-Dai languages could take place no more than 4000 years before the present (BP)20. Despite ambiguities on the Kra-Dai language divergence time, the recent phylogenetic studies of the surrounding language families such as Austronesian21 and Sino-Tibetan23 illustrate the vitality of language evolution in MSEA and adjacent Southern China region 5000–6000 years ago. Accordingly, most Chinese linguists favor that in the same geographic area, the initial divergence of Kra-Dai languages could be traced back to at least 5000 years BP18.The second issue is the time-depth of the Kra-Dai language divergence. The demographic documents can only provide clues about the Kra-Dai language dispersal from the Southern China region to Thailand and Laos in the past 750 yearsUrheimat inference in linguistics, the Kra-Dai dispersal can originate from the Guizhou inland of China which exhibits the highest linguistic diversity among the present Kra-Dai languages18. And then, the Kra-Dai languages dispersed southward into Guangxi, Hainan Island, and MSEA; and simultaneously westward into Yunnan inland and further into MSEA. This scenario can be summarized as the Inland Origin Hypothesis of Kra-Dai languages , with a 95% HPD interval range of approximately 2700 to 5500 years ago 38. The best model with the highest Bayes Factor (BF = 42.67) strongly supported the scenario that no transitions occurred between non-adjacent areas geographically and none between MSEA and Hainan Island , coupled with the initial divergence of Kra-Dai languages, a nearly unchanged population size, decreasing archaeological sites, survival probabilities of rice, and temperature. The second one was the “recovery period” after the 4.2 K event (3500–2300 years BP), corresponding to the early divergence events of Kra-Dai languages, a more temperate climate than before, and a steady increase of archaeological sites and population size. The third one was the “prosperity period” (2300 years BP—the present), which witnessed a rapid increase in language numbers and population size  was likely the dispersal center of Kra-Dai languages, and profiled the north-south and east-west dispersal routes which were consistent with previous genetic and cultural evidence46. Furthermore, our interdisciplinary alignment revealed that the Kra-Dai language dispersal might be associated with environmental change and demographic activities in East and Southeast Asia. Overall, our findings offered a new perspective on the evolutionary dynamics of Kra-Dai languages and their contributions to shaping language diversity in East and Southeast Asia. By studying the evolutionary history of Kra-Dai languages, we could gain more insights into the present socio-cultural landscape and better understand the prehistory of these regions.Studying the spatiotemporal evolution of Kra-Dai languages is crucial for comprehending the demographic activities and socio-cultural development in East and Southeast Asia. In this study, we employed Bayesian phylogenetic methods to reconstruct the linguistic relatedness of the five branches in accordance with that proposed by Ostapirat47. These possibilities have been regarded as deep problems with linguistic analysis that undermine Bayesian phylogenetic methods48  of Kra-Dai-speaking populations. In contrast, during the “prosperity period”, the long-term stable temperature and food production allowed the number of Kra-Dai languages and the size of Kra-Dai-speaking populations to increase spontaneously, contributing to more frequent demographic activities such as population expansions and interactions. . Based on our interdisciplinary analysis, the Kra-Dai language and agricultural spread might be driven by the climate fluctuating and the survival probability of rice declining dramatically42 , following the ethical research principles of the Ministry of Science and Technology of the People’s Republic of China . Study staff informed potential participants about the goals of the project, and the individuals who chose to participate gave informed consent consistent with broad studies of population history and human variation and public posting of anonymized data. There were no rewards for participating and no negative consequences for not participating; all participants signed or affixed a thumbprint to the consent form reviewed by Fudan University.https://starling.rinet.ru/cgi-bin/main.cgi), several previous research reports, and first-hand language documents from our linguistic fieldwork  to reconstruct the phylogenetic tree of Kra-Dai languages and estimate the divergence time71   on 1000 trees. The 1000 trees were randomly resampled in all posterior sample trees generated by BEAST after a burn-in of the first 10% of samples. Given the trees, we reconstructed the ancestral area for the dispersal center of proto languages geographically. To explore the dispersal routes among different areas, we tested five models and used the Bayes Factor to choose the optimum one 38, where the approach was run for 55,000,000 iterations, sampled in every 5000 iterations with a burn-in of the first 1000 samples. Finally, we obtained 10,000 posterior samples. Specifically, to find out the most probable homeland for Kra-Dai languages, we used paired one-side Wilcoxon signed rank test to find whether there are significant differences among the probabilities of the five distinct areas in the posterior samples , Hainan Island, and MSEA . Accordingly, each language sampled in our study was assigned to a definite geographical area. According to the available historical ethnic recordsles Fig. . For parTo study the population expansion of Kra-Dai-speaking populations, we reconstructed the maternal demographic history using the mitochondrial DNA (mtDNA) data, representing maternal inheritance of the Kra-Dai-speaking populations. Six Kra-Dai-speaking populations were included and 22 representative mtDNA lineages were identified in this study. Related samples in China consisted of 27 Dong, 19 Zhuang, and 35 Dai individuals, whereas related samples in MSEA consisted of 12, 56, and 266 Kra-Dai-speaking individuals in Laos, Vietnam, and Thailand, respectively , and was carried out following the approved guidelines. We followed the recommendations provided by the revised Helsinki Declaration of 2000. The participants responded to community advertising for our investigation on local ethnic groups and were recruited at the local study sites. All the samples included in this study were maternally unrelated and all of their parents were confirmed Dong or Zhuang people, respectively. The sample donors aged from 18–60 years old, and all of them were informed of an overview of the investigation and signed written informed consent before participating in the study. The details of mtDNA library preparation, sequencing, assembly, variant calling, sample selection, and sequence processing were demonstrated in Supplementary Information section 75  were identified as the representatives of Kra-Dai language expansion  drawn from Hosner et al. 78. The period was from 6000 to 2000 years BP. We counted the number of archaeological sites whose time range covered a specific time point of the year. And then the same smoothing approach was applied to obtain the trend curve. For genetic data, we used our Bayesian skyline plot reconstructed upon mtDNA data without any smoothing process. For paleoecologic data, we used the data derived from Gutaker et al.’s work in which the percent probability of tropical rice was displayed in the thermal niche (assuming a requirement of 2900 growing degree days at 10 °C bases) over time39. For paleoclimatic data, we used the Chinese Holocene Temperature Series which was synthetically reconstructed based on about 1397 temperature records during the Holocene in China42.To depict the global picture of Kra-Dai language divergence and dispersal, we put the linguistic, archaeological, genetic, paleoecologic, and paleoclimatic data together to align this cross-disciplinary evidence in the temporal domain (Supplementary Data Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Supplementary Data 10Supplementary Data 11Supplementary Data 12Supplementary Data 13Reporting Summary


Cystic fibrosis (CF) is a rare genetic disease characterized by life-shortening lung function decline. Ivacaftor, a CF transmembrane conductance regulator modulator (CFTRm), was approved in 2012 for people with CF with specific gene mutations. We used real-world evidence of 5-year mortality impacts of ivacaftor in a US registry population to validate a CF disease-progression model that estimates the impact of ivacaftor on survival.CFTR gating mutations by combining parametric equations fitted to historical registry survival data, with mortality hazards adjusted for fixed and time-varying person-level characteristics. Disease progression with standard care was derived from published registry studies and the expected impact of ivacaftor on clinical characteristics was derived from clinical trials. Individual-level baseline characteristics of the registry ivacaftor-treated population were entered into the model; 5-year model-projected mortality with credible intervals (CrIs) was compared with registry mortality to evaluate the model’s validity.The model projects the impact of ivacaftor vs. standard care in people with CF aged ≥6 years with Post-calibration 5-year mortality projections closely approximated registry mortality in populations treated with standard care (6.4% modeled [95% CrI: 5.3% to 7.6%] vs. 6.0% observed) and ivacaftor (3.4% modeled [95% CrI: 2.7% to 4.4%] vs. 3.1% observed). The model accurately predicted 5-year relative risk of mortality (0.53 modeled [0.47 to 0.60] vs. 0.51 observed) in people treated with ivacaftor vs. standard care.Modeled 5-year survival projections for people with CF initiating ivacaftor vs. standard care align closely with real-world registry data. Findings support the validity of modeling CF to predict long-term survival and estimate clinical and economic outcomes of CFTRm. Cystic fibrosis (CF) is a genetic disease affecting more than 80,000 people worldwide –3. CF is®, manufactured by Vertex Pharmaceuticals) is a CFTRm that facilitates chloride transport by potentiating the channel-open probability (or gating) of the CFTR protein at the cell surface [G551D-CFTR mutation [Ivacaftor  face validity, (2) internal validity, (3) cross-validity, (4) external validity, and (5) predictive validity . In the Years of accumulated real-world experience with ivacaftor in the United States now enables testing for predictive validity of the CF-PSM. Specifically, in this study, model projections from the CF-PSM were compared with outcomes observed among people treated with ivacaftor and a matched comparison group not treated with a CFTRm in the US Cystic Fibrosis Foundation Patient Registry (CFFPR) .The CF-PSM was developed to project long-term clinical and economic outcomes of ivacaftor treatment based on data from randomized controlled trials and real-world sources that characterize CF survival, disease progression, and the efficacy of ivacaftor. While the model has been updated and adapted for subsequent CFTRm, the core model framework  Fig 1)  Fig 1) hStaphylococcus aureus, Burkholderia cepacia), percent predicted forced expiratory volume in 1 second (ppFEV1), annual number of acute pulmonary exacerbations (PEx) requiring treatment, and weight-for-age z-score [1, reduction in the rate of ppFEV1 decline over time, and reduction in the number of PEx requiring treatment, as well as improvement in nutritional status in terms of weight-for-age z-score  who received standard care but not a CFTRm, with a Cox proportional hazards (CPH) equation developed from US CFFPR data by Liou et al. in 2001 that links 5-year survival in CF to several key demographic and clinical characteristics , 36. The z-score , 36. The z-score . The imp z-score  [10, 13,CFTR genotype) [1), improved nutritional status, lower frequencies of PEx and hospitalizations, lower probability of lung transplant, and higher probability of transplant-free survival over up to 5 years of follow-up [The ivacaftor post-authorization safety study had a follow-up time of 5 years, allowing for evaluation of the effects of ivacaftor treatment on disease progression and clinical outcomes . The stuenotype) . Longituollow-up .The objective of the current analysis was to assess the predictive validity of the CF-PSM by comparing outputs of this simulation model over a 5-year horizon with real-world 5-year survival data from the US CFFPR as reported in the Volkova et al. study .Our examination of predictive validity focused first on the prediction of survival in the CF population not receiving CFTRm and then on the impact of ivacaftor on survival; we intended to examine and revise methods as necessary to replicate observed data. Several model inputs and assumptions were of particular interest for evaluation due to their importance in estimating survival, including accounting for individuals’ characteristics at baseline, the estimation of the reference survival curve for CFTRm-untreated people with CF, the use of the CPH equation  to adjusThe validation process comprised a stepwise approach. First, de-identified baseline profiles for the 805 individuals treated with ivacaftor in the 5-year disease-progression cohort from Volkova et al.  were runThis study was reviewed and exempted by the Advarra Institutional Review Board (IRB). The IRB waived the requirement for informed consent.Among the 805 baseline individual patient profiles from the Volkova study provided by the US CFFPR, 178 observations (22.7%) were missing 1 or more covariate values. Where sufficient data were available, missing baseline values were imputed using multiple imputation by chained equations, in which values for missing covariates are imputed using non-missing covariates , 43. ThiWhen comparing the 5-year survival in the modeled CFTRm-untreated cohort with the observed 5-year survival in ivacaftor-ineligible individuals from the Volkova study, we initially found that the CF-PSM over-predicted mortality in the CFTRm-untreated group by over 5 years (17% modeled vs. 6.0% observed). On close examination, we found that calculating the baseline mortality risk for each simulated patient based on individual clinical characteristics at baseline using the CPH equation and reference survival curve identified individuals with extremely high mortality hazards at baseline due to concurrent risk factors and the multiplicative nature of CPH. A revision was consequently made to instead assign all people with CF an age-specific mortality risk at baseline derived from the reference survival curve and not to adjust for baseline clinical characteristics. Instead, the CPH equation was used only to adjust mortality hazard in each subsequent post-baseline model cycle. This revision improved model fit, but the model continued to slightly over-predict 5-year mortality in CFTRm-untreated people with CF (9.7% modeled vs. 6.0% observed). To more closely match the observed 5-year mortality rate in CFTRm-untreated individuals from the US CFFPR, the reference survival curve was shifted to increase the median survival from 39.7 years, which reflected survival in the historic population from which the survival curve was derived , in fullWe next generated model survival projections for an ivacaftor-treated cohort and found that the CF-PSM accurately replicated the observed ivacaftor treatment effect in the post-authorization safety study, estimating mortality of 3.4% (95% CrI: 2.7% to 4.4%) vs. the observed 3.1% . The modCFTRm such as ivacaftor are projected to change the trajectory of CF disease and extend survival by upwards of 30 years based on a published patient-level lifetime simulation model –32. WithTo validate the CF-PSM, we used a stepwise approach in which we first ensured that the model accurately predicted survival in the absence of CFTRm treatment. Consistent with modeling guidelines, we calibrated the model to achieve the best fit by adjusting inputs to replicate real-world target data as closely as possible . RemovinA key finding of this exercise is that, although the Liou CPH equation was developed in a population entirely unexposed to CFTRm, the changes in clinical outcomes as a result of CFTRm treatment combined with the CPH equation generate 5-year survival estimates that closely match the impact of ivacaftor observed in the real world. This result suggests that models using this equation and applying similar methods can provide plausible estimates of treatment impact and outcomes, and can be used with confidence to extrapolate beyond clinical trial data to provide informative predictions of long-term benefits of CF treatments, based on currently available evidence.1 for every age), all modeled baseline hazards were calculated relative to the reference population averages . This approach led to many younger-than-average modeled people with CF appearing unusually healthy while those older than average appeared unusually ill, with their age-specific hazards adjusted accordingly. Moreover, when individual baseline hazard predictions were examined, it was apparent that, for a small number of people with CF who had multiple risk factors at baseline, the CPH model generated extremely high mortality hazards at baseline due to the multiplicative nature of the CPH model. Removing this adjustment assumes that the baseline hazard for modeled people with CF is the same as that for people with CF in the reference population of the same age. This simplification resulted in model output that was a much better fit to the observed 5-year US CFFPR mortality rates.The change in the baseline application of the Liou CPH equation was an unexpected outcome of the calibration process. Prior to this validation study, the CF-PSM adjusted an individual’s baseline age-specific mortality hazard with reference to the average characteristics of people with CF in the background population  . This baWe further note that the increase in median survival for the reference survival curve as a result of calibration is consistent with epidemiologic data from the US CFFPR that show an increase in median survival over the past several decades. The predicted life expectancy from the most recent registry report  is basedBecause calibration changed the 5-year survival estimate in modeled people with CF treated with ivacaftor and standard care, we re-examined previous published analyses in which the CF-PSM was used to assess outcomes among populations with a range of ages over a lifetime horizon . When anWhile the availability of published data from a large real-world data set provided the unique opportunity to validate the CF-PSM, our study is subject to a number of limitations. The validation sample comprising individuals with complete baseline profiles used in the model was a subset of the ivacaftor-treated cohort in the Volkova study ; although baseline characteristics were similar to the overall sample, any bias introduced by using this subset is unknown. Similarly, bias introduced by imputation of missing data cannot be quantified. In addition, although the comparator group in the Volkova study was matched to treated individuals at baseline, loss of individuals from the comparator arm due to availability of a novel CFTRm may have selected for remaining individuals who were healthier at baseline. Thus, underestimation of the ivacaftor survival benefit used as a validation target may have occurred. Considering the close match between the treatment effect in Volkova et al. and that of the calibrated model, this would suggest that any underestimation of the ivacaftor survival benefit in Volkova et al. may also be present in the model.We further note that the available data for validation included only 5 years of follow-up in a US population of people with CF treated with a single CFTRm; however, Liou et al. demonstrated that the model developed in a historical population accurately predicted survival over a 25-year study period, despite changes in standard care and expected survival over this period. It will be of interest to continue validating and refining the model as longer-term data and real-world data for additional CFTRm and broader populations become available.1, that drive CF-PSM projections. However, because the median survival in CF is now predicted to be >50 years and therapeutic options and standard care are rapidly evolving in CF, the scope of the current CF-PSM may be subject to future revision. Changes may be needed to incorporate as yet unknown lifetime impacts, such as differential outcomes for subgroups, positive or negative effects of long-term treatment on non-respiratory organ systems, and adverse outcomes of CF in an aging population. These remain rich areas for future study.Finally, the CF-PSM structure is flexible to incorporate additional evidence as it becomes available for new treatment modalities, long-term adherence, and durability of clinical benefits, which are likely to impact the clinical characteristics, such as ppFEVThis study demonstrates the successful validation of a long-term survival model in CF by comparing modeled results to real-world, observed, 5-year survival in a US registry. These results show that, with appropriate model assumptions, disease progression and CFTRm clinical efficacy data in combination with the Liou CPH equation provide valid estimates of long-term survival and impact of CFTRm. The validation of the modeling methods, including use of the Liou CPH equation to estimate survival in both treated and CFTRm-untreated people with CF, should encourage confidence in lifetime projections of CF population survival, both for ivacaftor and for other CF treatments generated using similar modeling methods.After calibration, modeled 5-year survival projections for people with CF with gating mutations initiating ivacaftor and standard care tracked closely to trends observed in real-world registry data. These findings support the validity of modeling CF using the described approach to predict long-term survival and estimate clinical and economic outcomes attributable to CFTRm.


Aneurysm formation on the tumor-feeding artery is rare, and its treatment strategies are not yet settled. We herein report the case of a 49-year-old female with a large subependymoma in the left lateral ventricle presenting remote intracerebral hemorrhage at the left posterior cingulate gyrus. Digital subtraction angiography (DSA) revealed the presence of a 5.5 mm fusiform tumor-feeding artery aneurysm on the left parieto-occipital branch of the posterior cerebral artery, considered to be the source of the hemorrhage. Three months after total tumor resection, the aneurysm subsequently disappeared on the follow-up angiography. Subependymomas are generally known as tumors with low vascularity and seldom present with symptoms such as intracerebral hemorrhage. From the subsequent disappearance of the aneurysm after the complete tumor resection, the pathophysiological cause of the aneurysm formation is assumed to be flow-related hemodynamic vessel wall stress of the feeding artery. Tumor resection alone may be a favorable first treatment strategy to avoid unnecessary brain damage since subsequent disappearance of the aneurysm can be expected. The coexistence of feeding artery aneurysms should be kept in mind, especially in cases with remote hemorrhage. Brain tumors sometimes have coexisting intracranial aneurysms ,2, but bA 49-year-old female with no notable previous medical history presented with sudden dysmnesia and right sensory disturbance. The patient had not experienced any noticeable symptoms before the onset. Physical examination showed no physical function deficit. However, the patient had short-term memory and working memory disturbance. The Mini-Mental State Examination (MMSE) score was recorded at 27 points. Magnetic resonance imaging (MRI) revealed an 8 cm mass in the left lateral ventricle and an intracerebral hemorrhage at the left posterior cingulate gyrus Figure -1d. The We performed a two-stage tumor resection because of the tumor size and location. A transcortical approach from the left superior frontal gyrus was taken as the first stage, with a transcortical approach from the left superior parietal lobule as the second stage after a one-month interval. We only removed the tumor, and cingulate gyrus hematoma was not observed during the two surgeries. Gross total resection of the tumor was achieved, and the pathological diagnosis was subependymoma, World Health Organization grade 1 . AlthougThe coexistence of primary brain tumors and intracranial aneurysms is reported at an incidence of 1.1%-5.4% , but mosHemodynamic vessel wall stress is considered a primary cause of flow-related aneurysms in AVMs. Notably, distal flow-related aneurysms are assumed to have a strong association with vessel wall stress and a higher probability of regression after removal of the nidus . In our There is no unified view about treatment strategies for brain tumor-feeding artery aneurysms . NotablyBrain tumors with coexisting tumor-feeding artery aneurysms are rare. However, even if brain tumors have low vascularity, the coexistence of feeding artery aneurysms should be kept in mind, especially in cases with remote hemorrhage. Subsequent regression of the aneurysm may be expected with tumor removal, so aneurysm trapping may be proscribed if the immediate re-bleeding risk is considered to be low.


The depth-predicting score (DPS) was proposed based on conventional white-light imaging (C-WLI) endoscopic features of early gastric cancer (EGC) to determine the invasion depth of the neoplasm. However, the effect of DPS on training endoscopists remains unclear. Therefore, we aimed to investigate the effect of short-term DPS training on improving the diagnostic ability of EGC invasion depth and compare the training effect among non-expert endoscopists at different levels.In the training session, the definitions and scoring rules of DPS were instructed, and classic C-WLI endoscopic example graphics were exhibited to the participants. Another C-WLI endoscopic images of 88 cases of histologically proven differentiated EGC were selected as an independent test dataset for evaluating the training effect. Each participant was tested, and the diagnostic accuracy rate of invasion depth was calculated differently one week before the training and after the completion of training.P = 0.001). No significant difference between the trainee group and junior endoscopist group was observed for pre-training accuracy. The overall diagnostic accuracy of invasion depth was improved significantly after completing DPS training compared with before . In the subgroup analysis, the post-training accuracy was higher than the pre-training accuracy, but significant improvement was observed only in the trainee group . In addition, no significant difference in post-training accuracy between the two groups was observed.A total of 16 participants were enrolled and completed the training. Participants were divided into a trainee group and a junior endoscopist group according to the total number of C-WLI endoscopies performed. The total number of C-WLI endoscopies performed showed a significant difference between the trainee group and junior endoscopist group (350 vs. 2500, Short-term DPS training can improve the diagnostic ability of the invasion depth of EGC and homogenize the diagnostic ability of non-expert endoscopists at different levels. The depth-predicting score was convenient and effective for endoscopist training. Gastric carcinoma (GC) is one of the foremost prevalent malignant neoplasms of the digestive tract and is the fourth leading cause of cancer-related deaths worldwide –3. EarlyThe selection of curative endoscopic resection for EGC depends on the differentiation, size, and depth of invasion of the lesions , 10. TheBecause of its accessibility and convenience, C-WLI endoscopy is more commonly used to predict the invasion depth of EGC in clinical practice . The depEligible participants were gastrointestinal endoscopists from the Second Affiliated Hospital of Anhui Medical University who had not accepted a professional training course about EGC diagnosis and did not know DPS before this study. Participants gave their informed consent prior to starting the survey. All participants were asked to complete a survey questionnaire including age, gender, years of experience in C-WLI endoscopy, and the number of C-WLI endoscopies performed. Exclusion criteria were incomplete questionnaires, unwillingness to participate, and had accepted a professional training course about EGC diagnosis before this study.The training program and assessment consisted of three steps. Stage 1 (pre-training test): All participants were asked to take the diagnostic test by the test dataset first before the training. Stage 2(short-term training): One week after pre-training test, the definitions and scoring rules of DPS were introduced and explained, and classic C-WLI endoscopic example graphics were exhibited to the participants. Stage 3 (post-training test): All participants took the second diagnostic test by the same test dataset immediately after training. The diagnostic accuracy rate of each participant was calculated separately before the training and after the completion of training as evaluation metrics. Using the histological invasion depth as gold standard, the diagnostic accuracy was obtained by dividing the number of cases judged correctly before or after training by the total number of cases in the test dataset. Because SM2 patients can be excluded from absolute indication of ESD, specificity to predict M-SM1 may be more important in this situation. The specificity to predict M-SM1 was also calculated separately before and after the training. Using the histological invasion depth as gold standard, the specificity was obtained by dividing the number of M-SM1 EGC cases judged correctly before or after training by the actual number of M-SM1 EGC cases.The dataset of the diagnostic test comprised C-WLI endoscopic images of 88 cases of histologically proven differentiated EGC with complete medical record materials. Participants were blinded to the clinical and pathological information. We used the same 88 cases in the same order for the pre-training test and post-training test, and this information was not disclosed to participants. All lesion images were collected with close-up and distant views. All endoscopic images were taken by C-WLI endoscopy  during actual clinical practice.DPS includes four endoscopic metrics: remarkable redness, uneven surface, lesion size more than 30 mm and margin elevation. Each endoscopic metric was assigned a certain number of points. Remarkable redness or uneven surface was assigned 1 point, and lesion size more than 30 mm or margin elevation was assigned 2 points. The resulting DPS was measured by a 6-point scale. A total of 3 points was defined as the cut-off value between pathologic M-SM1 and SM2. Written definitions and assigned points of each endoscopic feature were made available to all participants during DPS training and the post-training test . The Shapiro–Wilk test was used to detect the normality of variables. Data were expressed as percentages or mean ± standard deviation, medians and interquartile ranges  as appropriate. Fisher’s exact test and Wilcoxon rank-sum tests were used to compare inter-group differences in demographic data. For normally distributed data, inter-group differences were compared using t-test for independent samples. Paired t tests were used to compare pre- and post-training differences. All statistical tests were two-sided, and P < 0.05 was considered statistically significant.A total of 16 participants were enrolled and completed the training. Participants were divided into a trainee group and a junior endoscopist group according to the total number of C-WLI endoscopies performed. Trainees were defined as residents-in-training who had completed < 400 previous C-WLI endoscopies. Junior endoscopists were defined as those who had completed 2000 to 3000 C-WLI endoscopies. The 16 learners included 8 trainees and 8 junior endoscopists. Participants in the trainee group were on average 26 years old , the majority were female (75%), and they had less than 1 year of operator experience. Participants in the junior endoscopist group were on average 33 years old , all were female (100%), and 37.5% had more than 3 years of operator experience. The total number of C-WLI endoscopies performed in the career was significantly different between the trainee group and the junior endoscopist group . The demographic information and characteristics are summarized in Table The pre-training test accuracy was described as a baseline level. The overall diagnostic accuracy was significantly higher than the baseline after DPS training , with a mean difference of 7.17%   and post-training accuracy  . In the subgroup analysis, the post-training specificity was also higher than the baseline both in the trainee group (72.54±11.89% vs. 62.12±9.92%) and the junior group (73.68±9.65% vs. 60.04±16.99%), but with no significant differences.In this study, the value of short-term DPS training for endoscopists to improve the diagnostic ability of the invasion depth of EGC during C-WLI endoscopy was evaluated. We found that the overall diagnostic accuracy improved after training. It is indicated that short-term DPS training can improve endoscopists’ diagnostic ability of EGC invasion depth, which may be helpful to select the correct treatment strategy for EGC. To the best of our knowledge, this is the first report to apply DPS in endoscopist training and evaluate the effect of DPS training on improving the diagnostic accuracy of invasion depth of EGC.DPS was proposed upon logistic regression analysis of four main endoscopic features summarized by three expert endoscopists, and the accuracy of the DPS was 82.5–84.8% in the validation set . AlthougIn our study, participants were further divided into two groups according to the total number of C-WLI endoscopies performed. We aimed to observe the training effect among non-expert endoscopists with different levels.On the one hand, we found that prior to training, the junior endoscopist group did not show higher accuracy in determining the invasion depth of EGC than the trainee group, although they performed more total C-WLI endoscopies. This suggested that the total number of endoscopies performed without special training was not correlated with the diagnostic ability of the invasion depth of EGC. Additionally, it was reported in previous studies that training plays an important role in improving the detection rate of EGC , 18. TheOn the other hand, post-training accuracy in the two groups increased after short-term DPS training, and showed no statistical difference between the two groups. In addition, the standard deviation of the post-training accuracy narrowed after the training. This shows that short-term DPS training helped to achieve homogeneity in the diagnostic abilities of invasion depth of EGC among non-expert endoscopists at different levels.Although the accuracy of both groups increased after training, a statistically significant difference was not found in the junior group. The reason may be that greater inter-individual variation existed among junior endoscopists before training, as evidenced by the larger standard deviation. Studies have shown that the diagnostic accuracy of invasion depth may largely depend on the knowledge and experience of the endoscopist , 20. ForFinally, we believed that the main contribution of this study is to show that DPS short-term training can be effective, improve swiftly the diagnostic ability of invasion depth for differentiated EGC among non-expert endoscopists, and have the superiority of being low-cost, simple, and convenient.The present study has several limitations. First, this was a study performed at a single institution. Second, assessments of the endoscopic staging were performed based on recorded still images rather than real-time endoscopic observation. Third, short-term training was designed in our study. Further studies are needed to observe the effect of using a systematic training atlas and long-term DPS training.Short-term DPS training can improve the diagnostic ability of the invasion depth of EGC and homogenize the diagnostic ability of non-expert endoscopists at different levels. The depth-predicting score was convenient and effective for endoscopist training.


There is a lack of attention to drive-thru services in the community pharmacy setting, particularly during the COVID-19 period in Malaysia. The main objective of this study was to assess the public awareness, attitudes, and perceptions towards drive-thru community pharmacy services among during COVID-19 in Malaysia.A cross-sectional study was conducted using a self-administrated, web-based survey (Google form) among the public in Malaysia between May and June 2022. Descriptive statistics were used to summarize the socio-demographic characteristics of the participants. Associations between the socio-demographic characteristics of the participants and the use of drive-thru community pharmacy services were assessed using a chi-square test. Regression analyses were carried out to determine whether the socio-demographic characteristics of the participants were associated with perceptions towards drive-thru community pharmacy services.A total of 565 (70.6%) of the general public completed the survey instrument. The median age of study participants was 40.0 (IQR = 36.0) and about half of them were males . Although 18.6% (n = 105) of the participants reported the presence of DTCPS in their cities, only 9.0% (n = 51) reported having used this service. Most of the participants were supportive to establish drive-thru services at community pharmacies in the country. Most of the believed advantages among participants were that DTCPS are helpful during COVID-19 and quarantine time 48.0% (n = 271) by enhancing social distancing and reducing the spread of the COVID-19 virus 48.5% (n = 274). Among sociodemographic factors, non-Malaysian nationality (p<0.001), and age above 55 years (p = 0.01) were found to negatively affect participants’ perceptions towards drive-thru community pharmacy services.This study showed positive awareness, attitudes, and perceptions toward drive-thru community pharmacy services during COVID-19 in Malaysia among the public. The participants believed that those services were helpful during COVID-19 to enhance social distancing and to reduce the spread of the COVID-19 virus. The World Health Organization (WHO) declared that Coronavirus disease COVID-19) constituted a Public Health Emergency of International Concern (PHEIC) on January 30, 2020 9 constit.Drive-thru is a way of receiving services or products without leaving the vehicle . The driIn the 1990s, to improve the provision of healthcare services for less mobile senior citizens, the first drive-thru pharmacy service was initiated by Walgreens pharmacy, in the United States of America (USA) . LikewisDrive-thru pharmacy is one of the services that was provided by the Malaysian Pharmaceutical Services Division, Ministry of Health Malaysia in 2003 as part of the Pharmacy Value Added Service (VAS) . For exaFurthermore, the Queen Elizabeth Hospital (QEH), Kota Kinabalu offered the drive-thru pharmacy service in 2015. Siung Liew et al. demonstrated that patients who used the drive-thru pharmacy service at (QEH) were satisfied with the service . AnotherDrive-thru pharmacy Griffith has been providing time-saving pharmacy services in Australia to the community since 2010 . SimilarDuring COVID-19 some countries-initiated drive-thru pharmacies for the first time to ensure the safety for pharmacists and consumers . For exaThe COVID-19 pandemic led to some economic benefits for pharmaceutical services, in which more sales and production of medications were needed; and fast-serving pharmaceutical services such as drive-thru service were highly desired by consumers . The gaiThere is a lack of attention to drive-thru services in the community pharmacy setting, particularly during the COVID-19 period in Malaysia, since the previously conducted studies focused on the drive-thru pharmacy in government hospitals and before the COVID-19 period ,15, and The first community pharmacy that provides drive-thru services was initiated by Superbig Kubang Kerian Pharmacy on 5th February 2022, as a need during the COVID-19 pandemic since customers are more likely to visit community pharmacies to get vitamins, hygiene products, masks, and even their regular medications to avoid going to the hospitals and clinics and to enhance social distancing . This coA descriptive, cross-sectional study was conducted in Malaysia between 19th May 2022 until 22nd June 2022. A non-probability convenience sampling method was used for data collection. A self-administered instrument using an online Google form was utilized as a data-collecting tool and was optimized as well to be easily filled using computers and smartphones. Was then distributed to the participants by research assistants  via social media platforms such as WhatsApp, Instagram, and Telegram applications. The time taken to complete answering the instrument was approximately 5 to 7 minutes. Study approval was obtained from the Human Research Ethics Committee of Universiti Sains Malaysia (USM) (Reference code: USM/JEPeM/21110755). Moreover, the participants who agreed to take part in the study signed the consent form electronically before proceeding to the first section of the instrument.ε is the margin of error, n is the population size, and The sample size was determined using the following formula  which re service , and unlA self-administered web-based survey (Google form) instrument was used for data collection. The instrument has been designed, validated, and presented in English based on an extensive literature review ,17,19,24The instrument design was divided into three main sections with forty-nine items. This included participants’ demographic information, attitudes toward DTCPS, and perceptions towards DTCPS. The objectives of this research study had been covered in these three sections. Items for the first section and second section which are socio-demographic information and attitudes of the general public towards DTCPS respectively were designed in open-ended questions and multiple-choice questions. Items for the third section discussed the perceptions, believed advantages, and believed disadvantages towards DTCPS, were designed in Likert Scale-type questions , and a reverse coding considered for negative statements and for the disadvantages , to indicate the degree of agreement or disagreement of participants after they went through the respective statements in this section. A total score of perceptions out of 140 was calculated. The higher the mean perceptions score the more positive the participants perceptions towards DTCPS.Descriptive statistics were used to summarize the socio-demographic characteristics of participants. Categorical variables data were presented as frequency and percentage. The mean, standard deviation (SD), median, and interquartile range were reported for continuous data. To assess the normality, Kolmogorov–Smirnov test was performed. Since the data did not support parametric assumptions, the Kruskal-Wallis test and Mann-Whitney U test were performed when applicable. For multiple group comparisons, Bonferroni correction was applied, and the results were evaluated accordingly. To assess the association between categorical variables chi-square test was performed. Multiple linear regression was carried out to determine whether the socio-demographics of participants  were associated with the perceptions of participants . Variables significant at a p-value of less than 0.10 were included in the multiple linear regression analysis. Preliminary analyses ensured that there were no violations of assumptions of normality and multi-collinearity before fitting the multiple regression model (VIF for each variable was < 10).  The internal consistency of the instrument scales was assessed using Cronbach’s alpha. A p-value of 0.05 was used for all statistical tests, all statistical tests were two-tailed. Analyses were performed using IBM statistical package for social science (IBM SPSS) version 28 .Eight hundred instruments were distributed to the public in Malaysia. A total of 565 (70.6%) of the general public completed the survey instrument. The median age of the participants was 40, with an interquartile range (IQR) of 36, and about half of them were males (50.6%). Briefly, the majority were single (46.9%), have no children (51.9%), Malay (94.2%), from Penang (17.2%), had a bachelor’s degree (49.4%), and were non-employed (42.8%). Less than half of the participants revealed that they don’t know about the presence of DTCPS at their city 44.8% (n = 253), only 9.0% (n = 51) of them used this service previously, and 6.7% (n = 38) evaluated their experience with using the service as a good experience. Most of the participants visited only one community pharmacy during the last month . Buying COVID-19 prevention supplies such as masks and hygiene products was the most common reason for the participants to visit the community pharmacies , followed by buying over-the-counter (OTC) medications . Most participants reported that they did not know any information about DTCPS . On the other hand, the internet and friends or colleagues were the main sources that triggered their awareness about the presence of the DTCPS ,  respectively. Pharmacy staff also played their roles in spreading information concerning drive-thru community pharmacy towards the participants contributing to the . A positive attitude was found with regard to participants ‘attitudes towards DTCPS in Malaysia . Most ofA positive perception was found with regard to participants’ perceptions towards the DTCPS . More thThe strongly agreed advantages of the DTCPS as believed by 48.5% of participants as it enhances social distancing and reduces the spread of the COVID-19 virus, followed by being helpful during COVID-19 time and quarantine time . On the other hand, strongly agreed disadvantages of drive-thru services were restricting the opportunity for interaction with the pharmacist , followed by not being convenient services for providing drug information/counselling to patients  . All other related advantages and disadvantages are presented in Face and content validity of the study instrument were satisfactory as found by the expert panel. The Cronbach’s alpha coefficient for the total perceptions score was 0.84 , which sThe relationship between the socio-demographic factors and participants’ total perceptions score towards DTCPS is shown in The relationship between the socio-demographic factors and participants’ perceptions towards DTCPS is shown in Tables Multiple linear regression analysis for factors associated with perceptions towards drive-thru community pharmacy services among the general population. These statistically significant variables predicted the perceptions score, F  = 1.867, p = 0.004, R2 = 9.2.This study describes the Malaysian public awareness and perceptions of DTCPS during COVID-19. During COVID-19, people in many counties were obligated to get their needs including medications through online platforms such as in Brazil, China, Germany, Italy, the Republic of Korea, the Russian Federation, South Africa, Switzerland, Turkey, and the United Arab Emirates ,29 or thDespite the high awareness among Malaysian hospital pharmacy consumers towards hospital pharmacy drive-thru services , it was The main sources that triggered participants’ awareness in this study about the presence of DTCPS were the internet, friends or colleagues, and pharmacy staff. Surprisingly, however, only 14% of the participants in the current study reported doctors as sources that triggered their awareness towards the presence of DTCPS. It demonstrates a need to raise publics’ awareness by doctors towards this service. Same results were presented among Jordanian drive-thru community pharmacy consumers .Participants showed positive attitudes towards the need for this service, as most of them expressed their support to establish drive-thru service at community pharmacies during COVID-19 time or later 44.8% (n = 253) and all over Malaysia 49.0% (n = 277) and believed that this will benefit all population 75.8% (n = 428). Same results were revealed in Saudi Arabia as customers of community pharmacies believed that drive-thru service will limit the risk of the COVID-19 pandemic and will be helpful for all population . This coParticipants of this study preferred to request an order at a community pharmacy using drive-thru services through a drive-thru window and to receive the counselling while using this service briefly through the drive-thru window as well. This could be justified by a previously reported study in Taiwan that the drive-thru area allowed minimal noise and distraction, and the quality of the service could be fruitful .As for participants’ perceptions towards the image of pharmacists following the introduction of the drive-thru service, about half of the participants believed that community pharmacists will have a good balance between the health of patients and the business side of their work. Nonetheless, the results of this study were consistent with a previous study conducted in Jordan, where it was found that 20.9% of drive-thru pharmacy consumers believed that community pharmacists will have a good balance between the health of patients and the business side of their work . This isThe participants of this study perceived some differences between drive-thru and in-store drug refills. About half (51.1%) of the participants believed that the prescription might be filled more quickly in the drive-thru compared to in-store pharmacy services. Same results were documented previously ,18,24. HThe study revealed that most of the participants had positive perceptions towards this service during COVID-19 time. Participants believed that this service has several advantages during COVID-19. They believed that this service is helpful during COVID-19 to promote social distancing and to reduce the spread of the COVID-19 virus. Only one study discussed this service during COVID-19 in Saudi Arabia . MoreoveThis study showed some socio-demographic factors that are associated with the publics’ use of drive-thru services during COVID-19. People with age above 55 years were more likely to use this service, in contrast to Lee and Larson , where yThis is the first study that assessed the socio-demographic factors associated with the participants’ perceptions towards DTCPS during COVID-19. Non-Malaysian nationality and age above 55 years negatively affected participants’ perceptions towards DTCPS, further studies are needed in this regard.There is a shortage of prior research studies about the use of DTCPS and its disadvantages ,34. The There were several limitations of our study. Firstly, to prevent the spread of COVID-19, the instrument was distributed online, and the participants filled it out via Google Forms, responses from the areas without the accessibility of the internet may not be captured. This may lead to demographic selection bias. Secondly, the instrument relied on participants’ self-rated assessment of their own perceptions, which may have resulted in an overestimation of the results. Furthermore, there is a lack of prior research studies on the DTCPS as previous studies only centered on Malaysian public hospitals such as Queen Elizabeth Hospital and Hospital Raja Perempuan Zainab II ,15.This study showed positive awareness, attitudes, and perceptions towards DTCPS during COVID-19 in Malaysia among the general population, where they believed that those services were helpful during COVID-19 to enhance social distancing and to reduce the spread of the COVID-19 virus. However, concerns about poor communication between the pharmacist and the patient represented the main believed disadvantage of the use of DTCPS. This research provides insight into the impact of instilling drive-thru pharmacies in Malaysia including the accessibility, workflow, pharmacist-patient interaction, and the general image of pharmacists depicted by the public. Further studies are needed to assess the use of DTCPS during COVID-19 on a larger scale public and as well from the healthcare perspective. Furthermore, further studies on the full cost of DTCPS implementation are needed including the medicine preparation, manpower, and additional equipment to help the decision-makers in better assessment whether the DTCPS would make a worthwhile contribution to pharmaceutical care without having to expand the budget allocation.


The present study analyzes the effects of each containment phase of the first COVID-19 wave on depression levels in a cohort of 121 adults with a history of major depressive disorder (MDD) from Catalonia recruited from 1 November 2019, to 16 October 2020. This analysis is part of the Remote Assessment of Disease and Relapse-MDD (RADAR-MDD) study. Depression was evaluated with the Patient Health Questionnaire-8 (PHQ-8), and anxiety was evaluated with the Generalized Anxiety Disorder-7 (GAD-7). Depression’s levels were explored across the phases  according to the restrictions of Spanish/Catalan governments. Then, a mixed model was fitted to estimate how depression varied over the phases. A significant rise in depression severity was found during the lockdown and phase 0 (early post-lockdown), compared with the pre-lockdown. Those with low pre-lockdown depression experienced an increase in depression severity during the “new normality”, while those with high pre-lockdown depression decreased compared with the pre-lockdown. These findings suggest that COVID-19 restrictions affected the depression level depending on their pre-lockdown depression severity. Individuals with low levels of depression are more reactive to external stimuli than those with more severe depression, so the lockdown may have worse detrimental effects on them. In January 2020, the World Health Organization declared a novel severe acute respiratory coronavirus disease caused by SARS-CoV-2, the main etiological factor of the disease called COVID-19, Coronavirus Disease 2019 . The SpaThe benefits in the control of the infection may have been at the expense of significant psychological impact . In partHowever, many of the reported studies lack data referring to the pre-pandemic levels of symptoms ,23,24,25To adequately understand the impact of the pandemic on depression severity, we need longitudinal studies able to include people with a recent history of MDD. Two previous publications ,42, usinhttps://www.radar-cns.org/ accessed on 8 February 2023), which developed the open-source mHealth platform RADAR-Base [The present study focuses on one site/country of the RADAR-MDD project to make more detailed analyses on how specific containment measures affected the mental health of individuals with a history of MDD. We aim to explore the impact of the COVID-19 control restrictions on mental health in individuals with a history of MDD in Catalonia (the Spanish sample of the RADAR-MDD study was recruited in this Autonomous Community), adopting a new ‘pandemic-centric’ perspective. RADAR-MDD is part of the RADAR-CNS  consortium (DAR-Base  to collehttps://www.radar-cns.org/ accessed on 8 February 2023) including people with a history of MDD who were evaluated from November 2017 to March 2021. All participants fulfilled the following inclusion and none of the exclusion criteria. They had at least one MDD episode within the last 2 years and at least 2 lifetime episodes of MDD and were over age 18 only. Furthermore, they were able to complete self-reported assessment via smartphone. Exclusion criteria: dementia, pregnancy, major medical disease, an alcohol or drug dependency within the past 6 months, or lifetime history of other mental disorders .The present study is based on data gathered in the RADAR-MDD project, refs ,48 a mulThe study was co-developed with service users in our Patient Advisory Board (PAB), who were involved in the choice of measures, the timing, and issues of engagement and have also been involved in developing the analysis plan, and a representative is an author of this paper and critically reviewed it.In this manuscript, we focused our analyses on the 155 participants with a history of MDD according to the DSM-5 criteria evaluated by clinicians from Catalonia and analyzed the data during the first wave of COVID-19, between 1 November 2019, and 16 October 2020. Of those 155 participants, 22 were not recorded during the periods of interest (from 1 November 2019 to 16 October 2020), and 12 missed records during the pre-lockdown period (2 participants died of COVID-19 infection).Thus, the final sample included 121 participants.p > 0.05) as compared to those without any missing data.Additionally, some participants had missing values that referred to certain phases see the . No diff▪Pre-lockdown (from 1 November 2019 to 10 March 2020);▪Lockdown (from 11 March 2020 to 26 April 2020);▪Later restrictions were lifted gradually through four phases of the post-lockdown:▪Phase 0 (from 27 April 2020 to 10 or 17 or 24 May 2020 *);▪Phase 1 (from 11 or 18 or 25 May 2020 to 24 May or 7 June 2020 *);▪Phases 2 and 3 (from 25 May or 8 June 2020 to 18 June 2020 *);▪“New-normality” (from 19 June 2020 to 16 October 2020).The phases and restrictions had a longer duration or were applied later depending on the location . The dates of the restrictions are shown in * Depending on the locationsDepression severity was assessed through the PHQ-8  . ParticiSeverity of anxiety was assessed using the Generalized Anxiety Disorder-7 item (GAD-7) scale , which wGender, comorbidity with medical conditions, age, marital status, number of people living with, employment, income, and age of finishing education were also collected at baseline.First, we described the sociodemographic characteristics of the sample and computed a descriptive analysis of the PHQ-8 values among the different lockdown phases. We then calculated each participant’s baseline depression and anxiety severity by taking the average of the PHQ-8 and GAD measures in the pre-lockdown period. Participants were grouped based on their pre-lockdown depression (PHQ-8 ≥ 10 or <10) and their pre-lockdown anxiety levels (GAD-7 ≥ 10 or <10). We analyzed the variations of PHQ-8 in the different phases of the pandemic using the paired Wilcoxon text. In those participants with more than one PHQ-8 evaluation in the same phase, we computed the mean values and used them for the following analyses. We also applied a linear mixed model to analyze the course of depression levels over phases. To identify the covariate variables (sociodemographic variables) that would be included in the linear mixed model, we computed the forward stepwise method implemented with R based on the best Akaike information criterion (AIC) value. We also evaluated the effect size on pre-lockdown depression levels of each variable and added to the model those not selected by the stepwise procedure but with at least a medium effect . To investigate how the pre-lockdown depression levels associated with variations in depression severity across phases, the model was stratified by the pre-lockdown depression levels (PHQ-8 cutoff ≥ 10). Random effects of participants were incorporated as well, and the model was adjusted using the maximum likelihood (ML) method, which provided correct estimation when data were not completely missing at random . All staMost of the participants were females (66.9%); the median age was 58 years (IQR 52–64) . More thp ≤ 0.05) and new-normality (p ≤ 0.01) phases, while a significant decrease was found in new-normality as compared with lockdown (p ≤ 0.05).Using the paired Wilcoxon test, a statistically significant increase in the depression levels (PHQ-8 score) was observed in phase 0 compared to the pre-lockdown (p ≤ 0.001) and phase 0 (p ≤ 0.001), as compared with the pre-lockdown phase. Depression severity was higher in patients with high pre-lockdown anxiety (GAD-7 ≥ 10)  than in those with low pre-lockdown anxiety symptoms (GAD-7 < 10) over all phases. p ≤ 0.022), three , or four or more  people was found to decrease the depression severity. No significant effects were found for what concerns the other covariates.A linear mixed model  was fittWe then stratified participants into two groups: one contained subjects with high pre-lockdown depression (82 participants and 1048 observations); the other included subjects with low pre-lockdown depression (39 participants and 547 observations). In this way, we aimed to verify whether the association between the phases and depression severity depended on the pre-lockdown depression severity see .p ≤ 0.001) and phase 0 , as compared to the pre-lockdown levels. Moreover, we found a significant decrease in depression levels during the post-lockdown .According to the first model , particip = 0.015) and phase 0 , as compared with the pre-lockdown, consistent with the previous model. Instead, we found a significant increase in depression levels during the post-lockdown . The coefficients of the stratified model adjusted for the covariates are represented in As regards the second model , particiWe observed that the participants with a history of MDD were mainly affected during the lockdown and the early phase of post-lockdown, when their depression severity increased as compared to pre-lockdown levels. These findings should not be surprising, since the most stringent measures were imposed during these phases.In previous literature, some works ,24 showeFurthermore, in these two studies ,22 patieMoreover, other studies  were “loSecond, the model suggests that in the “new-normality” period those subjects with high pre-lockdown depression experienced a decrease in depression severity.These results come out as partly divergent from those found in previous studies, but these discrepancies might again rely on the above-discussed differences in the division into phases of our common period of interest. Indeed, Leightley et al. 2021) included in their post-lockdown phase also the period that in the current study was studied as the phase 1 of the post-lockdown, when some social restrictions were still in place. Thus, as we based our analyses on different divisions of time, slight differences in findings are understandable. However, many studies 21 includ,40,53,54These results provide unprecedented points of reflection, soliciting the proposal of possible explanations. There is no doubt that the COVID-19 pandemic produced a contraction of the habitual interactions of the individual with the stimuli produced by the external world. Thus, we might suppose that the psychological impact of such everyday life upheaval must have been more critical on those patients whose pre-lockdown depressive and anxiety symptoms were not severe to such an extent that their psychological wellbeing could be influenced by relevant changes in the external world. In other words, high levels of depression before the pandemic might have made individuals less responsive to the stress  and prevHowever, given the scarcity of past references on the theme, these hypotheses need to be further proved by future analyses on the possible role of pre-pandemic levels of depression and anxiety as predictors for the impact of severe social restrictions on the course of depression severity in patients affected by MDD.First, the study of the effects of restrictions on depression severity in a clinical population of people suffering from MDD must be regarded as providing a relevant clinical implication. The results of the current work about the specific psychological impact of the different measures imposed a long time and could inform authorities in planning a future policy of restrictions in the case of new pandemics, taking into account more accurately and from several points of view the benefits and disadvantages of applying social restrictions.For example, analyses on our sample suggest that during the lockdown and the early phase of the post-lockdown, when only some outdoor sport activities were allowed, the levels of depression severity remained high, whereas a drop was registered during phase 1, when some restrictions of social activities were slightly eased. Thus, it is hoped that, in case of future pandemics, this specific vulnerability to restrictions that we described in depressed patients will induce authorities to, for example, exempt them from the strictest forms of lockdown, compatibly with the epidemiological context.In future research, we want to extend our study of the variations of depression levels across phases of pandemics to different waves of the COVID-19 pandemic and to different countries. To analyze this, it will be mandatory to take again into account the specific restrictions imposed across time and different areas.Some limitations must be considered. The assessment of severity was self-reported and may have lower reliability than heteroadministered  questionnaires or tests. Furthermore, some subjects missed data referring to certain phases.Strengths of the study include a homogeneous sample in terms of geographical origin, and we were able to analyze lockdown measures in detail; however, this limits the generalizability of the findings. Since we considered the differences between different areas within Catalonia, each group of patients corresponding to a specific phase contained only participants who were imposed under the same restrictions at the same time. Moreover, thanks to the length of our period of assessment, for each participant several observations have been available, referring to the pre-lockdown, lockdown, and post-lockdown phases. Thus, the present research went beyond the “lockdown-centric” perspective of the previous literature and has to be considered the first one assuming a “pandemic-centric” point of view, which not only considered the acute impact of the most severe restrictions but also aimed at detecting the subtle variations in depression during the post-lockdown phases.Furthermore, due to the employment of RMT and the prospectively planned, longitudinal design of our study, recall bias was completely prevented. Also, our database allowed us to distinctly study the role of pre-lockdown levels of depression and anxiety as predictors for variations in depression severity during lockdown and post-lockdown.Future studies will have the possibility to refer to the present work as a significant milestone of this new “pandemic-centric” perspective, based on which not only an impairment in depression severity during the first phases of pandemic was found, but also a more severe impact of the pandemic was described on those patients suffering from milder forms of MDD. Thus, our results emphasize the strong need for attentive care that these specific populations run under social restrictions, hopefully contributing to the process of turning the spotlight on the psychological impact of the pandemics.


Gene therapy has its intellectual origins in the context of treating Mendelian disorders,,Regenerative medicine is an attractive application of gene therapy because most of the morphogens and growth factors of interest for this purpose are difficult to deliver to sites of tissue damage and have very short biological half-lives. Gene transfer offers the prospect of providing these proteins by local synthesis in a sustained fashion at concentrations sufficient to drive a robust regenerative process. Problems in bone healing provided the earliest application of this concept,Although bone is one of the few organs in the human body that can regenerate scarlessly, in about 5%–10% of cases, fractures fail to heal, leading to non-unions. These are recalcitrant clinical problems. The 1965 publication by Marshall UristRecombinant, human BMP-2 (rhBMP-2) is available for clinical use in the United States and Europe as part of a product known as Infuse™ Bone Graft, whose approved indications are high-energy open tibial fractures, spinal fusions, and certain oromaxillofacial applications. Additionally, it has wide off-label useDespite much promise, the clinical efficacy of rhBMP-2 has been disappointing. In attempts to improve outcomes, very high amounts of the protein are used, but these provoke numerous adverse side effects, some of them severe.in vivo for a short period of time.Molecular Therapy – Methods and Clinical Development, Chris Evans and his team investigated why this was so.Genetic delivery of BMP-2 indeed proved superior to protein delivery of rhBMP-2 in pre-clinical models of bone healing, but in an unexpected way—efficient healing via gene therapy occurred when only small amounts of BMP-2 were expressed in vitro study focused on BMP-2 signaling via SMAD phosphorylation using reporter murine mesenchymal cells where firefly luciferase expression is driven by elements of the Id1 promotor, an early response gene activated by BMP-2.This Co-culture experiments demonstrated that cells expressing BMP-2 were able to induce luciferase expression in adjacent naive cells. When BMP-2 cells were co-cultured with naive cells but separated by a permeable insert, there was no activation of the naive cells, suggesting that close proximity and possibly cell-to-cell contact was necessary for this to occur. To confirm that these data were not a result of using an established cell line, osteogenesis of primary cultures of human mesenchymal stromal cells was demonstrated.In summary, the authors explain the superior activity of transgenic BMP-2 by its ability to elicit powerful autocrine and paracrine effects in a protected pericellular environment. Although the study focused on SMAD signaling via the BMP type 1A receptor, the authors left open the possibility of additional pathways, including intracrine signaling based on the observation that a substantial portion of transgenic BMP-2 remained cell associated.These data provide mechanistic support for the use of gene therapy-based approaches to regenerating bone and provide, for the first time, a plausible explanation for the observation that gene transfer is superior to a bolus of protein in healing bone. The efficiency of tiny amounts of transiently expressed, endogenously synthesized BMP-2 in healing bone suggests novel opportunities beyond traditional gene therapy. Recently, messenger RNA (mRNA) has shown remarkable success in pre-clinical models of bone healing.


Incidence, health consequences, and social burden associated with child maltreatment appeared to be borne disproportionately by very young children. We conducted a population-based data linkage study to explore child- and family-level factors that affect receiving different diagnoses of maltreatment injuries and investigate excessive mortality throughout toddlerhood.We conducted a retrospective cohort study comprising 2.2 million infants born in 2004–2014 in Taiwan. Incident cases of child maltreatment were defined by hospitalization or emergency department visits for three heterogeneous diagnostic groups of maltreatment-related injuries  within 12 months after birth. The generalized linear model and landmark survival analyses were used to evaluate risk factors.An estimated 2.9‰ of infants experienced at least one maltreatment-related injury, with a three-year mortality rate of 1.3%. Low birthweight was associated with increased risk of receiving the diagnosis of three maltreatment injuries, particularly maltreatment syndrome . Socially advantaged family condition was inversely linked with receiving the diagnosis of maltreatment syndrome and assaults , yet positively linked with undetermined cause . For infants exposed to maltreatment, low birth weight and non-attendance of postnatal care were highly predictive of fatality; low birthweight served as a vital predictor for premature death during toddlerhood .Raising awareness of maltreatment-related injuries in infancy and predictors should be a priority for appropriate follow-up assessment and timely intervention.The online version contains supplementary material available at 10.1186/s12889-023-17180-8. Child maltreatment has emerged as a challenging issue for pediatric care providers and child protection practitioners worldwide. A recent systematic review of multinational surveys from 96 countries indicated that half of children aged 2–17 years experienced some form of violence each year globally, with an estimated 68% in Asia –6. To prCumulative evidence indicated that incidence, health consequences, and social burden associated with child maltreatment were disproportionately borne by very young children , 7. The Clinically, identifying child maltreatment is challenging because the diagnosis relies on an array of clinical features, and evaluation often requires collaborative efforts , 13. In In the present study, we focused on three distinct injuries of child maltreatment, with the overarching aim to examine the extent to which family- and child-level characteristics may differentially affect the diagnosis associated with child maltreatment taking place before the first birthday and to evaluate the factors accounting for excess death in maltreated infants throughout toddlerhood.The present study used data from the 2004 to 2015 National Health Insurance (NHI) program, birth registration (BR), household registration (HR), and 2004–2018 death registration (DR) see . The NHIIn this retrospective cohort study, we initially limited our study subjects to those born between January 1, 2004, and December 31, 2014  in Taiwan. To avoid duplication resulting from the episode-based inter-hospital referral for the same case , we focused on the medical records for only the first maltreatment episode .The present study has ascertained infant maltreatment via maltreatment-related injury and marker condition , 26. ForFor infants with any positive record of child maltreatment injury (n = 6297), the occurrence of death in the following three months and three years after the index hospital visit for infant maltreatment was confirmed by the national death registration. For comparative purposes, deaths among non-maltreated infants who survived beyond the age of six months  were obtained. Infant abuse-related severe injuries are recognized as “catastrophic illnesses,” for which co-payment will be exempted for treatment and prolonged hospitalization.For each child, information concerning infant-level characteristics  was retrieved from the household registration records. Birthweight was categorized into three subgroups . Family characteristics included family structure  and socioeconomic condition . Given that (i) the mother is often the primary caregiver in the study population and (ii) children are more likely to be linked with their mothers , we systematically retrieved socioeconomic characteristics from maternal records. Since the national health insurance premium is calculated mainly by the monthly income of insurance enrollees  and enrollee category , we used premium as a proxy for family income, subgrouping children into high-income, middle-income, and low-income/poverty . MaternaCross-tabulation was first conducted to analyze the distribution of child- and family-level characteristics, together with infant maltreatment and death. Given that the occurrence of diagnosed infant maltreatment injury was rare, the present study turned to the generalized linear models in zero-inflated Poisson distribution with robust variance estimation to evaluate the association estimates for possible predictors. The incidence in person-months was calculated for infants using the number of births for each study month as the denominator. The follow-up period ended either on the first birthday, date of death, or treatment seeking for probable infant maltreatment, whichever came first. The incidence rate ratio (IRR) and adjusted incidence rate ratio (aIRR) for the occurrence of the first diagnosis were the ratio of incident infant maltreatment divided by the total person-month at-risk. The 95% confidence intervals (CI) for the association estimates were also provided to facilitate interpretation.Regarding the investigation of the prognostic factors for fatal maltreatment and long-term mortality, considering that the hazard function fluctuated over the course of a three-year follow-up and the survival probability was primarily conditioned on the length of index treatment see , we deciNearly 7% of the infants born between 2004 and 2014 weighed less than 2500 g at birth  were not only associated with an increased risk of maltreatment-related injury in infants , 29–32 bDisadvantaged socioeconomic status has been long indicative of increased maltreatment-related hospitalization or death in infants , 37–39. In Taiwan, the well-childcare has been fully covered by governmental funds, providing a comprehensive assessment of a child’s growth, developmental check-up, and vaccination. Extending prior studies that indicated under-immunization was a risk factor for child maltreatment in young children , our anaSome potential limitations of this study should be considered. First, since the database is designed for reimbursement purposes, the validity and completeness of coding for child maltreatment (ICD 9 CM-code:995·5) in the NHI claims data may not be satisfactory , 46. FurNotwithstanding these limitations and methodological issues, our findings are an important addition to the literature because only a few population-based studies have documented the incidence of child maltreatment in the first year of life, especially in regions where the economic burden of child maltreatment is substantial . Second,Our observed findings with respect to the predictors for maltreatment injury and subsequent excess death may be worthy of future attention in relation to preventive implications in infant maltreatment in the context of healthcare system , 49. EviBelow is the link to the electronic supplementary material.Supplementary Material 1


Bilayer graphene is a contender of interest for functional electronic applications because of its variable band gap due to interlayer interactions. Graphene growth on Cu is self-limiting, thus despite the fact that chemical vapor deposition (CVD) has made substantial strides in the production of monolayer and single-crystal graphene on Cu substrates, the direct synthesizing of high-quality, large-area bilayer graphene remains an enormous challenge. In order to tackle this issue, we present a simple technique using typical CVD graphene growth followed by a repetitive wrinkling-etching-regrowth procedure. The key element of our approach is the rapid cooling process that causes high-density wrinkles to form in the monolayer area rather than the bilayer area. Next, wrinkled sites are selectively etched with hydrogen, exposing a significant portion of the active Cu surface, and leaving the remaining bilayer areas, which enhance the nucleation and growth of the second graphene layer. A fully covered graphene with 78 ± 2.8% bilayer coverage and a bilayer transmittance of 95.6% at room temperature can be achieved by modifying the process settings. Bilayer graphene samples are examined using optical microscopy (OM), scanning electron microscopy (SEM), Raman spectroscopy, and an atomic force microscope (AFM) during this process. The outcomes of our research are beneficial in clarifying the growth processes and future commercial applications of bilayer graphene. Monolayer graphene is a desirable material for future electrical and photonic devices due to its extremely high carrier mobility and distinct one-atom-thick layered structure . However2O-assisted CVD technique. In order to facilitate the nucleation and growth of the bilayer beneath, the H2O strategically etches the edge of the top graphene layer, slowing the growth of the top layer ..2/Si subWe devised a G-rW-rE-rG method to produce large-area bilayer graphene films, which exhibit bilayer coverage of 78 ± 2.8% and a transmittance of 95.6% at room temperature. The formation of large-area bilayer graphene can be attributed to edge attachment and diffusion-based growth mechanisms. Following wrinkling and selective hydrogen etching, the coverage of the top monolayer graphene decreases while a portion of the bilayer area remains, and this facilitates the nucleation and growth of the bilayers during the regrowth process. By combining the G-rW-rE-rG strategy with other methods to induce the formation of bilayer graphene with a specific stacking order in the initial growth step, it is anticipated that substantially larger or even wafer-sized homogeneous-stacked bilayer graphene may be produced. We are certain that our work will help in understanding the growth mechanisms of CVD-derived bilayer and multilayer graphene on Cu, as well as clear the way for the mass production of bilayer graphene for industrial applications.


The COVID-19 pandemic has presented a unique opportunity to understand how real-time pathogen genomics can be used for large-scale outbreak investigations. On 12 August 2021, the Australian Capital Territory (ACT) detected an incursion of the SARS-CoV-2 Delta (B.1.617.2) variant. Prior to this date, SARS-CoV-2 had been eliminated locally since 7 July 2020. Several public health interventions were rapidly implemented in response to the incursion, including a territory-wide lockdown and comprehensive contact tracing. The ACT has not previously used pathogen genomics at a population level in an outbreak response; therefore, this incursion also presented an opportunity to investigate the utility of genomic sequencing to support contact tracing efforts in the ACT. Sequencing of >75% of the 1793 laboratory-confirmed cases during the 3 months following the initial notification identified at least 13 independent incursions with onwards spread in the community. Stratification of cases by genomic cluster revealed that distinct cohorts were affected by the different incursions. Two incursions resulted in most of the community transmission during the study period, with persistent transmission in vulnerable sections of the community. Ultimately, both major incursions were successfully mitigated through public health interventions, including COVID-19 vaccines. The high rates of SARS-CoV-2 sequencing in the ACT and the relatively small population size facilitated detailed investigations of the patterns of virus transmission, revealing insights beyond those gathered from traditional contact tracing alone. Genomic sequencing was critical to disentangling complex transmission chains to target interventions appropriately. After the emergence of SARS-CoV-2 in late 2019 in Asia, the Australian Government declared a human biosecurity emergency on 18 March 2020 and closed its international borders to non-permanent residents and non-citizens on 20 March 2020 . During 2) enclave within New South Wales (NSW) in south-eastern Australia with a population of approximately 453 558 people. During the first half of 2021, there were no local COVID-19 restrictions  and an elimination strategy  was in place pending the rollout of vaccines to the eligible population. Pathogen genomics was of interest, but had not previously been used for large-scale outbreak investigation in the ACT and it was not known how this may complement traditional epidemiological contact tracing.The ACT is a small  or Lunascript (New England Biolabs) reverse transcriptase for cDNA synthesis . For PCRSequencing turnaround time was estimated by calculating the time from sample collection to preliminary reporting of the sequencing results to ACT Health. Since no time stamp was available for sample collection, we used 12:00.https://www.geneious.com/). The alignment was curated manually to check for gaps and misalignments. Sequences were assigned to ACT genomic lineages and sublineages based on neighbour-joining phylogenies (≤10% ambiguous bases only) constructed using the Tamura-Nei model as implemented in the Geneious Tree Builder in Geneious Prime 2021.2.2 and/or on manual exploration of the alignments at lineage-defining sites. Lineage assignment was revised daily during the COVID-19 response, based on the incorporation of new sequences and in conjunction with epidemiological information. Comparisons to publicly available Australian and international sequences were performed with the UShER webserver .Fi.Fi27].Incorporating this genomic contact tracing, we were able to identify transmission at certain exposure sites and implement enhanced infection control measures in these settings. For example, genomic information showed that a case cluster at a social housing complex was the result of several incursions over a 3-week period, rather than a single superspreading event which was assumed based only on case interviews and contact tracing information . While iA key feature of the Delta (B.1.617.2) SARS-CoV-2 outbreak in the ACT was the very high proportion of cases for which sequencing was attempted  with exceptional short turnaround time from sample collection, leading to a detailed and timely understanding of local transmission networks and number of distinct incursions. The ACT is one of the few jurisdictions globally that was able to maintain such high rates of SARS-CoV-2 sequencing for an extended period –30. The Through our genomic analyses, we were able to determine that the ACT Delta ‘wave’ was driven by two separate dominant incursions, ACT.19 and ACT.20. This insight allowed us to explore the demographic factors contributing to the ‘success’ of these incursions compared to incursions that did not transmit extensively. Both dominant incursions particularly impacted vulnerable groups within the community, where crowded living arrangements and lower health literacy were contributing factors; however, the specific demographic characteristics of these vulnerable populations were distinct. The two major incursions varied considerably in the demographic groups affected, and subsequently in duration and total number of cases. Several studies have already demonstrated a disproportionate risk of SARS-CoV-2 infection in socioeconomically disadvantaged groups and in ethnic minorities –35, and Interestingly, the first cases of ACT.20 were detected on 15 August 2021, 3 days after the detection of ACT.19. Yet cases only began to increase exponentially in early September and there was very limited genetic diversity in the month following detection, with the first mutation detected in samples collected on 11 September 2021. It is possible that there was undetected transmission of ACT.20 after the first detection, although testing rates were high, there was exhaustive contact tracing of cases, and strict lockdowns were in place. Alternatively, there may have been a separate introduction of a genetically identical virus. Our findings constrain the lower bound of the number of possible incursions. The genetic diversity of SARS-CoV-2 circulating in Australia during mid-2021 was limited, since all local cases arose from a single point source outbreak in Sydney, NSW, on 15 June 2021 . Indeed,Notably, the two dominant incursions during the resolution of ACT.20 (i.e. ACT.37 and ACT.38) disproportionately affected incompletely vaccinated younger persons, with transmission occurring at (i) a large private party, and (ii) within a school setting, demonstrating the marked impacts of even superspreading events. Of relevance, schools reopened between 5 October and 1 November and vaccines were not available for those aged 16–18 years until 1 September, and for those aged 12–16 years until 13 September. The cohorting strategy applied in the school setting as students and teachers returned to on-campus learning, in addition to other COVID-safe measures such as masking, was very effective in limiting the extent of community transmission associated with this incursion.While we observed complete replacement of ACT.19 by ACT.20, and near-complete replacement of ACT.20 by ACT.37 and ACT.38, these replacements were not a consequence of enhanced epidemiological fitness of any of these viruses. Indeed, the ACT.20 founder sequence had only five non-synonymous changes relative to the ACT.19 founder sequence across all coding sequences, only one of which was in the spike protein, while ACT.37 and ACT.38 each had two non-synonymous changes relative to ACT.20, none of which were in the spike protein. The spread of SARS-CoV-2 Delta in the ACT is a clear example of repeated founder effects, because of the extensive mitigation measures and the stochastic nature of transmission of SARS-CoV-2 (overdispersion) . This coBy 11 November (the end of the study period) all incursions were considered to be sufficiently controlled and population vaccination coverage was high, leading to the lifting of most restrictions. Notably, during the study period, there were 11 COVID-19-related deaths. The extensive contact tracing and case follow-up employed in the ACT likely facilitated the early identification of those cases eligible for enhanced treatment, such as monoclonal antibody therapies, which became available in Australia from late August. The high vaccination rates achieved in the ACT and good compliance with public health social measures during the outbreak period likely contributed to the low observed mortality .Our study revealed that pathogen genomics could be rapidly implemented during a large-scale infectious disease outbreak and highlighted the utility of intensive genomic sequencing to support traditional contact tracing and other public health interventions in real time. We determined that the ACT Delta outbreak was driven by several independent incursions, with successive waves impacting different vulnerable groups. However, by deploying enhanced interventions  into these at-risk communities, timely public health measures were successful in mitigating these incursions and most importantly, in preventing severe clinical outcomes. The combination of stringent and timely public health interventions, exhaustive contact tracing  and high levels of viral sequencing close to real time effectively limited the two major Delta incursions, ACT.19 and ACT.20, within a 3-month period.