Schistosoma, is a neglected disease with high global prevalence, affecting more than 240 million people in several countries. Praziquantel (PZQ) is the only drug currently available for the treatment. S. mansoni NTPDases  are potential drug targets for the discovery of new antischistosomal drugs. In this study, we screen NTPDases inhibitors from Centella erecta (Apiaceae) using an ultrafiltration combined UHPLC-QTOF-MS method and potato apyrase, identifying asiaticoside as one of the apyrase-binding compounds. After isolation of asiaticoside from C. erecta extract, we assessed its in vivo antischistosomal activities against Schistosoma mansoni worms and its in vitro enzymatic apyrase inhibition. Also, molecular docking analysis of asiaticoside against potato apyrase, S. mansoni NTPDases 1 and 2 were performed. Asiaticoside showed a significant in vitro apyrase inhibition and molecular docking studies corroborate with its possible actions in potato apyrase and S. mansoni NTPDases. In mice harboring a patent S. mansoni infection, a single oral dose of asiaticoside (400 mg/kg. p.o.) showed significantly in vivo antischistosomal efficacy, markedly decreasing the total worm load and egg burden, giving support for further exploration of apyrase inhibitors as antischistosomal agents.Schistosomiasis, caused by parasites of the genus  Schistosoma, is a neglected tropical disease with an estimated global prevalence of 240 million infected people in 78 countries and significant mortality and social and economic consequences − adduct ion in the negative mode with m/z 993.4827. The other fragment ions were at m/z 487.3400 [M-H-2Glc-Rha]− and at m/z 469.1537 [M-H-2Glc-Rha-H2O]−, which are in accordance with MS/MS studies and fragmentation pattern previously reports to asiaticoside − found; 993.4831 [M + Cl]− calculated. Purity of the isolated asiaticoside was estimated to be higher than 95% by 1H NMR data analysis.As asiaticoside  was identerature : 1H NMR 50 value, asiaticoside inhibited potato apyrase activity by approximately 43% at 100 µM, while at 50 µM potato apyrase activity was inhibited by about 33%  against potato apyrase was investigated. Asiaticoside was able to decrease the enzymatic activity of potato apyrase . Althougbout 33% .To investigate possible modes of asiaticoside interaction with target enzymes at the active sites, we performed molecular docking simulations. As shown in p < 0.001) in comparison with control group (p < 0.001) (p < 0.001) after treatment with a single oral dose of asiaticoside (400 mg/kg) in comparison with control group (p < 0.001) and 93.6% (p < 0.001) after the oral treatment with asiaticoside and PZQ, respectively (p < 0.001) and 94.4% (p < 0.001) after the oral treatment with asiaticoside and PZQ, respectively  showed a significant total worm burden reduction of 65.4% (ol group . PZQ (40< 0.001) . In addiol group . In feceectively A. Additiectively B.S. mansoni [S. mansoni, also being effective in inhibiting both potato apyrase and SmNTPDases [C. erecta showed as a potential source of apyrase inhibitors (data not shown). Based on these findings and corroborating with the high homology degree between SmNTPDases and potato apyrase [C. erecta extract.The discovery of new antischistosomal compounds is crucial in terms of public health, since schistosomiasis remains as the second most important human parasitic disease ,4,41. Du mansoni . AdditioNTPDases . In addi apyrase , we usedC. erecta extract were carried out for identification of apyrase ligands. The particular ligands from C. erecta extract may bound to the apyrase sites, forming enzyme-ligand complexes during the incubation process, which, due to their macromolecular size, would remain in the ultrafiltration membrane when centrifuged, while the unbound small compounds could pass through the membrane [C. erecta were subjected to UHPLC-ESI-QTOF-MS identification. According to UHPLC-ESI-QTOF-MS analysis, the ultrafiltration process produced an ultrafiltrate enriched mainly with three compounds (peaks 1–3) with binding affinity for apyrase, although a little difference between the LCMS chromatograms of the crude C. erecta extract and the ultrafiltrate was observed. These main compounds in ultrafiltrates were analyzed by their mass spectrometry data, accurate molecular weight, and fragment ions and annotated as asiaticoside (peak 1), madecassic acid (peak 2), and asiatic acid (peak 3). Although other possible ligands might be presented in the C. erecta extract (such as in Rt= 9.61 min), only three target compounds were annotated as potential apyrase inhibitors. Among them, asiaticoside was the only successfully isolated from C. erecta extract by chromatographic fractionation and further chemically identified by 13C- and 1H- NMR data analysis. Centella species consist mainly of triterpenoid saponins, in which asiaticoside is one of most studied compounds [Typically, an affinity ultrafiltration LC-MS screening involves three steps: incubation, screening by ultrafiltration and characterization by MS . It is kmembrane . After, ompounds .However, binding of a ligand target compound to the enzyme does not mean that it is an enzyme inhibitor, due to the possibility of non-specific binding of the compound to non-functional sites on the enzyme . Then, iAdditionally, to investigate how asiaticoside might bind to the active sites of NTPDases enzymes, asiaticoside was docked to active pockets of potato apyrase and SmNTPDases 1 and 2. Our results indicate that asiaticoside is able to interact with important catalytic residues of apyrase and SmNTPDases, providing a possible inhibition of these target proteins. In addition, we also observed that asiaticoside preferably interacted in the positive electrostatic region of the catalytic site from target proteins, similar that results obtained to cardamonin .S. mansoni mouse model. The in vivo treatment with asiaticoside was performed in mice harboring patent S. mansoni infection. To our knowledge, asiaticoside has not been yet evaluated against Schistosoma sp. After oral treatment at a single dose (400 mg/kg) of asiaticoside, worm burden was significantly reduced in comparison with the control group. Additionally, when compared to the reference drug PZQ (400 mg/kg), which reduced the parasitic burden in ~90% [Centella extracts (with high concentration of asiaticoside) did not show any adverse effects by oral administration in mice at least up to 1 g/kg [C. asiatica extract (containing ~46% of asiaticoside) exhibited LD50 > 2 g/kg by oral administration in rats [Then, considering the importance of apyrase inhibitors as potential antischistosomal compounds and keeping in view the above-mentioned experimental approach, we evaluate the in vivo activity of asiaticoside in the  in ~90% , asiatico 1 g/kg ,45. Furt in rats .Actinopyga echinites and Holothuria polii have interesting in vitro schistosomicidal activity [Vernonia amygdalina (Asteraceae) were in vitro and in vivo evaluated against S. japonicum [Pulsatilla chinensis (Bunge) Regel, exhibits antischistosomal properties in vivo against S. japonicum and S. mansoni [Moreover, there are few reports in the literature regarding the schistosomicidal activity of saponins. Previous studies showed that saponins from the marine organisms activity . Also, iaponicum , showing mansoni .S. mansoni to the organism are due to eggs in the host’s tissues, triggering inflammatory reactions and granuloma formation [S. mansoni. After the treatment with asiaticoside the number of eggs per gram (OPG) was also markedly reduced in feces examined by the Kato–Katz method. Moreover, the oogram pattern showed that the oral administration of asiaticoside produced significant reductions in immature and mature eggs. Taken together, our results on egg burden agree with the noticeable ability of asiaticoside in reducing adult worms of Schistosoma.In addition, it is known that the main lesions caused by ormation ,52. Thenormation , the theSchistosoma tegument is the interface between the environment and the parasite, achieving vital functions for the worms, such as nutrient absorption, excretion, protection against the host’s immune system [Schistosoma tegument are some essential enzymes, such as the SmNTPDases, which are important for the survival of Schistosoma worms inside the host [The e system . In Schithe host ,31,53. Tthe host , other mthe host  should bC. erecta extract. Madecassic and asiatic acids were also annotated as apyrase-binding compounds by this method. Asiaticoside was isolated from C. erecta extract and further in vitro assays confirmed its potato apyrase inhibitory activity. Additionally, molecular modeling and docking assays were used to evaluate the interaction of asiaticoside with the active sites of SmNTPDase 1, SmNTPDase 2, and potato apyrase. In addition, our findings demonstrated, for the first time, that asiaticoside has markedly in vivo antischistosomal efficacy in mice harboring a patent S. mansoni infection, markedly decreasing the total worm load and egg burden. However, more clinical studies are needed to validate the use of asiaticoside as a therapeutic schistosomicidal agent. Additionally, future studies are also in progress to evaluate the antischistosomal properties of madecassic and asiatic acids.In this study, an ultrafiltration combined with UHPLC-QTOF-MS approach was used to identify the asiaticoside as a potential apyrase inhibitor from


Splenic cysts are classified on the basis of epithelial lining, either primary or secondary. Primary cysts are further divided as parasitic and nonparasitic. The secondary cysts are usually post traumatic or after a splenic extension of pancreatic pseudocyst. However, not all pseudocysts are associated with trauma. Mostly, they are asymptomatic (30%–60%) and usually grow in size to cause compressive symptoms. Splenic pseudocysts should be differentiated with other malignant and nonmalignant pathology, specifically hydatid cysts, in order to manage them correctly. The walls of pseudocysts may be degenerative or calcified, which may resemble hydatid cysts. Here, we present a case of a non‐traumatic splenic cyst masquerading as a hydatid cyst preoperatively. The patient was taken up for surgery and intraoperatively noted to be a hemorrhagic cyst with a non‐splenic cyst wall. We decided to preserve the spleen with marsupialisation of cyst and omentoplasty. On histopathology, the diagnosis of a pseudocyst of spleen was made in view of absent epithelial lining. We would like to report this case because of the diagnostic dilemma, its clinical rarity and, even more, in the absence of any history of trauma. Splenic pseudocyst Only 800 cases are reported in the world literature, and one of the rarest forms is the pseudocyst of spleen.2A 34‐year‐old lady under evaluation for recurrent vaginal discharge was referred by the department of Gynecology for a splenic cyst, likely a hydatid cyst detected on ultrasonography, as an incidental and sole finding. The patient also complains of left upper abdominal pain, early satiety, and heaviness in the epigastric region. There was no history of fever, cough, jaundice, and symptoms of upper GI bleeding. There was no history of any abdominal trauma, contact sports previously, or previous surgery. She was a non‐smoker and non‐alcoholic and was under the empirical treatment for vaginal discharge. On examination, she had no pallor and was non‐icteric. The abdomen was soft and non‐tender with no palpable abdominal mass. Contrast enhanced computed tomography of the abdomen and pelvis showed a well‐defined, round large cystic lesion measuring 7.8 × 8.8 × 9.6 cm (AP × TR × CC) seen in the hilar region of the spleen extending into the upper pole. The cystic lesion showed multiple thin walled septations and calcifications within. There were subtle hyperdense contents in the dependent part of the individual loculi within the cyst. Medially, the lesion abutted the pancreatic tail and cardia of the stomach with a maintained fat planes in between. The lesion appeared to compress the stomach cardia supero‐medially and compress the splenic vein inferiorly. There was no wall calcification, no surrounding collection, or fat stranding. Following the contrast administration, the cystic lesion, including the hyperdense content, thin septa, and cyst wall, did not show any significant enhancement Figures , 2, 3. THematological and biochemical parameters were under normal limits. Echinococcus IgG ELISA was negative. Preoperative preparation included Nil per oral protocol for 8 h, preoperative prophylactic iv. antibiotics one hour prior to incision and underwent an elective exploratory laparotomy under general anesthesia. Intraoperative findings noted was a large 10 × 8 cm splenic cyst with soft to firm walls, originating from the upper pole of the spleen. The outer wall of the cyst was separate from the splenic tissue, and the splenic vein did not pass through the cystic wall. The cyst was firmly adherent to the medial walls of the spleen Figure .Cyst was first aspirated to see the content that was altered/old blood without clots Figure . Marsupi3Splenic cysts are a very rare entity, and only around 800 cases have been reported as per the literature. The first classification of splenic cysts was done as true or false cysts. True cysts were either parasitic or non‐parasitic. Almost three fourths of all the non‐parasitic cysts are comprised by pseudocysts. They are mostly due to trauma, infarction, or infection.4Splenic pseudocysts are benign entity often secondary to the rupture  and hematoma resorption. Pre‐operatively its very difficult to differentiate They are usually asymptomatic but symptomatic forms need surgical treatment. Splenic preservation techniques are preferred over a radical approach unless very large entirely spleen covering or hilar cyst.Govinda Prasad Tiwari: Conceptualization; formal analysis; resources; writing – original draft; writing – review and editing. Ramchandra Poudel: Resources; writing – review and editing. Sabin Nepal: Resources. Sushil Dhakal: Resources.None.Non conflict of interests.This is the retrospective case report and no sampling was used. The ethical approval can be waived.Written and informed consent was obtained from the patient for the publication of the case report and is available for the review by the editor of the journal.


M = 14.8 ± 1.91) completed the IUS-R and measures of internalizing and externalizing symptoms, and psychological well-being. To test the factor structure of the IUS-R, one-factor, two-factor, and bifactor models were compared; measurement invariance, reliability, and validity were also addressed. Results showed that the bifactor model outperformed alternative factor models, and a general factor was needed to model the IUS-R. Bifactor model indices supported using the total score to assess IU reliably. Configural and metric invariance by age and sex were fully supported, while the IUS-R achieved partial scalar invariance. Significant correlations emerged for the IUS-R total score with psychopathological constructs, while no relationships with psychological well-being were found. Compared to adult normative data, higher total IUS-R scores were detected, suggesting that IU may be a phase-specific characteristic of adolescence. Our findings support using the IUS-R to measure IU across the lifespan. The recommended use of the total score and its associations with psychopathological dimensions highlight the transdiagnostic nature of IU in adolescence. Therefore, including IU when implementing interventions to prevent maladaptive outcomes in teenagers would be beneficial.Intolerance of Uncertainty (IU) is a transdiagnostic factor involved in several psychological disorders. Adolescence is characterized by elevated uncertainty and psychopathological vulnerability, but insufficient attention has been paid to IU at this age. This study aimed to investigate the factor structure and psychometric properties of the Intolerance of Uncertainty Scale-Revised (IUS-R) in Italian preadolescents and adolescents. 862 Italian students (57.3% girls) aged 11–17 (The online version contains supplementary material available at 10.1007/s10802-022-00944-y. Intolerance of Uncertainty (IU) is a trait-like disposition reflecting “the tendency to be bothered or upset by the (as yet) unknown elements of a situation, whether the possible outcome is negative or not” , an issue rarely explored in the literature. Although measurement invariance is frequently assumed, it is also often overlooked, even though it is a prerequisite for comparing group means. If measurement invariance is attained, the IUS-R items have the same meaning for preadolescents, early and middle adolescents, or girls and boys; consequently, the construct can be measured with the same measurement unit, baseline level, and precision across age and sex groups. Lastly, we expected the IUS-R to show moderate-strong correlations with internalizing symptoms and weak negative or no significant correlations with measures of general psychological well-being . Of these, 24.7% were recruited from lower secondary schools and 75.3% from upper secondary schools located in midsize cities in northern Italy. Specifically, with regard to the former, 30.5% attended the Italian first class (6th grade), 37.1% the second class (7th grade), and 32.4% the third class (8th grade). Among high school students, 14.6% attended the first class (9th grade), 22.7% the second class (10th grade), 31.4% the third class (11th grade), and 31.2% the fourth and fifth classes (12th grade). Participants were asked whether they had ever experienced psychological problems for which they requested professional help. Among those who responded (n = 808), 11% reported current or past psychological difficulties, such as anxiety, depression, eating disorders, learning difficulties , and family or school problems. The mean total score obtained by participants on the IUS-R was 31.8 (SD = 9.25), while the mean scores on the other administered questionnaires are presented in the Supplementary Information (Table S1).The sample was made up of 862 Italian youth  aged 11 to 17  signed a written informed consent form, and students gave their assent. No incentives or rewards were offered for participating. There were no exclusion criteria. An ad hoc online survey – composed of a socio-demographic form and a battery of self-report tools (see list below) – was administered to the sample during regular school hours. Students filled in the survey in their school’s computer room, and it took approximately 45/50 minutes in each class. Confidentiality of data treatment was guaranteed by using an identification code created by each student at the beginning of the survey.The online survey included the following questionnaires:not at all agree, 5 = completely agree). The Italian version showed an excellent internal consistency  and a good one-month test-retest reliability (r = 0.74) in undergraduate and adult samples . Problem behaviors are identified through eight syndrome scales: anxiety/depression, withdrawal/depression, somatization, social problems, thought-related problems, attention problems, aggressive behavior, and rule-breaking behavior. These scales can be grouped into problem areas like internalizing  and externalizing . In the present study, Cronbach’s alpha coefficients for each scale ranged from 0.69 to 0.90, consistently with the original Italian version of the tool (Cronbach’s α ranging from 0.83 to 0.91)  and internal consistency (Cronbach’s α > 0.85) . Although the P Scale evaluates different aspects of positivity , its unidimensionality has been supported in adolescent samples  . The factorial structure of the PWB has been also supported with Italian adolescents, and the tool showed adequate psychometric properties (Cronbach’s α ranging from 0.60 to 0.70)  and Inhibitory IU (items 8–12). Item responses are thought to reflect relatively independent common factors. As above, uniqueness reflected a mixture of random and systematic error variance. Most IUS-R research supported a bifactor model with three orthogonal factors: Inhibitory IU, Prospective IU, and General IU. The former two factors parse random error variance from the variance common to specific groups of items. The general IU factor reflects the common variance to all items and represents the trait underlying core IU beliefs above and beyond Prospective and Inhibitory IU.The “lavaan” and “semTools” packages for R were employed to estimate parameters and test hypotheses about CFA models  support using the IUS-R total score to measure IU in youth.The usefulness of a bifactor model for understanding the properties of a psychometric scale can be assessed using specific indices , early adolescent (14 and 15 years), and middle adolescent (16 and 17 years) groups. Girls and boys were compared in a subsequent set of analyses. First, we tested whether the equality in factor structure is supported in other groups . Provided a good fit for the configural model, we tested whether the factor loadings were equal across groups (Metric Invariance). Then, we constrained both the factor loadings and item intercepts to be equal across groups . Each level of invariance can be compared to the next using the chi-square difference test. Invariance is supported if the test turns out not statistically significant. However, because trivial differences might yield statistically significant differences, Chen  recommenr correlations were computed with the SAFA-A and YSR scales to test the convergent validity of the IUS-R. With regard to divergent validity, Pearson’s r correlations were calculated between the IUS-R and the PWB and P Scale. Following Cohen’s , casting doubts on the substantial independence of the two latent variables . Five items out of five loaded significantly on the Inhibitory IU factor (λrange = 0.13 –0.54). The ECV equal to 0.75 achieved Rodriguez et al. . For Prospective IU, the ωh was remarkably lower than the corresponding ω . Likewise, the ωh was lower than ω for Inhibitory IU . These findings showed that the Prospective and Inhibitory IU factors did not contribute substantially to the total score’s reliability. Conversely, the reliability of the subscales was mostly due to the General factor variance, especially for Prospective IU. Collectively, these findings show the IUS-R total score is viable and preferable to subscale scores.The standardized factor loading matrix is reported in Table 2. All items significantly loaded on the General factor, which explained 32% of the total variance. Except for item #3, all factor loadings for the General factor were in the 0.42 – 0.73 range. The Prospective and Inhibitory factors accounted for smaller fractions of the total variance . Only three items out of seven significantly loaded the Prospective IU factor  and age group , whereas the interaction was not significant. Girls’ scores  were significantly higher than boys’ scores . Gabriel’s post hoc test  showed that the preadolescent group  obtained higher IUS-R scores than early  and middle  adolescents, although mean differences were only marginally significant in the second comparison . No differences were found comparing the two adolescent groups (pGabriel = 0.565). The following The ANOVA of the IUS-R total score showed statistically significant differences by sex  aimed at testing the measurement invariance of the IUS-R. Fit indices and hypothesis testing are reported in Table 3.p < 0.10) in the analysis by age, and the differences in other fit indices were below the thresholds for concluding no substantial loss of fit .The inspection of the model modification indices revealed that the source of the misfit in the analysis by age was due to the non-invariance of a few item intercepts  between the preadolescent group and the two adolescent groups. Likewise, noninvariant item intercepts  produced misfit in the analysis by sex. Allowing those intercepts to be freely estimated for the preadolescent and female groups supported the partial scalar invariance by age and sex , but the latent mean of the early adolescent group did not differ from the reference group .Partial scalar invariance is deemed insufficient for interpreting latent mean differences without ambiguity  and with the YSR anxiety/depression, withdrawal/depression, social problems, thought-related problems, and internalizing problems scales (Pearson’s r ranging from 0.30 to 0.51). Low and positive correlations were detected with the YSR somatic complaints, aggressive behavior, attention problems, and externalizing problems scales (Pearson’s r ranging from 0.10 to 0.28), while the only non-significant correlation was observed with the rule-breaking behavior scale. Then, pertaining to divergent validity, no significant correlation emerged considering the PWB total score, while a small negative correlation was found with the P Scale. Regression analyses (Supplementary Table S3) showed that the SAFA-A and YSR scales accounted for 34% and 28% of the variance in IUS-R scores, respectively. The SAFA-A generalized anxiety and YSR anxiety/depression scores were the single best predictors of IU scores. The variables used to assess the divergent validity accounted for a minimal amount of the IUS-R variance (3%) and only the P Scale was significant. Thus, regression analyses confirmed the convergent and divergent validity of the IUS-R.The correlation matrix between the IUS-R and the other administered questionnaires is displayed in the Supplementary Information (Table S2). As regards convergent validity, moderate-to-high positive correlations emerged with all the SAFA-A scales , so it seems to imply deliberative processes to cope with uncertainty. Frontal and prefrontal brain areas - responsible for planning complex cognitive behaviors, rational decision-making, and moderation of social conduct - are still underdeveloped during adolescence Spear, , thus reItem loadings on the group factors also align with previous research in adult samples. Our study showed that some IUS-R items failed to load significantly on the Prospective IU factor , and the rest had small loadings . Similarly, all studies using a bifactor model to address the factor structure of the IUS scales in adulthood found that the Prospective IU domain was weak  failing to yield a statistically significant loading. Nevertheless, in line with previous research, this factor only accounted for a small portion of variance, far less than the General factor . Accordingly, a recent cluster analysis study showed that hedonic and eudaimonic well-being could be dissociated . In particular, the thought-related problems scale of the YSR encompasses a wide range of symptoms, such as self-harming behaviors, obsessive-compulsive symptoms, psychotic symptoms, and sleep problems. Overall, these results are consistent with the literature; in fact, although IU is notoriously a factor spanning internalizing symptoms (Carleton, In conclusion, because the multidimensionality of the IUS-R does not appear to be substantial in adolescents, the present study recommends researchers to model the factorial structure of the IUS-R according to a bifactor approach and urges clinicians to use the total score instead of subscale scores when assessing adolescents. Since IU can be a defining characteristic of adolescents and simultaneously a transdiagnostic vulnerability factor, it seems crucial to rely on the IUS-R to determine its clinical levels and further investigate IU’s relationships with psychopathological constructs in this age group. In particular, IU is a well-known risk factor for the onset of anxiety disorders, which are the psychological problems with the highest prevalence rate among teenagers (e.g., De Girolamo et al., Below is the link to the electronic supplementary material.Supplementary Material 1


Though targeted testing for latent tuberculosis infection (“LTBI”) for persons born in countries with high tuberculosis incidence (“HTBIC”) is recommended in health care settings, this information is not routinely recorded in the electronic health record (“EHR”). We develop and validate a prediction model for birth in a HTBIC using EHR data.In a cohort of patients within Kaiser Permanente Southern California (“KPSC”) and Kaiser Permanent Northern California (“KPNC”) between January 1, 2008 and December 31, 2019, KPSC was used as the development dataset and KPNC was used for external validation using logistic regression. Model performance was evaluated using area under the receiver operator curve (“AUCROC”) and area under the precision and recall curve (“AUPRC”). We explored various cut-points to improve screening for LTBI.KPSC had 73% and KPNC had 54% of patients missing country-of-birth information in the EHR, leaving 2,036,400 and 2,880,570 patients with EHR-documented country-of-birth at KPSC and KPNC, respectively. The final model had an AUCROC of 0.85 and 0.87 on internal and external validation datasets, respectively. It had an AUPRC of 0.69 and 0.64  on internal and external validation datasets, respectively. The cut-points explored resulted in a number needed to screen from 7.1–8.5 persons/positive LTBI diagnosis, compared to 4.2 and 16.8 persons/positive LTBI diagnosis from EHR-documented birth in a HTBIC and current screening criteria, respectively.Using logistic regression with EHR data, we developed a simple yet useful model to predict birth in a HTBIC which decreased the number needed to screen compared to current LTBI screening criteria.Our model improves the ability to screen for LTBI in health care settings based on birth in a HTBIC. Mycobacterium tuberculosis infection is spread from person to person through the air. Most persons exposed to M. tuberculosis will develop latent tuberculosis infection (LTBI), an asymptomatic infection, but have an estimated lifetime risk of 5 to 10% of developing active TB disease from reactivation of their LTBI [Active tuberculosis (TB) disease causes substantial morbidity and mortality. eir LTBI .In 2020, 71% of TB cases in the US occurred among non-US-born persons . An estiLTBI screening in persons born in high TB incidence countries (“HTBIC”) has historically been implemented through the public health sector and focused on those newly arrived to the US –9. HowevUnfortunately, country of birth is not routinely recorded in the electronic health record (EHR). With health systems and providers struggling under the burden of multiple conflicting priorities and new demands brought about by the pandemic, it is currently not feasible to enact a comprehensive system to reach out to patients collect this information or to require physicians to ask this question during office visits for millions of patients. As such, this information is often missing for a large percentage of patients, greatly diminishing the effectiveness of the LTBI screening guidelines.In the absence of information on country of birth, information on preferred spoken language, already routinely collected in most medical records, has been used as a proxy to determine country of birth. However, with many non-US-born persons preferring to use English when seeking medical care, this indicator may greatly under-detect the true number of persons born in HTBICs . As suchWe aimed to predict birth in a HTBIC in a cohort of patients within Kaiser Permanente Southern California (KPSC) and Kaiser Permanente Northern California (KPNC) between January 1, 2008 and December 31, 2019. KPSC and KPNC are large, integrated health systems serving over 9.2 million racially and socioeconomically diverse members in 489 medical offices , 16. AppThe KPSC Institutional review board approved this study and granted a waiver of informed consent, as the data-only research activities were determined to pose minimal risk.Patients were required to be 18 years or older at some point during the study period and to have at least one year of continuous membership with a 45-day enrollment gap allowed at either KPSC or KPNC.The outcome of interest was birth in a HTBIC, using the definition defined for adult LTBI screening by the CDPH . SpecifiPredictors for birth in a HTBIC were determined through input from clinicians and subject matter experts. Individual level predictors came from the EHR, and included whether the patient indicated that they preferred to speak a language primarily spoken in a HTBIC per the CDPH definition (“preferred language”), whether they wanted an interpreter at office visits (“needs interpreter”), the patient’s self-reported race/ethnicity , whether the patient received a Bacillus Calmette–Guérin vaccination in the past for tuberculosis (“BCG vaccination”), and whether the patient was screened at any point for hepatitis B (“screened for HBV”).As an area-level predictor, we included the percent non-US born population living in patient’s residential census tract as reported by the 2019 American Community Survey (“percent non-US born in census tract”). The vast majority of non-US-born persons in California are born in HTBICs according to our definition .Due to the larger number of patients missing race/ethnicity who were also missing country of birth in the EHR, those missing race/ethnicity were categorized into the Other/Multiple/Unknown categories. There was no imputation of missing data for other predictors where missingness was rare. Patients with these missing predictors were not included in model development; however, once the final model was chosen, predictive performance for sub-models estimated using the predictors available for each patient was evaluated to understand if useful screening recommendations could be made for these patients.Detailed definitions and rational for inclusion of these predictors are provided in The KPSC dataset was split into training and testing components in a 75/25 split. The relationships between predictors and outcomes, as well as selection of final model predictors and estimates for parameter values were assessed using only the training dataset at KPSC. Once a final model was chosen and fit, performance was evaluated using the KPSC test data , and then the KPNC data .To maximize interpretability and facilitate implementation in the clinical setting, we derived a prediction model using logistic regression. Categorical variables were entered into the models in categories specified above, while percent non-US born in census tract was included in models as a continuous variable.We assessed model performance by evaluating the following 3 metrics on the KPSC training dataset: area under the receiver operator curve (AUCROC), Brier score, and the area under the precision (PPV) and recall (sensitivity) curve (AUPRC) . AUCROC Using the final model, we explored various cut-points by calculating the proportion with a positive result with that cut-point for which the true condition is positive , proportion of the individuals for which the true condition is positive receiving a negative result , sensitivity, and specificity using the internal and external validation datasets. Cut-points explored were chosen so that the number screened would match the estimated percentage (based on non-missing country of birth data in the EHR) of members born in HTBICs at KPNC (19%), KPSC (24%), as well as the percent non-US-born in California (27%) and Los Angeles County (34%) . ResultsTo better understand how this model might be directly useful to LTBI screening, we also applied these cut-points to screen patients with positive LTBI and TB diagnoses. LTBI diagnoses were made using tuberculin skin tests (TST) or interferon gamma release assays (IGRA), while active TB was diagnosed using polymerase chain reaction (PCR) and culture methods. Since patients with LTBI diagnoses do not have symptoms and only a portion of patients are currently screened in KPSC and KPNC health systems, we were only able to evaluate the models’ use for LTBI for those screened for LTBI at any point in the study period. As TB is symptomatic, we evaluated TB screening for the full population, regardless of LTBI screening. We examined the number screened, true positive rate , absolute number of LTBI or TB diagnoses captured for each cut-point, and the number needed to screen  to explore how various cut-points might capture LTBI and TB diagnoses and minimize screening. This was done in the internal and external validation datasets, as well as for patients missing information on country of birth in the EHR.The study adhered to the TRIPOD  statement for reporting . All modThe KPSC cohort had 7,482,417 people, of which 496,257 (6.6%) had documentation in the EHR of birth in HTBIC, 1,540,143 (20.6%) had documentation of showing they were not born in a HTBIC, and 5,446,017 (72.8%) were missing information on country of birth in the EHR . EHR docThe KPNC cohort had 6,190,227 patients, of which 559,865 (9.0%) had EHR documented births in HTBICs, 2,320,705 (37.5%) had documentation showing they were not born in a HTBIC, and 3,309,657 (53.5%) were missing information on country of birth in the EHR. Patterns between categories were similar to KPSC . The patOf the 7,482,417 patients in the KPSC cohort, 2,036,400 (27.2%) had information on country of birth. An additional 11,791 (0.6%) were excluded for having incomplete covariate information, leaving 2,024,609 patients in the dataset for prediction. This was split further into a 75/25 training/test split for model development. Cut-points were chosen using population percentages in the training dataset. For LTBI, the NNS for selected cut-points ranged from 7.1–8.5 persons/positive LTBI diagnosis, compared to 4.2 for EHR-documented birth in HTBIC and 5.8 for persons/positive LTBI diagnosis for language. Using any of these cut-points would lead to an improvement from the NNS of 16.8 persons/positive LTBI case which has resulted from current LTBI screening criteria at these health systems. For active TB, the cut-point which screens 19% of the population achieved the NNS closest to EHR-documented birth in HTBIC (721.8 persons/positive TB case vs. 641.7 persons/positive TB case). Among those without information on country of birth at both KPSC and KPNC, the final model using these cut-points showed similar true-positive rates and patterns for NNS compared with the language only model, thought the NNS was higher across for all metrics for the active TB outcome. .Using a logistic regression model with only 3 variables available from the patient’s EHR, we were able to predict birth in a HTBIC with an AUCROC over 0.85 on both internal and external validation datasets and an AUPRC of 0.69  and 0.64  for internal and external validation datasets, respectively. While a model using language only had very high PPV compared to our final model using pre-specified cut-points, the true positive rate was only 0.37 and 0.29 in internal and external validation datasets, respectively. Although language preference is an accurate predictor of birth in a HTBIC when a patient prefers to speak a language other than English, it misses a great deal of individuals born in HTBICs who prefer to use English when receiving medical care. The true positive rate of our final model reaches about 0.65 for both internal and external datasets when using a cut-point that would screen a number similar to the estimated percentage of those born in HTBIC at both KP sites, close to twice the true positive rate when using language alone.Importantly, when using predictions for birth in HTBIC from the final model to screen for positive LTBI or TB, the true positive rate was similar to using EHR-documented birth in a HTBIC, even using the most conservative cut-point explored. Choosing less conservative cut-points brought the true positive even closer to that of EHR-documented birth in HTBC, with the sacrifice of more patients screened. However, while the NNS was lower for EHR-documented birth in HTBICs than all cut-points for both these endpoints, they were comparable in magnitude. The NNS estimates using the selected cut-points on those with information on country of birth ranged from 7.1–8.5 persons/positive LTBI diagnosis, all an improvement from the current NNS of 16.8 persons/positive LTBI diagnosis which has resulted from current LTBI screening criteria at these institutions; similar results were observed using the algorithm to screen those missing information on country of birth in the EHR.The improvement in performance metrics seemed smaller after moving beyond a cut-point that would result in screening more than 24% of the population. Targeting a cut-point which may screen around 24% of the patient population, close to the estimated percentage of non-US-born residents in California, may be a good default rule of thumb in an urban Californian setting. However, it is important to note that the selection of optimal cut-points with application to LTBI or TB screening will depend on resources and priorities for case capture at each institution. The current study did not take directly take into account health effects nor costs related to screening for LTBI or acquiring active TB in evaluating various cut-points. Studies directly incorporating these inputs, which may vary by institution, may reach different conclusions. Relatedly, receiving a positive LTBI and TB diagnosis is a relatively rare event, and though birth in a HTBIC is a strong predictor, the overwhelming majority of persons in California born in HTBICs do not have LTBI, and even fewer have LTBI which will progress to active TB. Thus, it is to be expected that screening all patients that any model predicts are born in HTBICs could result in exceeding desired screening resources in some health systems, even in the event of perfect prediction. Future predictive models incorporating predicted birth in HTBIC along with other elements that may predict TB activation  may improve the predictive power and reduce the number needed to be screened overall. Even testing only 25% of eligible patients in primary care encounters could prevent a substantial amount of TB cases [Though performance metrics such as AUCROC, AUPRC, and Brier were similar between the internal and external validation datasets, calibration plots for KPNC data showed the model tended to overpredict birth in HTBIC. However, using the pre-established cut-points, the overprediction did not seem to be problematic and resulted in similar performance metrics for birth in a HTBIC as KPSC. This model could easily be implemented for use at other health-care centers in California, particularly those serving more diverse, urban dwelling patients such as those at KPSC and KPNC. Further validation should be explored if this will be used outside of California, or in non-urban areas.This study has several limitations. Though missing race/ethnicity was included as a separate category in prediction, patients missing information on preferred language or census tract were excluded from predictions for birth in HTBIC in the final model. For those missing information on country of birth, 5.2% were missing information on language preference or census tract information at KPNC, while 5.6% were missing this information at KPSC. However, patients who were missing this information can have birth in a HTBIC predicted using alternative models including available variables only (such as only race/ethnicity and language). Performance metrices showed these sub-models still showed satisfactory predictive performance for patients missing information on preferred language and/or census tract. A strength of this model is strong predictive ability, using very few predictors, which make missing data less of an issue than more complicated models requiring an extensive number of covariates which collectively could make the missing rate quite high.Additionally, those missing information on country of birth in the medical record may be systematically different than those who have this information in the EHR. However, the model performed similarly predicting active TB among patients with both complete and missing information on country of birth in the EHR, indicating the percent of the population born in HTBICs is not extremely imbalanced between the populations. Additionally, the percent of active TB cases detected by our definition ranged between 65–80% in both these populations, which is also in line with the actual percentage seen in California and the US in previous studies.Finally, it should be noted that LTBI tests have imperfect sensitivity and specificity, and in particular TST may be more likely to give false positive results in the case of prior BCG vaccination . TST madIn conclusion, the prediction algorithm developed in this manuscript improved our ability to screen for LTBI using birth in a HTBIC. The model identified thousands more positive LTBI cases compared to using language alone, and resulting sensitivities for LTBI and TB were comparable to using EHR-documented birth in a HTBIC. Using the developed model also resulted in a lower NNS compared to the NNS using current LTBI screening criteria at these institutions. This predictive algorithm could be used to efficiently prompt providers to consider LTBI screening during primary care visits, or as part of a larger, targeted external outreach program prompting patients to come in for screening. While this predictive algorithm has direct benefit for LTBI screening, it may also be relevant for screening of other diseases with higher burdens in patients born in high TB incidence countries.S1 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.


Many studies have shown low birth weight is associated with psychopathology later in life, particularly attention-deficit/hyperactivity disorder (ADHD). The association is well-replicated, independent from a variety of potential familial confounds, and follows a dose–response curve (decreasing birth weight linked with increasing odds of disorder). However, the specificity of the association to attention problems is called into question by the extent of comorbidity in ADHD, and recent findings that the association is stronger for autism than ADHD. We test the relative dose–response strength of birth weight on multiple aspects of behavior to explore specificity of the effect to attention problems. We also test recent suggestions that the association between birth weight and attention problems is driven by males. Our sample consisted of 9,076 children aged 9–10 from the United States (Adolescent Brain Cognitive Development study). Outcomes included 9 problem-scales and the total problems scale from the Child Behavior Checklist (CBCL). Attention problems were the most strongly associated with birth weight after controlling for gestational age, potential familial confounds, and multiple testing, supporting the outcome-specificity of this association. Contrary to recent registry-based findings, an association between birth weight and an autism scale was not observed. Sex moderated the effect of birth weight on total problems, attention problems and aggressive behavior such that these inverse associations were strongly driven by males. Our findings have strong implications for sex-specific prediction and etiological models of childhood psychopathology.The online version contains supplementary material available at 10.1007/s10802-021-00859-0. Attention-deficit/hyperactivity disorder (ADHD) and its symptoms have been the most extensively studied psychological effects of low birth weight. A recent meta-analysis of 88 independent studies , showing an increased risk of psychopathology amongst these groups  or experiencing intra-uterine growth restriction (IUGR). Low birth weight can result from early gestational age, slow foetal growth rate, or both. While early gestational age and reduced foetal growth share some risk markers/factors  it is important to parse their respective contribution to effects of birth weight as they remain distinct biological paths with partially unique risks factors  and childhood mental health in a large population-based sample of 9–10-year-old children from the United States . We aimed to investigate: (1) the specificity of the association of birth weight across 10 different outcome scales generated from the CBCL (Child Behavior Checklist) including an attention problems, autism spectrum disorder (ASD) and total problems scale; (2) whether any of these associations were moderated by sex; (3) which associations were better explained by gestational age Vs foetal growth.https://doi.org/10.15154/1520466).This was a secondary analysis of data from the Adolescent Brain Cognitive Development (ABCD) study, a prospective cohort study of children, aged 9–10 at baseline and recruited from 22 study sites across the United States. Children were born between 2006 and 2008. The data used in this study were drawn from ABCD Release 3.0 and can be found via our registered NDA study page  Institutional Review Board was responsible for the ethical oversight of the ABCD study. The secondary analysis of the data was approved by the Research Ethics Committee for the Royal College of Surgeons in Ireland.Of the 11,875 participants in the ABCD baseline sample, we selected only the 9,612 singleton-born individuals given systematic differences in birth weight and gestational age of multiple births. Presence of a twin  OR presence of a co-twin/triplet within the ABCD study (rel_relationship) was used to define non-singleton. Siblings who did not share a womb were retained in the sample, with siblings statistically nested within the family unit . Mean age was 9.88 years (SD = 0.62). In 85% of cases, the primary respondent, henceforth referred to as "the parent”, was the biological mother of the child . The final sample size used in the analysis was 9,076 (M1) which was reduced in the fully-adjusted model .Birth weight was reported by the parent in pounds and ounces, which was converted to kilograms. In some cases (N = 1622), pounds were reported whilst ounces were not— in these cases it was calculated to the nearest kilogram using pounds alone. Some cases (N = 683) reported in ounces but not in pounds—these values were very small and unlikely to be the true birth weight therefore were removed. One individual was removed from the analysis due to improbable birth weight for their gestational age (born 6 weeks early at 6.7 KG).Children born with less than 28 weeks gestation were excluded from participating in the ABCD study. Further, parent-reported gestational age was only provided for children born earlier than 40 weeks, therefore we could not identify late-term or post-term births. We created 4 gestational age groups from the data available: full-term , early-term (37–38 weeks), late preterm (34–36 weeks), and early-moderate preterm (33 weeks or less). Maternal retrospective recall of birth weight 9 years after the birth has been found to align closely with medical records  group “prematurity” into late-moderate preterm (32–37 weeks), very preterm (28–32 weeks) and extremely preterm (< 28 weeks) .The CBCL  depression, (2) problems with nerves, (3) mania, (4) psychosis, (5) drug abuse, (6) alcohol abuse (7) antisocial behavior, or whether a family member had (8) attempted/committed suicide. The specific wording of questions probing family history are provided in Supplementary Material. “Blood relatives” included biological parents & siblings, half-siblings, biological grandparents & aunts/uncles. We created summary variables such that, if there were 1 or more familial instances of an issue mentioned above, that type of issue was given a 1. That is, each mental health issue had a maximum score of 1. These were then summed to form a total score with a min–max of 0–8. This variable was thus a cumulative score of discrete mental health conditions in the extended family.Three socioeconomic factors were included in this study: household  income, parental education level and single parenthood. Household income was captured by 10 annual income brackets from $5,000 or less to $200,000 or more. Parental educational level was captured by 7 levels from incomplete high school to doctoral degree. Dual-parenthood was qualified by presence of a partner who helped in raising the child and is involved in 40% or more of the child’s daily activities. Note this partner could be a spouse, boyfriend/girlfriend, relative or friend of the parent. Single-parenthood was the absence of such a partner. Greater detail on these variables is provided in Supplementary Material.All analyses were performed in R  was run with birth weight as a continuous predictor, CBCL score as continuous outcome and covariates adjusted for in consecutive models: M1 adjusted for sex only, M2 added race/ethnicity and socioeconomic factors , while M3 added family history of mental illness. All 3 models were re-run adjusting for gestational age (aim 3).Specificity of the association between birth weight and mental health to attention problems (aim 1) was explored both visually, by plotting relative effect sizes of birth weight on all 9 CBCL sub-scores, and with inferential statistics by testing whether attention problems were significantly predicted by birth weight after correction for all covariates and multiple testing (Bonferroni corrected p = 0.05/9 = 0.006).To test whether the effect of birth weight on mental health was moderated by sex (aim 2) we included a birth weight*sex interaction term to the fully-corrected model (M3) for the CBCL total problem score and all 9 sub-scores.Given that some studies have found links between large birthweight and psychotic disorders  is very similar that of Momany et al.’s on parent- and teacher-rated inattentive symptoms  both in terms of magnitude and variance. We extended this pattern to two more outcomes: aggressive behavior and general mental health , is more susceptible to prenatal insults (e.g. ischemia-hypoxia) in males compared to females. Rat experiments have shown that withdrawal of oxygen supply perinatally impairs behaviour and alters prefrontal dopamine (and other monoamine) levels in males more than females  implying this effect was driven largely by foetal growth, consistent with other findings  had the worst CBCL outcomes Table ; Fig. S1The strengths of this study include the large sample of children  within a narrow age range (9–10 years), the continuous rather than binary measurement of birth weight and mental health allowing us to capture smaller effects on a continuous scale, and the use of generalized linear model to predict non-normally distributed mental health scales. Limitations of this study include the non-generalizability of findings to multiple births and the reliance on parent-report only for both retrospective birth data and the child’s mental health status. The ASD scale constructed from CBCL items , rather than autistic traits. This contrasts with recent registry-based findings and highlights the importance of assimilating evidence from a variety of study designs to avoid sampling bias. Our data also suggests males are particularly vulnerable to the psychological effects of lower birth weight, particularly problems of attention and aggression. This, given further replication, may have strong implications for sex-specific mechanistic and prediction models.Supplementary file1 (DOCX 61435 KB)Below is the link to the electronic supplementary material.


Background Recent data suggest that dementia incidence is declining. We investigated whether similar secular trends consisting of increasing size of brain structures and improving memory performance could be simultaneously occurring as a possible explanation. Method The Framingham Heart Study is a 3 generation, longitudinal study that includes cognitive assessment and medical surveillance. This study cohort consisted of 4,506 unique, non-demented, stroke free, individuals with brain MRI, cognitive assessment, and demographic information spanning dates of birth from 1902 to 1985. Outcomes consisted of height, MRI, and memory measures. Covariates included age at MRI, sex, decade of birth, and all interactions. Models with neuropsychological outcomes also included educational achievement as a covariate. Results Height and intracranial (TCV), hippocampus and cortical gray matter volumes were significantly larger, and memory performance significantly better, with advancing decades of birth after adjusting for age, sex, and interactions. Sensitivity analysis using progressively restricted age-ranges to reduce the association between age and decade of birth, confirmed the findings. Mediation analysis showed that hippocampal volume mediated approximately 5–7% of the effect of decade of birth on logical memory performance. Discussion These findings indicate improvement in brain health and memory performance with advancing decades of birth. Although brain structures are under substantial genetic influence, we conclude that improved early life environmental influences over ensuing decades likely explain these results. We hypothesize that these secular improvements are consistent with declining dementia incidence in this cohort potentially through a mechanism of increased brain reserve. Individuals are also living longer resulting in an increasing percentage of the population at risk for Alzheimer’s disease and related dementias (ADRD). Recent data4, including from the Framingham Heart study5, however, finds that dementia incidence may be declining. While many factors such as greater educational achievement6 and medical management of vascular risk factors8 may explain part of this effect, early life environmental differences likely also contribute6.The overall health of the U.S. population has improved dramatically over the last 100 years. Infant mortality has declined precipitously due to improved sanitation, nutrition, and medical treatment9. The Framingham MRI study, initiated in 1999, has imaged 5,145 unique individuals across all three birth cohorts enabling age-specific comparisons of secular trends. We hypothesized that secular trends in improving early-life health, as observed in the general US population, would be accompanied by the expected increase in height as well as head and brain size. Given that increasing head size has been shown to be independently associated with improved later-life cognitive performance10 we further hypothesized that these secular trends would significantly associate with similar trends in improved levels of education and cognitive ability, and that increased hippocampal volume would partially mediate the impact of decade of birth on memory performance indicating a possible mechanism of brain reserve that could explain declining dementia incidence in this cohort.The Framingham Heart Study, initiated in 1948, consists of multiple generations of participants across more than 80 years difference in birth dates, making it ideal for investigating secular trends in cardiovascular and brain health12. In brief, the Framingham Heart Study is a community-based population study of over 15,000 individuals from the town of Framingham Massachusetts, USA spanning more than 75 years of observation. The Original cohort of the Framingham Heart Study included 5,209 participants who were enrolled into the study in 1948. At enrollment, the mean age was 44 years (range 28–62 years) and 55 per cent were female, the majority of whom were white and of middle socioeconomic class. Surviving members of this cohort are examined approximately every two years. The Offspring cohort included 5,124 offspring of the original cohort and their spouses enrolled in 1971 with examinations performed approximately every 4 years. At enrollment, the mean age was 36 years (range 25–70 years) and 52 percent were female. The 3rd generation cohort included 4,095 children of the Offspring cohort enrolled in 2002 and has completed 3 evaluations to date. At enrollment, the mean age was 41 (range 25–70 years) and 53 percent were female. In addition to repeated examination cycles, surviving participants are under constant surveillance for incident events such as myocardial infarction, stroke, and dementia as part of regular health evaluations that included assessment of vascular risk factors . As part of multiple large ancillary studies on brain structure and cognitive function, FHS participants were recruited, starting in March 1999, to undergo MRI of the brain and to be administered an neuropsychological test battery as previously described14.The general design and demographics of the Framingham Heart Study (FHS) have been previously describedTo examine secular trends in MRI and cognitive performance, initial visits where MRI and neuropsychological testing were performed were selected. This resulted in a cohort of 5,145 individuals with a mean age of 58 (range 25–98 years) who were born between 1900 and 1990. MRI was performed between the years 3/18/1999 and 11/15/2019 on a total of 12 different MRI machines. Most participants, however, were scanned on only two different Siemens machines and to reduce the impact of machine variability on MRI measures, this subgroup of 4,643 individuals were selected for this study. Of the 4,643 participants, 137 were excluded due to prevalent dementia, stroke, or other Significant neurological disorder  at the time of MRI, reducing the final number for analysis to 4,506 see . All parHeight measurement in inches was obtained at the first FHS visit for each participant and was made at an average age of 35, 36 and 41 years across the 3 cohorts, and 37.7 ± 8.1 years of age on average, avoiding the impact of later life skeletal degeneration on the measure.15. For this study, intracranial volume was defined as the cranial vault above the tentorium. Image intensity correction was used to remove B1 inhomogeneity effects16 and segmentation of the image into 3 tissue classes used an algorithm optimized to improve precision of tissue segmentation . Additionally, hippocampal analyses were performed using an atlas based diffeomorphic approach18 with the minor modification of label refinement. Cortical thicknesses were measured using the DireCT method19. Cortical surface area was calculated using 3D cortical surface mesh reconstructions employing marching cubes20, a standard algorithm in computer graphics that has been used extensively in brain image analyses21. To estimate gray matter surface area in a region of interest in MRI native space, we compute the area of the polygonal facets lying within the ROI mask.Quantifications of MRI measures were obtained from high resolution 3D T1 weighted MP RAGE images using either a Siemens Magneton Expert or Siemens Avanto machine to obtain intracranial, cortical gray matter, hippocampal volume, and cortical thickness measures. Quantification included automatic removal of non-brain elements from the 3D T1 image volume using a robust and accurate method relatively insensitive to machine type or field strength22 where regional measures were calculated by back transformation of the atlas into segmented image native space. A voting scheme is used to assure precise labelling of each region after interpolation of the atlas into native space. Using this approach, we defined four measures for study: 1) Intracranial volume , 2) Total cortical gray matter volume (cc), 3) Hippocampal volume (cc), 4) Surface area in squared centimeters (cm2) and 5) Average cortical thickness in millimeters (mm). To correct for scanner effect, MRI measures were corrected using NeuroComBat, a robust method for reducing machine related differences in MRI data25 .Cortical parcellation used the Desikan-Killiany-Tourville Atlas26. Given that verbal memory performance is both sensitive to brain aging29 and predicts future clinically relevant cognitive impairment30, analyses for this study focused on verbal memory variables of immediate, delayed and the sum of both immediate and delayed recall.Neuropsychological assessment consisted of a battery of tests of verbal learning and memory , visual memory , abstract reasoning , language (Boston Naming Test) and executive functioning (Trails A and B) as previously described31. Systolic blood pressure  was taken as the average of the Framingham clinic physician’s two measurements. Educational level was defined as the obtainment of a college degree (yes or no).APOE genotype was determined with a standard TaqMan assay; APOE-e4 carriers were defined as those having at least one e4 allele. The Framingham Stroke Risk Profile (FSRP) score is a validated, widely-used composite measure of vascular risk factors that predicts the 10-year probability of a strokeTo understand the relationship of decade of birth with body height and cranial volume, we performed univariate associations between mid-life height, intracranial volume, and decade of birth, stratified by sex at birth.In addition, because there are striking sex-differences in both these features, we investigated possible sex-differences in these relationships using multivariate analyses to test the interaction between mid-life height, intracranial volume, decade of birth and sex at birth. Given that height and cranial volume are correlated, we also performed multivariate analysis to investigate if cranial volume differed by decade of birth adjusting for height.To understand the relationship of decade of birth with brain measures, we performed multi-variate linear regression analyses of TCV, cortical gray matter, cortical thickness, and hippocampal volumes with decade of birth including sex at birth, age at MRI, and all possible interactions as covariates.While many brain aging studies correct regional brain volumes for head size to reduce variance, we recognized that any secular differences in head size would influence our results. Consequently, we examined each brain region independently without adjusting for intracranial volume to avoid confounding by secular differences in intracranial volume (TCV).We used logistic and linear regression to examine the association between vascular risk factors, including ApoE genotype (E4 genotype yes/no), FSRP and, SBP with decade of birth including sex at birth, and age at MRI and all possible interactions as covariates. We then examined the influence of these factors on the association between decade of birth and brain measures by adding to the models described above ApoE genotype, FSRP and, SBP as additional covariates, as well as the interaction of these risk factors with decade of birth.Initial analysis included the full range of decade of birth and age at MRI. Given the likely association between decade of birth and age at MRI, sensitivity analyses were also performed to limit the impact of age at MRI on the observed associations. The first analysis limited the age range at MRI from 45 to 75 years in 3 ten-year epochs. A second analysis further limited the age range at MRI to 55–65 years of age.Given the known, strong relationship between educational achievement and memory task performance, we first used a logistic regression to examine the association between achieved college education with decade of birth including sex at birth, and age at MRI and all possible interactions as covariates.We then examined the impact of decade of birth on verbal memory performance  using multi-variate linear regression analyses with decade of birth, sex at birth, age at MRI, educational achievement, and all possible interactions as covariates.We further investigated the effect of brain measures on verbal memory performance using linear regressions with decade of birth, sex at birth, age at MRI, educational achievement, and all possible interactions as covariates.Given the likelihood that brain structure would increase, and verbal memory performance improve, we examined whether hippocampal volume might mediate the relationship between decade of birth and verbal memory performance. Causal mediation analysis was used to test the mediation effect of hippocampal volume on the relationship between decade of birth and memory performance adjusting for sex at birth, age at MRI, educational achievement, and all possible interactions as covariates using 5,000 bootstrap iterations for estimation.31.All MRI measures and neuropsychological test performances were converted to Z-score values for relative comparison of results across measures. Analyses were performed using R version 4.1.2. Mediation analyses used the R Mediation package which is based on methods of Imai et al32 as determined by clinical examinations an average of 1.1 ± 0.9 years around the time of MRI. The average time interval between neuropsychological testing and MRI was 0.01 ± 0.09 years and ranged from 0 to 1.33 years with 90% of subjects receiving neuropsychological testing at the time of MRI. Significant differences by sex at birth were found among women for height (−2.74 inches), intracranial volume (−77.9 ccs), hippocampus (−0.32 ccs), total cortical gray matter (−27.0 ccs), immediate memory (0.51 points greater) and delayed verbal memory (0.55 points greater). The specific distributions of age for various decades of birth are also summarized in the Supplemental Materials to this manuscript.The final cohort sample is summarized in The results of our primary analyses are summarized in 1 All MRI measures were z-transformed for comparison of effect across regions and results presented as beta coefficients from regression analysis. age(yrs) = −0.021 versus βbirth decade = 0.008 z-scores), although opposite in sign (i.e. increasing age leads to atrophy whereas increasing birth decade leads to larger volume).N denotes numbers of participants at each level of sensitivity analysis. Age-range of restricted analyses is given as heading to that column.Logistic regression analysis of the prevalence of ApoE4 genotype by decade of birth was not Significant. Conversely, SPB measures declined significantly with advancing decade of birth (p < 0.0001). Both FSRP and SPB, however, strongly interacted with age and decade of birth (p < 0.0001) in predicting cortical GM volume outcomes, where the positive impact of decade of birth on brain structure was diminished by increased participant age (and consequently worsening FSRP or SPB) at the time of MRI.33, was associated with significantly smaller, hippocampal, and cortical gray matter volumes (p < 0.0001), that interacted modestly with decade of birth such that declining mean FSRP with increasing decade of birth was associated with increased volumes of these measures.Adding ApoE genotype to the MRI models resulted in a very small (0.03 cc), but Significant (p < 0.05) increase in TCV and cortical gray matter volumes for ApoE4 genotype carriers that did not modify the positive effect of decade of birth on these measures. Conversely, FSRP, a summary scale for stroke riskGiven the substantial association between age at time of MRI and decade of birth, two additional analyses, selecting more limited age-ranges, were performed. The decade of birth relationship is different for cortical thickness as summarized by 2 = 0.88, p < 0.0001). Similarly, for each cc in cortical gray matter volume, cortical surface area increased by nearly 3 cm2  but for each cc in cortical gray matter volume, cortical thickness increased by only 0.001 mm . Conversely, for each cm2 increase in cortical surface area, cortical thickness declined by 0.003 mm . These results indicate that the increased head size and gray matter accompanying advancing decade of birth led to increases in surface area that were greater per unit increase than cortical thickness, leading to the negative association of cortical thickness with decade of birth within similarly aged individuals.To further investigate this finding, we examined the relationship between intracranial volume, cortical surface area and cortical thickness, as cortical surface multiplied by cortical thickness should reflect cortical gray matter volume. For each cc in intracranial volume, there was 0.35 cc increase in gray matter . There also was a Significant 2-way interaction between decade of birth and sex (p < 0.01) where women had more rapidly increasing likelihood of achieving a college education with advancing decade of birth, particularly between decades of birth spanning 1910–1950.The impact of decade of birth on memory performance is summarized in Intracranial, cortical gray and hippocampal volumes were significantly associated with each measure of memory performance after adjusting for age, sex, and education (p < 0.001−<0.0001). Cortical thickness was not associated with memory performance. When intracranial, hippocampal, and cortical gray matter volumes were included in the same model, however, only hippocampal volume remained significantly associated with memory performance (p < 0.001−<0.0001). Finally, hippocampal volume remained significantly associated in a model that included the addition of decade of birth with age, sex, and education as predictors of memory performance (p < 0.0001).We assessed the possible mediation of hippocampal volume on the relationship between decade of birth and memory performance using causal mediation analysis. 35 and potentially greater “brain reserve”41 that could explain the declining incidence of dementia as previously reported in the Framingham Heart Study5 and further supported by improved verbal memory performance associated with greater hippocampal volume found in this study.Our results find Significant secular increases in height and brain structure along with improved memory performance with advancing decades of birth. Brain structures also appear to be increasing at a rate slightly greater than height. Further, improved memory performance accompanying advancing decade of birth appears to be mediated  by increasing hippocampal volume after adjusting for age, sex, and education. We hypothesize that larger brain volumes indicate improved “brain health”43 and it is tempting to postulate these major stressors as potential factors contributing to secular trends in hippocampal volume along with the many other differences lifestyle that occurred over the same time period. Finally, cortical thickness declined with decades of birth as well as advancing age. Declining thickness coincided with increasing TCV as well as cortical gray matter volume and surface area. Although cortical surface area was positively associated with cerebral gray matter, cortical surface area was inversely associated with cortical thickness. Rakic44 describes how the radial unit lineage model could explain evolutionary and developmental aspects of the cerebral cortex that would be consistent with our findings. In this model, expansion of the cerebral cortex occurs through increasing convolutions and expanding surface area while limiting change in cortical thickness. White et al45 extend this concept to “gyrification” of the cerebral cortex, which they note results in a surface area “1700 times larger [in humans] than in shrews, yet the thickness of the cortex is only six times greater”. Like Rakic44, White et al45 emphasize the computational utility of expanding surface area over thickness within the radial unit lineage model that allows for “an optimized compaction of neuronal fibers with an efficient transit time for neuronal signaling”. Consequently, increased cortical gray matter with advancing decades of birth results in increased surface area through increased gyrification. According to the radial unit lineage model, the increased gyrification (and consequent surface area) leads to subtle reductions in cortical thickness as seen in our data. Importantly, both Rakic44 and White et al45 emphasized regional differences in “gyrification” are likely under unique genetic influence46. Such an analysis is beyond the scope of this report but could add further information regarding the biology of cortical gray matter expansion with subtle reduction in cortical thickness as seen with our more global measures.Our findings of consistent enlargement of TCV, cortical gray matter and hippocampal volume with advancing years of birth spanned dates from 1902–1985. Confounding by association of age at MRI with decade of birth was addressed through sensitivity analysis. Results from these analyses found that stepwise restriction of the age of participants, first to 45–74, and then to 55–65 years, where age-related differences in brain volumes were minimal, showed consistent increases in brain size measurements with advancing decade of birth, even when the decades of birth were limited to 30 years as occurred with the most restricted analysis . Analysis of the age-range between 45 and 75 years of age  and covering 5 decades of birth, resulted in multiple unique findings –6. FirstMemory performance also improved with decade of birth and was significantly associated with all MRI measures except cortical thickness. Improved memory performance was dramatically associated with level of educational achievement, being nearly 1/3 of a standard deviation higher for college educated participants. Memory performance was nearly linearly associated with decade of birth for women but appeared to asymptote somewhat for men . This to50. TCV is highly associated with brain growth during normal development40, whereas aging or disease-related brain-volume decrease does not alter TCV. Thus, adult TCV is a stable valid measure for maximal attained brain size, widely used as a proxy for brain reserve51, and is an important predictor of cognition in old age41 including studies that used similar measures of memory performance51. Conversely, cross-sectional, and longitudinal age-related differences in brain volume measures associate with cognitive performance in aging and disease54. Hippocampal volume loss, in particular, is considered to be sensitive to early degenerative diseases such as Alzheimer’s disease56. Although absolute volumes do not associate with cognitive ability per se, as illustrated by the fact that women in this study had significantly smaller hippocampal volumes, but significantly better memory performance, loss of brain tissue within an individual is strongly indicative of pathological effects57, which, therefore, may be buffered by larger structural brain development. As noted in our data, while decade of birth had a Significant positive effect of cortical gray matter volume, the impact of aging remains essentially unchanged. Consequently, individuals in each decade of birth appear to atrophy at the same rate, but later decades start further away from any ‘threshold’ of atrophy that might manifest as clinical cognitive impairment. Alternatively, and more likely, larger structural brain development may be a surrogate for other environmental processes ongoing during development and early adult life such as increased brain connectivity58. Increased connectivity is consistent with the radial unit lineage hypothesis44 that enables increased neuronal connectivity through cortical expansion and gyrification45. Such increased connectivity could mitigate the impact of age-related diseases on cognitive performance and fits well with the scaffolding hypothesis of cognitive reserve59. Evidence supporting the notion that experience is associated with regional brain expansion can be seen among London taxi-cab drivers who have larger hippocampal volumes compared to same aged individuals60.How might these secular effects modify the likelihood of later life dementia? Brain growth begins in utero, increases throughout childhood, and reaches a maximum size in early adulthood,64, the timeline of effect found with our results indicate that early life environmental influences are also likely substantial contributors, particularly educational achievement6. Life course perspectives emphasize the impact of early life experiences on brain health66 that also translate into larger brain structures67 and reduced risk for later-life dementia through improved reserve68. Similarly, effort to improve cardiovascular health during adulthood71 are associated with reduced incidence of cognitive impairment72 and dementia73 indicating that modifying these factors could also serve to improve resistance to late-life dementia74.While head and brain size are under substantial genetic influence67 as well as secular improvements in modifiable dementia risk factors leading to better “brain health” and reserve32. While these effects are likely to be small at the level of the individual, they are likely to be substantial at the population level adding to growing literature that suggests optimized brain development and ideal health through modification of risk factors could substantially modify the impact of common neurodegenerative diseases such as stroke and Alzheimer’s disease on dementia incidence70. Moreover, taken together with increases in IQ throughout the 20th century75, these secular trends in brain volume may contribute to an overall more cognitively resilient and productive society.In summary, our results indicate that TCV and brain structures have increased over birth years ranging from 1902 to 1985. These differences are coincident and associated with improved memory performance that is partially mediated by larger hippocampal volumes. These findings likely reflect both secular improvements in early life environmental influences through health, social cultural, and educational factorsThe strengths of our study include the design of the Framingham Heart Study that began in 1948 and has followed a community of individuals with comprehensive health evaluations throughout much of their lifespan and across three generations. The addition of MRI beginning in 1999 enabled quantitative brain assessment across all three generations spanning more than 80 years difference in dates of birth. Obtaining contemporaneous MRI with neuropsychological assessment also enabled brain behavior associations. A high rate of participant participation resulting in a large sample size across decades of birth also enabled reliable sensitivity analyses that might not be accomplished with smaller studies. The duration of observation that includes younger individuals also suggests that this secular trend may be continuing. Finally, the fact that more than 80% of subjects were imaged on just two MRI machines also helped to reduce machine differences that were further reduced using the NeuroCombat statistical harmonization method.78 and health disparities2 which are more common among non-White individuals32 in the US may adversely affect brain health. Second, this is a cross-sectional study that has limited causal inference. Longitudinal analyses showing secular differences in rates of regional brain atrophy would further support evidence of increased brain reserve through resilience to age-related atrophy39. Similarly, we are not aware of another cohort spanning 3 or more decades of birth to validate these findings.This study, however, is not without limitations. First, and most importantly, the Framingham Heart Study is predominately non-Hispanic White, healthy, and well educated and, therefore, not representative of the more diverse US population. For example, current evidence indicates social-culturalDespite these limitations, we conclude that this study extends current knowledge by showing that secular trends in brain structure are occurring and are associated with improved memory performance. We hypothesize that the larger brain structure reflects improved brain health and is at least one manifestation of improved brain reserve that could buffer the impact of late life diseases on incident dementia. Our results also support the growing literature that emphasizes optimal brain development and ideal brain health as preventive measures to mitigate against rising dementia prevalence among our aging US population.


Chalinolobus gouldii, at two sites over two years and derived growth curves and age estimation equations which will be useful in the study of how intrinsic and extrinsic factors alter parental investment strategies. We found that male and female offspring did not differ significantly in their size at birth or their postnatal growth rates. Bats born in 2018 were smaller at birth but grew at a faster rate than those born in 2017. When date of birth was compared across sites and years, we found bats born in 2018 had a later median birthdate (by 18 days) and births were more widespread than those born in 2017. Cooler and wetter weather during late gestation (Nov) in 2018 may have prolonged gestation and delayed births. With many bats facing threatening processes it is important to study reproductive plasticity in common and widespread “model” species, which may assist in the conservation and management of threatened microbats with similar reproductive traits.Reproductive phenology, size at birth, and postnatal growth are important life history traits that reflect parental investment. The ability to document detailed changes in these traits can be a valuable tool in the identification and management of at-risk wildlife populations. We examined reproductive traits in a common, widespread Australian microbat,  Anthropogenic disturbance is a major threat to biodiversity , with wiPredicting the impacts of anthropogenic disturbance on wildlife is challenging without detailed knowledge of fitness-related traits, such as survivorship , reproduFor temperate-zone insectivorous bats , cold winters, and corresponding low insect availability can result in pregnancy and lactation being restricted to spring and summer , with thPteropus bats have been studied for juvenile growth, Pteropus poliocephalus , a common tree-roosting insectivorous species with a broad distribution encompassing most of the Australian continent . We developed equations for estimating age during the linear growth period (first 20 days) and produced best-fit growth curves on postnatal growth patterns in body mass and forearm length using three non-linear growth equations . Finally, we compared size at birth and postnatal growth across the 2017 and 2018 breeding seasons, and documented climate variables during both years.Here, we conducted a fine-scale study on the reproductive phenology and postnatal development of free-ranging Gould's wattled bats (ontinent . We surv history . We collC. gouldii) are widespread across Australia, historically roosting in tree-hollows but also actively utilizing artificial roosts. Mating occurs in the austral autumn (Apr–May) resulting in sperm storage in the female reproductive tract over winter (Jun–Aug) with ovulation and fertilization occurring at the end of winter  to twin pups (unpublished data).Gould's wattled bats  grassy woodland, 11 km north-east of the city center . Organ Pipes National Park (OPNP) is a 152.5-ha site, located 35 km north-west of the Melbourne city center along the peri-urban border . The predominant vegetation is river red gum interspersed with manna gum , with an understory of Acacia spp. and grasses. The site was revegetated in 1972 from heavily cleared farmland and consequently there are fewer hollow-bearing trees and a large uptake of artificial roosting boxes by C. gouldii  sampling on mothers and pups over two successive years (2017–2018). Bats were collected from boxes by hand during the day at regular intervals (averaging 3–4 days) during the first 60 days of the reproductive season and then every second month for 12 months, to encompass the reproductive period from parturition date throughout lactation, weaning, and independence. All bats within a box were placed into calico cloth bags until processing. Processing was done on-site to limit the time bats were kept out of their boxes. This protocol was employed to minimize the amount of disturbance to the mother and pups. Any female bat that was lactating was placed separately in a calico bag with her pups still attached and then following processing was returned to the roost box of collection.TM) (C. gouldii pups (from 256 mums) were PIT-tagged across the two sites during 2017 and 2018 . Of the 475 pups PIT-tagged, 56 were captured on their day of birth , with 36 pups having an attached umbilical cord and 20 within 1SD of mean forearm length across both sites in 2017 and 2018. Pups that were not found on day 1 had their birth dates back calculated from the regression equation produced from known-age young (described below).Pups with the umbilical cord (and sometimes placenta) still attached were presumed to be born that day . Pups thTM) . A totalEach pup's sex was determined visually by the presence/absence of a penis. Forearm (FA) measurements were taken of the length of the right forearm from three repeated measurements to the nearest 0.1 mm using digital vernier calipers (Carbon Fiber Composites). While it has been suggested that forearm length may not be a good indicator of body size, it is the only measure currently appropriate for small bats . The fouAll measurements of forearm length and epiphyseal gap throughout development were taken between November and March of 2017/2018 and 2018/2019, while body mass in bats that remained in the OPNP population was measured for the entire year following birth.n = 56). As mass was collected from only six pups on day of birth, a model for mass was not produced. Most day of birth measurements collected in 2017 were from NTWS (n = 18 vs. n = 4 at OPNP) and most collected in 2018 were from OPNP (n = 27 vs. n = 7 at NTWS). Given this unbalanced sample collection, we used year of sampling as a predictor variable in all analyses but note that year and site are confounded. Both models included maternal ID as a random effect. All data were tested for normality using the Shapiro test, and unequal variances tested using Bartlett's test.We used a series of linear and non-linear models to assess differences in size-at-birth between sexes and years and in growth curves between years. We used a linear mixed-effects model (LMM) to compare the forearm lengths and epiphyseal gap lengths on day of birth between sexes and years (Growth data of the 56 young bats (forearm length and mass) were fitted to three non-linear growth models: the Gompertz equation, the logistic equation, and the von Bertalanffy equation  as the response variable and the size variable as the predictor n = 56; . This moC. gouldii pups throughout their first year of life. Only pups that we collected repeated measurements on were included in the model, and two female pups were removed from the data set as their weights were very low for female late-Autumn (n = 136). They were assumed to be very late born pups (February), not comparable with the November–December born pups included in this analysis.We used a generalized additive model (GAM) to describe non-linear changes in body mass of male and female Daily rainfall and half-hourly dry-bulb temperature readings were obtained from the Bureau of Meteorology  for weatAll analyses were conducted in R version 4.0.3 . We usedOf the mothers captured with pups, 90% produced twins with parturition occurring between November and December in both years. At birth, neonates were naked with pink skin and their eyes closed , and 11 out of 12 recaptured females in the 2019 breeding season (Nov 2019–Jan 2020) were lactating or postlactating. One female was recaptured with an unfurred pup in February 2020 after being recorded as postlactating in January, suggesting a second pregnancy in her first year.Of the 110  pups marked at OPNP in Nov–Dec 2018, 29% remained in the population one year after birth , and 6% were recaptured 28 months after birth . This is consistent with longer term records, see additional demographic data on PIT-tagged pups at OPNP collected from 2014 to 2016 . At the  gouldii . Pups beThe median parturition date in 2017  forearm length of neonates captured on day 1 was 14.4 ± 1.0 mm . Neonates in 2017 had significantly longer forearms at birth than in 2018 .Body mass on day 1 ranged from 2.0–3.2 g (mean 2.4 ± 0.2 g), with each pup averaging 9–15% of mother's body weight. Length of the epiphyseal gap at birth ranged from 2.0–3.1 mm (mean 2.7 ± 0.04 mm). There was no difference in epiphyseal gap length at birth between years or sexes . There was no difference in the rate of epiphyseal gap fusion between years, or between sexes (P > 0.05).Bats born in 2018 had shorter forearms at birth but grew significantly faster over the first 20 days than bats born in 2017  than males across the first year of life from day 50 onwards  daily minimum and maximum temperatures differed significantly between years , and low levels of reproductive seasonality across the two years of the study (median birth date was 18 days later in 2018). At our study sites, November (late-gestation) 2018 was cooler and wetter than November 2017, so our results are consistent with past observations of links between cool and wet weather during pregnancy and delayed parturition and decreased prenatal growth in bats . Temperaturition , which mturition . Potentiturition .Chalinolobus gouldii pups were born smaller in 2018 but grew at a faster rate during the initial 20-day growth period. As described above, small birth size has been linked to cool and wet weather in late pregnancy (Tadarida brasiliensis (Pipistrellus subfalvus pups after a period of cold weather.regnancy . Evidencregnancy  with theiliensis . Howeveriliensis . Compensiliensis ; notablyC. gouldii reproduction could be due to site differences, rather than climatic conditions per se, due to the confounding effect of site and year in our study design. For example, unmeasured factors such as landscape structure and insect availability may influence reproduction in C. gouldii. Previous studies provide evidence for increased diurnal roost temperatures altering reproductive physiology, resulting in differences in timing of parturition, size at birth (C. gouldii. It is unknown whether a second pregnancy occurs in C. gouldii that primarily roost in natural tree hollows.Interannual differences in at birth , and posat birth . There aat birth . Consequat birth . Timber at birth . Thus, sat birth . Howeverat birth . UnfortuC gouldii on the day of birth, during the growth period, or when they reached full-sized adult dimensions. We did, however, find sex differences in body mass fluctuations across the first year of life, with females being on average heavier at all timepoints after the initial growth period. This is consistent with findings in C. gouldii at another location in suburban Melbourne (C gouldii appear to be reproductively active in their first year.The present study found no sex differences in forearm lengths of elbourne  and in telbourne . The heaelbourne . In addiC. gouldii parturition dates, size at birth, and postnatal growth. Given predictions of increased temperatures and reduced rainfall across the Victorian range of C. gouldii (C. gouldii in different climate zones of Australia, and extend the models developed in the current study as a powerful means of understanding climate influences on microbat reproduction and postnatal growth.Our results suggest that changes in weather during the breeding season may alter  gouldii , climate gouldii . Future obac030_Supplemental_FilesClick here for additional data file.