This paper summarizes the assessment of directional anisotropy in local mechanical properties for Laser Powder Bed Fusion (LPBF) IN-718 bulk samples via the use of miniature samples excised from the bulk for both as-deposited and post-treated states. The quasi-static tensile properties at room temperature are investigated at several different locations along the build direction and at different orientations for both considered states. A comparison between the excised miniature tensile specimens and standard-sized sample results have also been conducted and exhibit very good agreement. Significant anisotropy is present in mechanical properties at different build heights for the as-deposited state, while the post-treated material exhibited more homogenous properties, both along the height and for different sampling orientations. However, significant reductions  in the strength  along with a significant increase in the reduction in area at fracture is found for post-processed materials. Metallography and fractography analyses were conducted in order to begin to determine the source(s) of this anisotropy for the as-deposited state. While AM technologies are rapidly evolving, the field of potential applications for AM-produced components is also growing. However, implementation of AM for applications in strength/fracture critical components requires reliable performance . This caA better understanding of AM-deposited materials requires the ability to assess the location- and orientation-dependence of mechanical properties with the use of minimum material volume. While various small-sized testing methods are available for such assessments, instrumented hardness-based methods and/or Small Punch Test methods are difficult to correlate with mechanical properties that are applicable in design. Mini-Tensile Tests (M-TT) procedures were originally developed to assess the residual service life of power industry components while providing a broadly applicable test method that does not require correlation coefficients for each material investigated, such as that needed for the Small Punch Test method. The M-TT approach has been verified for multiple materials while the currently used test procedure was developed in previous works ,9,10,11 The present work was conducted to extend the expertise in the field of small-sized specimens testing for AM-deposited materials. The focus of the work presented here relates to the demonstration and quantification of local mechanical anisotropy in an idealized Laser Powder Bed Fusion (LPBF)-processed IN-718 rectangular bar. Bars in the as-deposited and post-treated states are reported here. Local mechanical properties in terms of quasi-static room temperature tensile tests along the build height were determined along with sampling various orientations over the build direction, at each height position. Metallography and fractography were also used in order to begin to understand the source(s) of such property variations.P and Q, in order to assess the effects of this post-processing treatment on property homogenization along the builds. Bars P and Q were adjacent bars on the same build plate and were prepared to also check for build homogeneity.An SLM 280 machine  was used to produce 10 mm × 20 mm × 120 mm length cuboidal bars using SLM Solutions pedigree gas-atomized IN-718 spherical powders with an average particle size of 15–45 µm. Chemical composition of the powder is shown in Sample designations in relation to the specific orientations, height positions and appropriate batches are summarized in −3 s−1 followed by basic parameter evaluations. Specimen thickness was measured with a micrometre while the other dimensions were measured using an optical measuring system. At least three M-TT specimens per location and orientation were tested with the use of a small-sized linear drive-based testing system  with 5 kN capacity and valid calibration certificate. Strain in the course of the miniature tests was measured using Mercury Digital Image Correlation (DIC) system . Prior to each test, strain calibration with certified calibration blocks was performed while sample preparation for DIC measurements consisted of several steps. Specimens were first cleaned using detergents, then a thin uniform layer of dull acrylic spray was used to cover the sample. A rough DIC pattern was achieved by “light” spraying of a black dull spray (graphite spray) that provided a high-contrast stochastic pattern with very small random distributed spots. In a few cases, full field measurement was performed with appropriate post processing to provide more detailed analysis on the strain distribution over the specimen active length. However, the DIC system was used in video extensometer mode and recorded the distance between two points representing the gauge length in order to enable large data processing. Three specimens per condition were tested, except the standard-size specimens where only one test was conducted due to space limitations. Then, 0.2% offset yield stress (YS), ultimate tensile strength (UTS), uniform elongation at maximum force (UE), elongation (EL) and reduction in area (RA) were evaluated. Dimensions post-test for reduction-of-area were determined with the use of a stereo-microscope. The data were evaluated in terms of engineering stress–engineering strain (here designated as extensometer strain).Details of the M-TT specimen geometry based on previous work ,8 are shComparison of standard sized round specimen with mini-M-TTs is demonstrated in 3. Imaging was conducted with the use of Zeiss Axio Observer.Z1m optical microscope  and with the use of a JEOL 6380  scanning electron microscope (SEM) that was used for microstructure observations as well as for fractography. Section cuts in XY, YZ and ZX planes were prepared by means of standard metallographic procedures and images are provided in The samples for microstructural evaluation were cut from tensile specimens’ shoulders with respect to the deposition planes. Samples were then embedded in resin and processed by means of standard metallographic preparation using grinding and subsequent polishing. The microstructure of IN-718 was revealed with electrolytic etching in an aqueous solution of 60% HNOThe 3D microscopy images in Fracture analyses on the IN 718 M-TT specimens typically revealed evidence of processed-induced porosity with fracture features centered on internal defects as shown in P and Q, with removal of the columnar structure  in the YZ and XZ plane. Section cuts in XY, YZ, and ZX planes are captured in 3D microscopy images in P heat-treated specimens, Coarse-grained microstructures resulted from the heat treatment of P and Q samples in the middle part of the bar are shown in The tensile test fracture surfaces of the Z-oriented heat-treated The scatter in the results could be attributed to both the difference between the cooling rates along the bar, samples’ orientation in the powder bed, and also to the microstructural features such as planar defects and entrapped pores. The effects of loading in the X and Z directions could include stress concentrations that provide an easier path for defect growth and coalescence during testing in the Z direction .The present work has built upon the significant previous work developed at COMTES FHT Inc. regarding the use of miniature M-TT samples to explore the location- and orientation-dependence of properties in a variety of AM-processed as well as conventionally-processed materials ,9,10,11 Post-treatment that included HIP homogenized the mechanical properties across all M-TT samples  and elimThe present work has successfully used miniature M-TT tension samples excised from bulk samples to investigate the location- and orientation-dependence of properties in comparison to bulk LPBF IN 718 in both as-deposited and post-treated conditions. This approach again provides a useful procedure to illustrate the potential differences in local properties from global properties obtained on bulk samples/parts.M-TT samples excised from the gage length locations of the bulk samples exhibited comparable results to standard bulk samples printed at the same time. The resulting offset yield strengths/UTS obtained for M-TT and standard specimens were 182/674 MPa and 182/660 MPa, respectively. These findings are consistent with previous work of this type on other AM-processed materials.The as-deposited material exhibited significant property anisotropy in the different orientations in addition to both location- and orientation-dependent properties. Difference between the tensile properties along the build direction and perpendicular to the build direction reached 15% for some locations, while samples excised along the build direction generally produced lower properties compared to those excised perpendicularly. Microstructure examination of the as-deposited material revealed coarsening of the columnar microstructure along the build direction, suggestive of heat accumulation during the build. This partially contributed to the differences in M-TT properties both along the build and in different orientations, although relatively homogeneous properties were obtained in the middle of the build. Fracture surface examinations revealed multiple examples of process-induced porosity at various locations on the fracture surface, although the remainder of the fracture surface exhibited very fine  dimples.Metallurgical examinations of the post-treated samples revealed preferential carbide precipitation along grain boundaries and removal of the columnar microstructure.The post-treated samples exhibited isotropic properties as well as significant reductions  in the strength  along with significant increases in the reduction in area at fracture for all of the M-TT samples in comparison to as-build state. The HIP post-treatment reduced the number of process-induced defects exhibited in the fractured samples, while the fracture surfaces continued to exhibit very fine (<1 micron) dimples.


Despite a growing body of research on the risks of SARS-CoV-2 infection during pregnancy, there is continued controversy given heterogeneity in the quality and design of published studies.We screened ongoing studies in our sequential, prospective meta-analysis. We pooled individual participant data to estimate the absolute and relative risk (RR) of adverse outcomes among pregnant women with SARS-CoV-2 infection, compared with confirmed negative pregnancies. We evaluated the risk of bias using a modified Newcastle-Ottawa Scale.We screened 137 studies and included 12 studies in 12 countries involving 13 136 pregnant women.Pregnant women with SARS-CoV-2 infection—as compared with uninfected pregnant women—were at significantly increased risk of maternal mortality ; admission to intensive care unit ; receiving mechanical ventilation ; receiving any critical care ; and being diagnosed with pneumonia  and thromboembolic disease .Neonates born to women with SARS-CoV-2 infection were more likely to be admitted to a neonatal care unit after birth ; be born preterm  or moderately preterm ; and to be born low birth weight . Infection was not linked to stillbirth. Studies were generally at low or moderate risk of bias.This analysis indicates that SARS-CoV-2 infection at any time during pregnancy increases the risk of maternal death, severe maternal morbidities and neonatal morbidity, but not stillbirth or intrauterine growth restriction. As more data become available, we will update these findings per the published protocol. Despite the ballooning literature regarding SARS-CoV-2 infection during pregnancy, it is difficult to synthesise the information and evaluate the overall quality of evidence given the heterogeneity in study design, selection of comparison groups, methods for assessing infection, population-specific baseline risks and definitions of key outcomes.Prior reviews based on published data have included limited data from low-income countries.We established plans for a sequential, prospective meta-analysis in April 2020 with a goal of better understanding the excess risks—or lack thereof—of COVID-19 during pregnancy.This individual patient data meta-analysis of unpublished and published data from a dozen studies includes more than 13 000 pregnant women and shows that COVID-19 during pregnancy increases the risk of maternal mortality, intensive care unit admission, receiving mechanical ventilation, receiving any critical care or being diagnosed with pneumonia or thromboembolic disease.Infants born to infected pregnant women were more likely to be admitted to the neonatal intensive care unit and to be born premature.In contrast to other reviews, we did not find any link between SARS-CoV-2 infection during pregnancy and an increased risk of stillbirth at or beyond 28 weeks’ gestation, nor any link with intrauterine growth restriction.Further, we include the first large set of pregnancy cohort data from sub-Saharan Africa.Global guidance has been equivocal on the potential risks of infection and benefits and safety of vaccination, and more than 80 countries do not currently recommend that all pregnant and lactating women should be vaccinated.Given the clear and consistent findings regarding the risk of COVID-19 infection during pregnancy, global effort to improve access to safe preventives and therapeutics is an urgent priority.Since early in the pandemic, a key question has been how SARS-CoV-2 infection affects pregnant women and pregnant people, given the physiological, immunomodulatory and mechanical changes that occur during pregnancy. A living systematic review published in February 2021 identified 47 studies comparing pregnant women with COVID-19 versus a contemporaneous or historical group of pregnant women without the disease.More recent electronic healthcare record studies from the USA and a multicountry cohort study found that pregnant women with SARS-CoV-2 infection had higher risks than uninfected pregnant women for pre-eclampsia, eclampsia, caesarean section, ICU admission, stillbirth, preterm birth and neonatal intensive care unit (NICU) admission.Despite the ballooning literature regarding SARS-CoV-2 infection during pregnancy, it is difficult to synthesise the information and evaluate the overall quality of evidence given the heterogeneity in study design, selection of comparison groups, methods for assessing infection, population-specific baseline risks and definitions of key maternal and child health outcomes.A unified, collaborative analytical plan is required to overcome many of these issues. Accordingly, we established plans for a sequential, prospective meta-analysis (sPMA) in April 2020 with a goal of better understanding the excess risks—or lack thereof—of COVID-19 during pregnancy.This analysis is part of a larger sPMA study that aims to answer epidemiological questions about COVID-19 and its association with maternal and newborn health by pooling data from independent studies using harmonised data definitions and an IPD meta-analytical framework to minimise data variability. The protocol for the sPMA was registered with PROSPERO (ID: CRD42020188955) on 28 May 2020; the full protocol has been published elsewhere.Eligible study designs included registries, single or multisite cohorts, or case–control studies enrolling pregnant women with suspected or confirmed COVID-19. To be eligible, studies must have had a defined catchment area, included at least 25 pregnant women with confirmed or suspected SARS-COV-2 infection and had a contemporaneously recruited comparison group of pregnant women who had not been diagnosed with COVID-19.Given the heterogeneity of study designs, we also applied participant-level inclusion and exclusion criteria. The SARS-CoV-2 infected group included pregnant women with a diagnosis during pregnancy or within 7 days of pregnancy outcome based on: (a) PCR testing or antigen testing; (b) WHO suspected case definitionFor this comparative analysis, we identified studies using two approaches. Studies were recruited into the sPMA via professional research networks and support from key stakeholder networks a priori,Data contributors shared deidentified IPD with the sPMA coordinating team based on a core variable list.10.1136/bmjgh-2022-009495.supp1Supplementary dataThe core variables for the larger sPMA study were established a priori along with the protocol.Data quality was assessed for each study by examining the distribution and frequency of each variable. We identified outliers and inconsistent values for key data points such as gestational age at birth, maternal age and neonatal birth weight and checked that the timing of outcomes was consistent with our definitions . For all published data, we also compared the distribution and frequency of outcomes to published manuscripts and resolved discrepancies through discussion with study investigators.We assessed the quality of individual studies, by outcome, based on criteria for participant selection and outcome ascertainment using an adapted Newcastle-Ottawa Scale.Critical care indicators included outcomes related to COVID-19 severity: admission to the ICU, receipt of critical care (defined as admitted to ICU or received ventilation or any site-defined indicator), any ventilation use and clinician-diagnosed pneumonia. Maternal mortality and morbidity outcomes included maternal death (due to any cause during pregnancy or 42 days post partum),Fetal and neonatal mortality and morbidity outcomes included stillbirth ,Adverse birth outcomes included combined extremely, very and moderate preterm birth , preterm birth , very low birth weight (<1500 g), low birth weight (<2500 g) and small for gestational age . Because we found very little difference in adjusted and unadjusted RRs within each site, we proceeded with the meta-analysis using unadjusted RRs to allow inclusion of studies with zero outcome event in either the exposed or unexposed group. We pooled the absolute risks of each outcome using the Freeman-Tukey double arcsine transformation with DerSimonian and Laird random-effects model; we calculated exact 95% CIs.2 statistic.We applied a two-stage IPD meta-analytical framework (accounting for site-specific clustering) to generate pooled absolute risks and relative risks (RR), along with 95% CIs, for each outcome. First, we estimated site-specific prevalence estimates for the infected and uninfected groups, as well as unadjusted and adjusted RR with 95% CIs. We originally produced unadjusted and adjusted RRs for each site contributing data. We adjusted for maternal age and, where available, pre-pregnancy obesity (pre-pregnancy body mass index (BMI) >30 kg/mIn cases of zero event for an outcome in the exposed or unexposed group, we applied a continuity correction of 0.5. Outcomes with zero event in both arms were omitted when estimating pooled absolute risk and pooled RRs because the infected and uninfected groups varied in size. All participants in a study were excluded from an analysis if more than 25% of participants were missing outcome information.Not all studies collected information about the date of COVID-19 onset (symptoms or test dates) and the date of each outcome; however, we performed a sensitivity analysis restricting the analysis to those studies with known date of onset as well as dates for three outcomes: preterm labour, preterm birth and moderate preterm birth. For preterm labour and preterm birth outcomes, we restricted the sensitivity analyses to women with gestational age of COVID-19 onset at less than 37 weeks and for moderate preterm birth by restricting the analyses to women with gestational age of onset at less than 34 weeks. For the outcome hypertensive disorders of pregnancy, we conducted a sensitivity analysis looking at diagnoses that occurred at or after COVID-19 diagnosis.To address concerns about the varying degree to which studies employed universal screening strategies and thus identified asymptomatic pregnant women, we conducted a secondary analysis restricting exclusively to symptomatic cases of COVID-19. Further, we conducted a sensitivity analysis comparing our results to those studies included in the PregCOV-19 Living Systematic Review that were eligible for the PMA but not successfully recruited to examine any major differences in results across seven common outcomes. Finally, we conducted a sensitivity analysis using different definitions of stillbirth to examine differences based on gestational age cut-offs. All analyses were performed using Stata (V.16), SAS (V.9.4) and R (V.4.2.0).Patients or the public were not involved in the design, conduct, reporting or dissemination plans of our meta-analysis. However, many contributing studies did involve patients and community stakeholders in the design and dissemination of their study results.et al’s living systematic review and identified five additional studies that were willing to participate in this round of the sPMAAmong the 26 studies that had prospectively joined the PMA study team, 16 had a study design that allowed for the comparison of SARS-CoV-2 infected and uninfected pregnancies. Six of these studies had completed data collection or were willing to contribute ongoing cohort data to the current analysis . We additionally contacted the corresponding authors of apparently eligible studies included in the Allotey We identified and deduplicated three participants who were included in both the current AFREhealth (Nachega) and PREPARE Uganda (Le Doare) data sets. No other overlapping participants were identified.12 studies conducted in 12 countries  ; Le Doare (2021) used the WHO case definition for probable cases of COVID-19 when testing was unavailable in addition to PCR and antibody testing at recruitment; and Ahlberg et alSelection of the SARS-CoV-2-negative group varied slightly between studies; seven studies defined SARS-CoV-2-negative pregnancy based on a single negative PCR test result , one study based the selection on repeated negative PCR tests throughout pregnancy (Akelo and Tippett Barr), two studies used a negative antibody test result  and one population-based pregnancy surveillance study ascertained SARS-CoV-2 infection using PCR and/or antibody testing at recruitment, followed by testing or assessment for probable diagnosis based on clinical concern (Le Doare). The timing of testing varied by study, but most studies included infections in all three trimesters  . Across the USA) .24 25 Acimesters .The pooled data included 1942 pregnant women with confirmed or probable SARS-CoV-2 infection during pregnancy or within 7 days of pregnancy outcome and 11 194 pregnant women who were either PCR negative at delivery ; antibody negative at delivery , both antibody negative and PCR negative at delivery ; antibody negative at an early ANC visit with PCR testing at delivery ; negative throughout pregnancy based on repeated PCR or antibody testing offered at ANC visits and delivery ; or who were antibody and/or PCR negative at recruitment in early pregnancy with no subsequent positive test  or clinical diagnosis of probable COVID-19  . The totICU admission  and ventilation . Across seven studies, about 4% of pregnant women with COVID-19 received any critical care (95% CI 0% to 13%) and they were more than five times more likely to receive critical care than their COVID-19-negative peers .Compared with pregnant women without infection, women with SARS-CoV-2 infection at any time during pregnancy had an increased risk of all outcomes related to critical care . The poomaternal death  as compared with uninfected pregnant women.While 10 studies collected data regarding maternal deaths, only three studies  recorded deaths during the study period and thus contributed information to the pooled estimate. All the remaining studies recorded zero death in both groups. Based on these three studies, women with SARS-CoV-2 infection had an increased risk of pre-eclampsia , pre-eclampsia or eclampsia  and thromboembolic disease  among pregnant women with SARS-COV-2 infection compared with those without. We also found an increased risk for hypertensive disorders of pregnancy  among pregnant women with SARS-CoV-2. Although most studies did not collect data on the timing of diagnosis of hypertensive disorders of pregnancy, we conducted this analysis again restricting to only those cases of hypertensive disorders of pregnancy diagnosed at or after a positive SARS-CoV-2 test; we found a similar increased risk but a wider CI . The risk for caesarean delivery was slightly higher among pregnant women with SARS-CoV-2 . While there was no significant difference in the risk of preterm labour across both groups overall, we find an increased risk of preterm labour  for pregnant women with SARS-CoV-2 onset before 37 weeks’ gestational age as compared with pregnant women without SARS-CoV-2 for those studies where data on gestational age at onset and preterm labour as a maternal morbidity are available . There was no difference between the two groups on the risk of other maternal morbidity outcomes .Regarding maternal morbidity, we found a greater risk for Among the five fetal and neonatal outcomes examined, we found an elevated risk only for NICU admission after birth among infants born to women with SARS-CoV-2 infection .preterm  and moderate preterm . A sensitivity analysis restricted to the seven studies recording the date of COVID-19 onset and preterm birth found a similar, although strengthened, link between SARS-CoV-2 infection and moderate preterm and preterm births. Infection during pregnancy was linked to a nearly threefold increased risk of moderate preterm birth  and a near doubling of the risk in preterm birth  (Infants born to women with confirmed or probable SARS-CoV-2 infection during pregnancy were more likely to be born to 2.29) . InfantsWe conducted a secondary analysis restricted to only symptomatic infections as compared with SARS-CoV-2 uninfected pregnant women; asymptomatic infections were excluded from this analysis. Similar to the primary analysis, we found that pregnant women with symptomatic infections were more likely than uninfected pregnant women to be admitted to the ICU, require ventilation or receive critical care. The risk of maternal death was also significantly higher. They were also more likely to be diagnosed with pneumonia, hypertensive disorders of pregnancy, pre-eclampsia, pre-eclampsia or eclampsia, or thromboembolic disease. They were more likely to experience preterm labour and to have a caesarean delivery or require an intrapartum caesarean delivery. Infants born to women with symptomatic SARS-CoV-2 during pregnancy were more likely to be born very low birth weight, low birth weight, moderate preterm and preterm; they were also more likely to be admitted to the NICU as compared with infants born to women without COVID-19 during pregnancy .Additional sensitivity analyses comparing the results of this meta-analysis to results of eligible studies in the PregCOV-19 Living Systematic Review and comparing pooled estimates among PMA studies using different definitions of stillbirth are presented in the We found the majority of included studies and outcomes to be at low risk of bias . Three sOur IPD meta-analysis confirms findings from a growing body of published literature that SARS-CoV-2 infection during pregnancy increases the risk of maternal death and imparts an increased risk for adverse health outcomes for both pregnant women and their fetuses and neonates.Compared with a contemporaneous group of pregnant women who tested negative for SARS-COV-2 infection, those with infection at any time during pregnancy had a higher risk for all critical care indicators, maternal mortality and several morbidity outcomes such as hypertensive disorders of pregnancy, pre-eclampsia or eclampsia, preterm labour and thromboembolic disease. Our findings are consistent with a living systematic review that included studies using concurrent or historical controls which found that women with COVID-19 during pregnancy had an increased risk of ICU admission and all-cause mortality.There is widespread disagreement about the biological plausibility that SARS-CoV-2 infection can induce hypertensive disorders of pregnancy, including pre-eclampsia. Some have hypothesised that altered ACE2 expression linked to COVID-19, or the systemic inflammation and hypercoagulable state common in COVID-19, may increase the risk of pre-eclampsia.Our analysis also revealed that neonates born to women with a SARS-CoV-2 infection had a significantly higher risk for a moderately preterm <34 weeks) or preterm (<37 weeks) birth, though we did not distinguish between spontaneous and iatrogenic preterm births. These findings are consistent with other studies. Based on 18 studies in the living systematic review, COVID-19 during pregnancy is linked with a 47% increased risk of preterm birth; SARS-CoV-2 infected women in the INTERCOVID study had a similar increased risk of preterm birth and 97% increased risk of having a medically indicated preterm birth. weeks orrisk of stillbirth at or beyond 28 weeks’ gestation, based on analysis of 78 cases of stillbirth (14 in the COVID-19 group). This is in contrast with the living systematic review that reported that women with COVID-19 had 2.84 times the risk of stillbirth as compared with their uninfected peers, although this was based on only 35 stillbirths (nine in the COVID-19 group).We did not find a link between SARS-CoV-2 infection during pregnancy and an increased Our study is intended to provide robust and high-quality estimates of the impacts of SARS-CoV-2 infection during pregnancy as compared with uninfected pregnancies. The IPD meta-analysis includes both unpublished and previously published data that were uniformly processed and analysed using a harmonised set of outcomes. We also included an expanded set of maternal morbidity outcomes that have not been extensively studied. The unpublished data include information from five countries in sub-Saharan Africa; no data from sub-Saharan Africa were previously available for inclusion in the current living systematic review.Our study is not without limitations. The possibility of selection bias remains, given that selection of pregnant women with a COVID-19 diagnosis depended on when and how the participants were tested for SARS-CoV-2; this changed over time across sites along with the availability of test kits. However, our risk of bias assessment carefully documents the methods for recruiting exposed and unexposed study participants and suggests that most participants across most studies were sampled in a representative way. Further, this analysis does not consider the differential impact of SARS-CoV-2 variants that have emerged since the onset of the pandemic because sequencing data was not available for individual patients in this study. Additionally, the majority of studies included in this analysis conducted recruitment only during a time period where a single variant was dominant at the national level . AnotherTaken together, this analysis of 12 studies including 13 136 pregnant women from 12 countries indicates that SARS-CoV-2 infection at any time during pregnancy increases the risk of maternal mortality, severe maternal morbidities and adverse newborn outcomes. These findings underscore the need for global efforts to prevent COVID-19 during pregnancy through targeted administration of vaccines and non-pharmaceutical interventions. Further efforts are needed to advance our understanding of the best clinical care and management strategies for SARS-CoV-2-infected pregnant women and their newborns. As more data become available, we will update these findings as per the published protocol.


Giant focal nodular hyperplasia (GFNH) is rarely seen in children, presenting complex diagnostic and management considerations. Pathognomonic radiographic findings can be absent in this population, and the nuances of pathologic examination are critical. We present a child with a GFNH involving the right side of the liver arising in the background of hepatic steatosis. The details of the diagnosis and therapeutic decisions involved in his treatment are discussed. Focal nodular hyperplasia (FNH) is an uncommon diagnosis in children . LesionsA 14-year-old boy presented with abdominal pain. Abdominal ultrasonography and CT scan revealed a liver mass 21 cm in greatest dimension . The rigHistologically, the non-neoplastic liver revealed moderate microvesicular and macrovesicular steatosis with mild inflammatory infiltrate. No significant fibrosis was noted. H&E-staining of the liver mass demonstrated proliferation of well-differentiated hepatocytes displaying nodular architecture separated by bands of fibrosis highlighted by trichrome staining. Endothelialization of sinusoids was observed with an incomplete pattern of CD34 staining. Reticulin staining emphasized that most cell plates were no more than 2–3 hepatocytes in thickness. Bile duct proliferation was noted at the periphery of the nodules, best appreciated with the aid of cytokeratin 7. Glutamine synthetase (GS) staining showed a geographic pattern with hepatocyte sparing close to the fibrous band . Glypica2). Given the patient’s symptoms and the possibility of malignancy, we proceeded with right hepatectomy. The patient was discharged home on post-operative Day 9 due to rehabilitation requirements, an episode of opiate-induced delirium and a surgical wound complication (seroma). Post-operative follow-up demonstrated a normalization of the patient’s AST and ALT. After unremarkable MRIs at 3 and 6 months, the patient was discharged from clinic.The geographic pattern of GS staining coupled with the imaging findings pointed to a probable diagnosis of FNH, although well-differentiated HCC and hepatic adenoma could not be entirely excluded. The etiology of the steatosis in this patient was unclear, but it may be associated with his increased body mass index  revealed a well-circumscribed nodular lesion  and declare no conflicts of interest.


It is unclear whether unintentional ingestion of povidone-iodine following its application to the oropharyngeal space could affect thyroid function.To examine thyroid function among individuals who regularly apply povidone-iodine throat spray for SARS-CoV-2 prophylaxis.We designed a case-control study to compare thyroid function among participants who received povidone-iodine throat spray three times a day for 42 days (‘cases’) and those who received vitamin C (‘controls’). Thyroid function was assessed by profiling serum TSH, free T3, and free T4; iodine status was estimated using serum thyroglobulin level, while infection status was determined by measuring anti-SARS-CoV-2 antibody against the nucleocapsid antigen. All measurements were performed in pairs, at baseline and 42 days later. Pre-post changes in thyroid function were compared between groups, before and after stratification according to baseline TSH quartiles.Post-hoc analysis did not reveal differences in thyroid function according to infection status.A total of 177 men (117 cases and 60 controls)  were included. Despite comparable demographics and clinical profiles, no clinically or statistically significant differences were observed in thyroid indices between ‘cases’ and ‘controls’ before and after stratification according to TSH quartiles. None of the participants developed symptomatic hypo- or hyperthyroidism throughout the study. Data from this study support the overall safety of povidone-iodine use in the oropharyngeal space for SARS-CoV-2 prophylaxis among individuals with normal thyroid function and subclinical thyroid disease. Povidone-iodine is a broad-spectrum antiseptic that is used to disinfect the oropharyngeal space . GuideliIodine is a micronutrient that is required for the synthesis of thyroid hormones . AlthougTo examine the impact of frequent and repeated use of povidone-iodine throat spray on thyroid function, we employed the resources of the DORM trial  and desiAll participants were men, aged between 21 and 60 years, who were recruited for the DORM trial during an outbreak of SARS-CoV-2 infection involving migrant workers in Singapore ,16. We i‘Cases’ were identified from participants randomized to receive povidone-iodine throat spray three times daily for 42 days who reported full compliance with the treatment protocol. At study entry, participants in this group were shown and taught the proper use of the throat spray before being dispensed a 50 ml bottle of povidone-iodine 0.45% . Compliance was assessed by examining self-reported compliance data captured using an encrypted mobile application (FormSG) developed by Singapore’s GovTech Data Science and Artificial Intelligence Capability Centre, and by measuring the residual volume of povidone-iodine at the end of the 42-day study. A participant was considered to be fully compliant if they did not miss any treatment dose and returned a bottle with <40% of the initial volume. ‘Controls’ were selected from age-matched participants who were randomized to receive an active comparator  but recorded a compliance rate of <30% which was verified by counting the number of unconsumed pills at week 6; the latter condition was added to derive expected thyroid function values that are closer to placebo estimates. The proportion of individuals with infection in both groups at baseline and on day 42 was balanced to account for possible confounding effects of acute infection on thyroid function. Blood pressure (mmHg) and heart rate (per min) were measured on recruitment. Body mass index (BMI) was calculated by dividing weight in kilograms by the square of height in meters. The trial was approved by the Domain-Specific Review Board, National Healthcare Group (2020/00561), the Ministry of Health, the multi-ministerial Joint Task Force, and was conducted under a Clinical Trial Authorization (CTA2000053) by the Health Sciences Authority which oversees all clinical trials in Singapore.Following absorption, iodine is metabolized to form thyroid hormones and excess iodine is stored as thyroglobulin . Serum Tn) and percentages (%). Continuous variables are expressed as mean (standard deviation) or median , and, based on their distribution, compared using the Student’s t-test and Mann–Whitney U tests as appropriate. Pre-post changes in individual thyroid indices were compared using the Mann–Whitney U test at baseline and on day 42, and in the adjusted analysis, through linear regression of the logged biomarkers or the logged ratio of final to baseline measurements. To examine whether individuals with subclinical thyroid disease could be more prone to changes in thyroid function, participants were categorized into quartiles based on their baseline TSH values. Changes in thyroid indices were compared between cases and controls within each quartile. To evaluate the impact of SARS-CoV-2 infection on thyroid function, participants were categorized into different seroconversion groups based on their baseline and day 42 serology status ; pre-post changes in thyroid indices were compared between these groups. Sample size calculation was not performed a priori and was based on pragmatic considerations, such as sample availability and funding. Post-hoc assessment of sample size demonstrated 75% power to detect a 20% difference in the geometric mean change in all four biomarkers over the study duration, which was an effect size considered to be clinically meaningful. SPSS Statistics version 27  and R 4.1.1 [Categorical variables are summarized as numbers . Despite regular use of povidone-iodine, no clinically and statistically significant differences were observed in thyroglobulin and in the pre-post changes in thyroglobulin between cases and controls   were included in this study. Cases and controls were comparable in terms of demographics, risk factors, clinical parameters, and SARS-CoV-2 infection status . The precontrols . None ofcontrols . Confidecontrols . No clincontrols .Findings from this study suggest that regular use of povidone-iodine for prolonged periods is not associated with thyroid dysfunction among individuals with normal thyroid function, nor in those with subclinical thyroid disease. No significant differences in thyroid function were observed between individuals according to their SARS-CoV-2 infection status. To our knowledge, this study is the first to rigorously examine the impact of povidone-iodine throat spray by measuring different indices of thyroid function and included individuals who were highly compliant with the treatment protocol and a control arm that would allow the detection of minor changes in thyroid function but within normal limits.Our findings are consistent with one study that observed no change in thyroxine levels among 24 men following irrigation of the oral cavity with povidone-iodine compared with those who irrigated with chlorhexidine gluconate . The lacThe SARS-CoV-2 virus enters cells using the angiotensin-converting enzyme type 2 (ACE2) receptor. This receptor is also highly expressed in thyroid glands, at levels greater than those present in lung tissues ,24. AmonSeveral limitations merit mention. First, the study included relatively young and healthy men but did not involve older individuals with various comorbidities which may affect iodine absorption and excretion. Second, although serum thyroglobulin did not differ between cases and controls, we are unable to definitively conclude that regular use of povidone-iodine did not result in iodine absorption as we did not directly measure levels of circulatory iodine. Third, we did not include those with symptomatic thyroid disease to conclude on the safety or harm associated with povidone-iodine use in these individuals. Data from this study support the overall safety of povidone-iodine throat spray for SARS-CoV-2 prophylaxis among individuals with normal thyroid function and those with subclinical thyroid disease.This study is a nested case-control study involving data from participants in the DORM trial. Consent was obtained before the participants entered this trial. The trial was approved by the Domain-Specific Review Board, National Healthcare Group (2020/00561), the Ministry of Health, the multi-ministerial Joint Task Force, and was conducted under a Clinical Trial Authorization (CTA2000053) by the Health Sciences Authority which oversees all clinical trials in Singapore.


The demonstration of the first enzyme-based electrode to detect glucose, published in 1967 by S. J. Updike and G. P. Hicks, kicked off huge efforts in building sensors where biomolecules are exploited as native or modified to achieve new or improved sensing performances. In this growing area, bionanotechnology has become prominent in demonstrating how nanomaterials can be tailored into responsive nanostructures using biomolecules and integrated into sensors to detect different analytes, e.g., biomarkers, antibiotics, toxins and organic compounds as well as whole cells and microorganisms with very high sensitivity. Accounting for the natural affinity between biomolecules and almost every type of nanomaterials and taking advantage of well-known crosslinking strategies to stabilize the resulting hybrid nanostructures, biosensors with broad applications and with unprecedented low detection limits have been realized. This review depicts a comprehensive collection of the most recent biochemical and biophysical strategies for building hybrid devices based on bioconjugated nanomaterials and their applications in label-free detection for diagnostics, food and environmental analysis. Since 1967, when S. J. Updike and G. P. Hicks showed the first enzyme-based electrode for glucose detection , sensorsModern biosensors are usually endowed with (i) efficient capturing of specific targets with no off-target interaction, (ii) transformation into electrochemical, electrical, optical, gravimetric or acoustic signals, (iii) sensitivity, short response time, reproducibility and low detection limit (LOD) and (iv) miniaturized components . In the To note, as the sensors get miniaturized to the nanoscale, the so-called “label-free” detection of molecular analytes becomes affordable. One first advantage of using labeled-free detection, indeed, is the possibility of revealing molecular targets which are not easy to tag or cannot be tagged at all; secondly, many targets cannot be labeled for they are found at very low amounts thus making labeling expensive and a nonsense strategy. In such a scenario, the interplay between biomolecules and nanomaterials becomes straightforward because at the nanoscale the interactions with the target analytes turn highly specific, strong and favored; moreover, these interactions become affected by other factors which are absent in bulk macroscopic devices, for instance the molecular conformation of the biomolecules. Under these conditions, the detection inspired by structure-based high affinity biorecognition turns on and accurate targeting of the analyte can be addressed with no off-target issues even at very low concentrations.−1–500 ng mL−1 with an LOD of 23 fg mL−1, which corresponds to target concentrations in the femto range (10−15 M) 3−/4-, thus leading to an increased or decreased rate of redox reactions on the electrode and therefore to changes in the voltametric and current signals. It must be noted that the signal changes that occur during the interaction between the aptamers and the sample containing the target molecules is very dependent upon the time, which usually spans within tens of minutes before reaching a stable signal. A main drawback that must be avoided comes from the non-specific binding of the target on the surface of the electrode via adsorption that leads to an increase in the background signal therefore decreasing sensitivity of the device. In all cases, this drawback is usually fixed by allowing blocking agent to adsorb over the electrode surface, e.g., surfactants, metal-specific thionic compounds as well as proteins such as casein and bovine serum albumin (BSA). This expedient allows the target molecules to interact exclusively with the aptamer probes.Most biosensors where aptamers and nanomaterials are physically integrated, so-called “aptasensors”, rely on an electrochemical setup that includes an electrode working as transducer and support for deposition of the nanomaterial, which improves the electrochemical properties of the underlying electrode and furnishes binding sites for the aptamers. In this configuration, the nanomaterial-bound aptamers act as receptors/probes that are freely accessible to capture and detect the target analytes. Namely, upon binding to the target the aptamers undergo a complementary base pairing that creates secondary structures, e.g., helical arms and single stranded loops, which in turn provide the information to form tertiary structures responsible for the high-specific and high-affinity binding to the target via weak forces. According to this structural plasticity, aptasensors show electrochemical performances that can be monitored via voltametric apparatus as, upon binding to the analytes, the molecular conformational change of aptamers alters the free surface of the electrode to accomplish an electron exchange, e.g., [Fe(CN)A representative example is reported by Beitollahi et al.  that dev2NPs to detect the epithelial sodium channel protein in urine [The aptasensor just described includes only three main components, i.e., the electrode (the transducer), a layer of NPs (the nanostructured support) and the aptamer . Likewise, the same simple though efficient configuration can be found in other devices having different components such as screen-printed gold electrodes coated with flower-like gold microstructures for detection of serpin in plasma  or coatein urine .2-IL and 2D nanosheets of molybdenum disulfide (MoS2), the former helping the nanomaterial to stabilize via electrostatic repulsion and avoiding aggregation. The resulting AuNPs-IL-MoS2 composite is dried onto a glassy carbon electrode and furnishes a proper binding surface for the adsorption of DNA nanotetrahedrons, which are obtained by warming and cooling of four different SH-DNA sequences. The thrombin analyte molecules can bind to the aptamers and the resulting thrombin-DNA nanotetrahedron complex is sandwiched using a third nanostructured composite AuNPs-Fe-MIL-88 where AuNPs are loaded onto hydrothermally synthesized Fe-MIL-88 metal-organic frameworks (MOFs), which in turn are labeled with a fifth DNA sequence recognizing the thrombin-DNA complex. The authors recalled two import features of such high-complex devices: first, in contrast to traditional stem-loop structures or linear DNA probes, the enhanced mechanical rigidity of the DNA nanotetrahedrons increases the accessibility of the target molecules and their loading amounts; secondly, this aptasensor results bind to many interfering substances even when their concentrations are greater than the concentration of thrombin. This aptamersensor shows an LOD of 56 fM within a large linear range of 0.298–29.8 pM thus representing a potential detector for ratiometric methods in clinical applications.As the complexity of the nanomaterial laying onto the electrode is increased or several nanomaterials are combined to produce more complex composites, the performances of the aptasensor are enhanced to reach very low LODs. For instance, the study by Xie et al.  is worth2-AuNPs for the double detection of zearalenone and fumonisin B1 mycotoxins in food [3O4-Cu2O and Ag-resorcinol-formaldehyde NPs-Ag nanodots (NDs) for detection of prostate specific antigen in serum [This aptasensor represents a prominent example of how nanomaterials, according to their intrinsic features such as hydrophilicity, high specific surface area and good electrical conductivity, can sensibly improve the electrochemical performances of detection without affecting the biochemical features of the attached aptamers. These properties reflect the high number of similar devices which have been realized in recent years. Additional examples where the glassy carbon electrode is coated with hybrid nanostructured composites include: reduced MoS in food ;  regulating the nucleation and growth rate of the Fe3+-catechol MOFs on the surface of the AuNPs. The presence of several -COOH groups within the framework complexes is exploited to link carcinoembryonic antigen-specific aminated aptamers (NH2-aptamers) via classical 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDS/NHS) crosslinking reaction to achieve a colloidal aptasensor with high sensitivity and selectivity, good biocompatibility and strong stability. These functionalized AuNPs can be used to coat a glassy carbon electrode and detect carcinoembryonic antigen in serum. Note that both the polydopmine moieties and the Fe3+-catechol MOFs accelerate the electron transfer on the electrode interface, increasing the amplification of the current signals and therefore enhancing the sensitivity of the device. The authors used such a complex aptasensor to detect the carcinoembryonic antigen in the concentration range of 1 fg mL−1–1 μg mL−1 with a LOD as low as 0.33 fg mL−1 and proposed it for detection of the carcinoembryonic antigen biomarker in early clinical diagnosis.In this context, another prominent example is reported by Li et al. for detecting carcinoembryonic antigen in blood serum . The apt4-2D-C3N4 photoanode with II type heterojunction to detect microcystin-LR in water [3-g-C3N4 heterostructures for detection of streptomycin in food [2-BiOI-BiOBr visible-light photosensitive material to detect streptomycin in food and serum [3O4 to detect organophosphorus pesticides in food [The aforementioned examples can be further extended if considering other types of electrodes, such as: screen-printed carbon electrodes coated with carbon-black NPs-AuNPs composites to detect Cd(II) ions in water  or coatein water ; Au elecin water ; indium  in food ; Au micr in food ; screen- in food ; indium nd serum ; screen- in food ; Pt elec in food ; Au elec in food ; Au elec in food ; Au elec in food ; and exo in food .In this context, an example of an aptasensor developed by Lv et al.  is signiAmongst biomolecules, proteins represent the larger and most intriguing group for their multiple biochemical, structural and functional features. Briefly, proteins, thanks to their self-recognition and self-assembling properties, can form macromolecular 3D globular or filamentous structures ranging from a few to millions in Daltons molecular weight . Solving2– groups located on the antibody surface. In these conditions, the polyethylene glycol molecules serve as spacers between the metal surface and the antibody molecules thus preventing nonspecific adsorption. As a voltametric apparatus, Au-screen-printed carbon electrodes are obtained through the electrodeposition of Au onto the surface of the carbon electrode followed by a coating with cysteamine, the latter acting as linkers between AuNPs and antibodies. The resulting modified electrode is used to detect via voltametric measurements the epithelial sodium-channel protein molecules in urine samples with an LOD of 2.8 × 10−1 ng mL−1 in the range of 9.375 × 10−2–1.0 ng mL−1 and is proposed as a biosensor for suspected hypertensive urine sample patients and an alternative method to replace the ELISA screening.Like aptamers, proteins have been widely exploited as probes in sensing applications where traditional voltametric electrode-based sensors are the most explored to reveal the presence of specific analytes. Antibodies are found in most of the biosensors reported in literature, which are referred to as “immunosensors”. The basic principle of immunosensors recalls the one exploited for aptasensors as it is based on a high specificity and high affinity binding to the target analyte that affects the electrochemical reaction rate on the electrode surface. Accordingly, coating the surface of the electrode with a blocking agent is one main condition to accomplish and to avoid non-specific adsorption and background noise. An example is shown by Hartati et al.  that rea2NPs-antibodies on screen-printed carbon, as follows: AuNPs electrodes for detection of Herceptin-2 in serum [3O4NPs-antibodies on Au electrodes to detect Siglec 15 protein in serum [2 nanoflake-chitosan composites functionalized with biotinylated antibodies for the detection of carcinoembryonic antigen in serum [This immunosensor involves the most common and simple configuration based on NPs to detect biomolecules and it is representative of many other similar studies such as those reporting on CeOin serum , divalenin serum , magnetiin serum , poly, where the high aspect ratio guarantees large, functionalized surfaces compatible with biological probes. In this regard, Zhao et al.  describee enzyme . Under tIn this context, other similar examples are worth mentioning for their use of both multi-walled and single-walled carbon NTs alone or complexed in nanostructures such as enzyme-immobilized single-walled carbon NTs for detection of glucose , vinyl f2 nanosheets  or surface-enhanced plasmonic resonance (SERS). Colorimetric, naked-eye methods able to detect analytes with good LOD values are also employed in these cases. Note that the labelling is required to detect the signal upon target engagement but exclusively regards the nanometric probes rather than the target analyte thus keeping the advantages typical of label-free methods.As a starting example, the study reported by Wang et al.  describeColloidal probes which tend to precipitate or aggregate upon binding to the target molecules represent a common strategy for being simple and easy to monitor with common lab equipment, such as fluorescence or UV-Vis spectrophotometers. In this regard, other similar aptasensors can be recalled, highlighting the importance and efficacy of such a strategy where the aggregation of nanomaterials triggers recordable optical changes upon detection of the analyte, for example AuNPs-mediated fluorescence of Rhodamine B to detect carbendazim in water  or sulfa−1  and 25.6 pg mL−1  within the detection ranges of 24–600 pg mL−1  and 64–1600 pg mL−1 . This strategy can be adapted to other nanomaterials such as the aptamer-coated AuNRs for enzyme-assisted detection of adenosine triphosphate, thus justifying its versatility and usefulness [In this context, another significative example is described by Yu et al.  that preefulness .−1 within a range 20–300 ng mL−1.Besides fluorescence-based detection to capture analytes through aptamers-functionalized nanomaterials, other easier colorimetric methods have been developed alongside allowing the biosensing without the need of instrumentations. Indeed, colorimetry detection comes with changes in the optical properties of nanomaterials when they move from non-aggregated, monodispersed nanometric objects to clusters that aggregate and/or precipitate in solution, which usually show a red shift of their absorbance spectrum in the visible region . As a tyBacillus carboniphilus on bacterial biofilms [Such an easy, simple method based on naked-eye colorimetric detection opens up many other possibilities as reflected by several similar examples published, e.g., AuNPs coated with unfunctionalized polyadenine aptamers to detect prostate specific antigen in serum ; AuNPs cbiofilms ; AuNPs cbiofilms ; and AuNbiofilms .2-aptamers targeting kanamycin to be linked to the NF membranes using amide bond. As a probe, AuNPs are synthesized chemically and coated via adsorption with a complementary single-stranded DNA (cDNA) of the kanamycin aptamer and left to hybridize with the functionalized NF membranes by taking advantage of the hydrogen-based hybridization between the two nucleic acid strands. Because the aptamer shows higher affinity to the kanamycin molecules than to its cDNA, in the presence of antibiotic the cDNA is replaced, leading to detachment of the cDNA-decorated AuNPs probes from the strips; bleaching is observed using a visual color inspection and UV-Vis spectrophotometry. The authors used this aptasensor to detect kanamycin residues in food samples with a tested LOD of 2.5 nM in the working range of 2.5–80 nM. Based on the same principle, similar devices can be found as those reporting on AuNPs functionalized with truncated aptamers run onto a nitrocellulose membrane through lateral flow assay to detect oxytetracycline in food [Other colorimetric approaches based on the aggregation of NPs have been developed to realize a ready-to-use and portable device with quick naked-eye recordable results. An example worth mentioning is the aptasensor proposed by Abedalwafa et al.  as the s in food  and AuNP in food .The ease of making these kinds of colloidal optical sensors comes along lower LOD values, therefore showing decreased sensitivity when compared with the electrochemical counterparts. However, like the electrochemical aptamers based on voltametric measurements, the combination of colloidal probes with more efficient nanomaterials helps improve the performance of the resulting aptasensor as reported in many cases. The first example to be recalled is described by Chinnappan et al.  and reli2 nanosheets-based AuNPs aptasensor to detect the activity of the alkaline phosphate and the mycotoxin ochratoxin A in food [3O4-GO-assisted AT-rich three-way junctions DNA-stabilized CuNPs to detect isocarbophos in food and water [Accordingly, several similar studies have been described showing the advantage of having a nanometric 2D platform for fluorescent or colorimetric detection of analytes, e.g., enzyme-assisted, MnO in food , Fe3O4-Gnd water  and zircnd water .As reported for aptamers, proteins, antibodies and enzymes are very attractive when combined to nanomaterials and build colloidal or soluble sensors with special optical features. If compared to single-stranded DNA or RNA probes, the protein surface available for functionalization is much more feasible and enables multiple chemical functionalities to coexist on the same molecule simultaneously. The surface versatility makes proteins ideal building blocks for realizing complex nanostructures soluble in hydrophilic media even when combined to hydrophobic nanomaterials.A proper example is described by García-Rubio et al.  where Au3O4NPs-antibodies for detection of glyphosate in tap water [3O4NPs-antibodies for detection of prostate-specific antigen in serum [According to the same simple strategy, many other nanometric immunosensors have been proposed that exploit NPs conjugated with antibodies, as for instance magnetic Au-coated Feap water ; AuNPs-ain serum ; AuNPs cin serum ; and magin serum .−1 with a working range of 0.15–10 ng mL−1, thus proving to be much more sensitive than commercially available immunoassays even without the use of fluorescence-enhancing nanomaterials.The enhancing or quenching effect of nanomaterials based on their plasmonic behavior is found to be useful, taking advantage of 2D nanostructures as reported for GO sheets. GO, similar to metal NPs, triggers significant changes on the physical properties of molecules thus becoming a suitable tool for detecting low concentrations of analytes via optical methods. In their study, Ortiz-Riaño et al.  demonstr2-Pt-Fe2O3 polycaprolactone micromotors for detection of bacterial lipopolysaccharides [The quenching effect induced via 0D and 2D nanomaterials is a very common tool exploited in immune-based sensing and is reported by other studies, as for instance antibodies-conjugated CdSe-ZnSQDs and AuNRs to detect porcine reproductive and respiratory syndrome virus in swine serum  and rhodcharides .2) shell to grow. The silica shell is obtained by mixing the nanobeads in ammonia and tetraethyl orthosilicate before the addition of (3-aminopropyl)triethoxysilane to produce the silica surface functionalized with -NH2 groups. Following the treatment with glutaric anhydride, the silica-coated nanobeads are decorated with many -COOH functional groups that allow sheep anti-human hemoglobin polyclonal antibodies to be covalently linked onto the surface through EDC/NHS crosslinking chemistry, resulting in the final fluorescent bioconjugated QDs sensors. The same procedure is applied to create fluorescent QDs conjugated to rabbit anti-deoxyribo-nucleoprotein antibodies. The authors prepared a complex device for lateral-flow immunoassay using a nitrocellulose membrane as a physical support, which is divided into a sample loading zone and a revelation zone. Both the bioconjugated QDs are sprayed onto the absorbent nitrocellulose membrane; similarly mouse anti-human hemoglobin and anti-deoxyribo-nucleoprotein-bovine serum album antibodies are adsorbed onto the revelation zone. In the presence of hemoglobin, a complex is formed with the QDs conjugated with anti-human hemoglobin polyclonal antibodies that leave the adsorption zone and migrate towards the revelation zone forming a sandwich-like structure with the mouse anti-human hemoglobin molecules and resulting in a clear luminescent band onto the nitrocellulose membrane; conversely, without hemoglobin molecules the sandwich structure is not formed and only the control luminescent band is revealed due to the formation of a complex between the QDs conjugated with the rabbit anti-deoxyribo-nucleoprotein antibodies and the anti-deoxyribo-nucleoprotein-bovine serum album antibodies adsorbed onto the revelation zone. The device has been proposed as a signal-amplification probe for a lateral-flow immunoassay biosensor for the accurate detection of biomarkers such as the glycated hemoglobin that is considered as the gold standard for long-term glycemic control in diabetes mellitus.Optical immunosensors based on papers and pads for visive colorimetric and/or fluorescence detection are also reported. An example describes how hydrophobic QDs can be coupled to protein moieties to increase both the solubility and fluorescence and detect glycosylated hemoglobin in blood samples as reported by Li et al.  Figure . In this3O4 NPs capped with a gold shell to detect the human growth hormone in plasma [Further examples must be recalled that take advantage of using proteins and nanomaterials coupled to optical methods and or characteristics to reveal analytes, as for instance those reported for a multiplex lateral flow lateral flow assay device onto nitrocellulose membrane and antibodies-functionalized AuNSs for detection of metalloproteinase-9, S100 calcium-binding protein B and neuro-specific enolase in blood , antibodn plasma  and antin plasma .Prevention of diseases in terms of tracing and monitoring represents the evergreen desire to take care of all pathological conditions, from cancers to infectious diseases, especially in cases of highly contagious organisms.In this context, the recent and still current diffusion of the SARS-CoV-2 virus and its derivatives that quickly spread worldwide from January 2020 represents the prime example for at least two main reasons: first, infectious diseases usually move across the early to late stages of development inside the host organism by initially being latent and difficult to detect before becoming aggressive and causing severe health conditions; secondly, the development of pharmaceutical therapeutics takes time, usually years, to achieve final approval by regulatory institutes. Throughout 2022, local, national and global initiatives such as the World Health Organization (WHO) have unceasingly expressed main concerns regarding the SARS-CoV-2 pandemics especially related to its variants. The WHO, for instance, placed the COVID-19 disease within the list for “Prioritizing diseases for research and development in emergency contexts”, which also include pathologies caused by Ebola and Zika amongst many others  while up2 by means of photolithography and containing micrometric channels. These electrodes are modified through dielectrophoresis with the semiconducting single-walled carbon NTs and annealed at a high temperature before the use. The carbon NTs act as supporting nanomaterials to covalently attach antibodies targeting the SARS-CoV-2 spike protein or the nucleocapsid protein to the FET via the EDC/NHS coupling chemistry. The authors tested the efficacy of both the spike- and nucleoprotein-specific immunosensors on remnant nasopharyngeal swab samples by adding a few microliters to the antibody-functionalized devices and incubating a few minutes before rinsing with water to remove unbound virus particles. To note, the electrochemical FET performances in water show that these immunosensors can detect SARS-CoV-2 viruses very quickly, within 5 min, using both the anti-spike and anti-nucleocapsid protein antibodies-functionalized carbon NTs in clinical samples without prior sample processing. To note, the FET device functionalized with the anti-spike antibodies performed better than those functionalized with anti-nucleocapsid antibodies in discriminating positive and negative nasopharyngeal samples and resulted in being less susceptible to nonspecific species. The authors reported very low LOD values of 0.55 fg mL−1 and 0.016 fg mL−1 for the anti-spike and the anti-nucleocapsid antibodies devices, respectively, therefore being suitable for clinical diagnostic applications in detecting virus particles or for realizing multiplex devices for other kinds of infections.In such a huge and broad context, it now becomes clear that methods for preventing highly diffusive diseases through detection and monitoring of the etiological agents, even at the very early stages, are the missing weapons we need to realize. The following examples are not intended to comprehensively cover the relentless growth of papers reporting on detection strategies for SARS-CoV-2 but instead aim at highlighting how very different detection strategies can be used for sensitive label-free detection based on bioconjugated nanomaterials. It must be noted that the examples described herein refer to devices able to detect whole, intact viral particles rather than single, isolated molecular components of the organism. A proper starting study is described by Shao et al. , a studyIt must be recalled that other carbon allotropes can be exploited as supports for antibodies bioconjugation and the realization of advanced immunosensor devices. Similar FET devices, for instance, can be obtained by taking advantage of 2D graphene sheets conjugated with antibodies and used to detect viral particles without sample pre-treatment or labelling .−1 and 0.77 fg mL−1, respectively, as well as in serum and blood with LOD of 7.60 fg mL−1 and 0.10 pg mL−1, respectively. The ability of this immunosensor to detect whole viral particles from saliva samples extruded from COVID-19 patients might hold promising potential for the sensitive screening of symptomatic and asymptomatic individuals during early infection.When speaking about ultrasensitive methods for detection of analytes, Raman spectroscopy stands as one of the most useful and straightforward technique. In this context, the SERS effect that occurs on nanostructured surfaces is highly desired, for it can amplify the signal coming from the analyte or the probes in contact with the nanostructure. A study relying on this approach is reported by Zhang et al.  that pre2 functional groups on the surface. Further treatment with silane and glutaraldehyde is required for a covalent immobilization of the while fiber structure. As plasmonic elements, AuNPs are chemically synthesized by reducing agents before being immobilized on the U-bent fiber optic probe and used as supports to covalently conjugate antibodies specific for the SARS-CoV-2 nucleoprotein. The antibodies-immobilized probes are then treated with bovine serum albumin to avoid nonspecific interactions between the viral particles and the AuNPs. In this configuration, when the AuNPs-bound antibody probes are exposed to the virus they undergo a modulation of the light transmitted and passing through the U-shaped fiber; during this process, the underlying AuNPs can amplify the interaction signal that enhanced light interaction resulting in an increase in intensity. The authors used their device to be very cost effective with minimum logistics and without the need of trained personnel. Furthermore, they argued that the device can be simply connected to a national health services system for reporting, self-analysis and limiting the COVID-19 outbreak.Accounting for the plasmonic behavior typical of metal nanostructured objects, other interesting approaches have been developed for the ultrasensitive detection of SARS-CoV-2 particles from real samples. Coupling between plasmons and optical fibers is one of the most intriguing as the light travelling across an optical fiber is not affected by electromagnetic interference and, as reported for U-shaped configurations, they are the most efficient to achieve localized plasmon response and provide the quickest response. In this context, the immunosensor described by Hadi et al.  and base−15 moles) of analyte to be detected. Many of the reported examples show very high selectivity for the target even when tested in real complex media such as serum, natural water, urine and saliva, and do not require pre-treatment of the sample therefore helping the realization of simple, ready-to-use devices; furthermore, many of these examples can realize detection within a few minutes.Label-free detection based on hybrid nanostructured materials where biological moieties are used as probes is rising as one of the main strategies to achieve this goal. This review summarized several examples of label-free devices containing biomolecular components, mainly nucleic acids and proteins, and nanostructured materials with 0D to 2D shape, highlighting how both the electrochemical and optical measurements can reach ultrasensitive detection, from molecules to whole organisms, even at very low LOD values down to femto moles (10Aptamers and immunoglobulins represent the two main biological components integrated in such devices for they highly ensure selectivity with no or little off-target engagement. It seems, however, that when approaching attempts of detection of viral particles, such as SARS-CoV-2, the immunosensors, i.e., devices using antibody probes, are the most exploited; conversely, aptasensors are still in a childhood stage and further development is needed even though examples are already demonstrating their efficacy for accurate and sensitive detection . HoweverNow as never before, the importance of detection, tracing and monitoring activities are particularly relevant and are targeted worldwide, and the interest in obtaining advanced devices to achieve the quick and highly performing sensing of molecules and organisms grows year by year. The environmental changes, pollution and the rising of new lethal diseases caused by etiologic agents represent primary challenges for many countries. As an example, the European Commission recently adopted a set of proposals known as “Green Deal” whereby healthy and affordable foods as well as fresh air, clean water, healthy soil and biodiversity represent some of the beneficial effects it wants to achieve by taking advantage of the “NextGenerationEU Recovery Plan” and the seven-year budget Research & Innovation programme 2021–2027 “Horizon Europe” . To get


Objective: Bimanual training has recently attracted attention as it achieves better outcomes as compared to repetitive one-handed training. This study compared the effects of three MI tasks with different visual feedback. Methods: Fourteen healthy subjects performed single hand motor imagery tasks while watching single static hand , single hand with rotation movement (rmMI), and bimanual coordination with a hand pedal exerciser (bcMI). Functional connectivity is estimated by Transfer Entropy (TE) analysis for brain information flow. Results: Brain connectivity of conducting three MI tasks showed that the bcMI demonstrated increased communications from the parietal to the bilateral prefrontal areas and increased contralateral connections between motor-related zones and spatial processing regions. Discussion/Conclusion: The results revealed bimanual coordination operation events increased spatial information and motor planning under the motor imagery task. And the proposed bimanual coordination MI-BCI (bcMI-BCI) can also achieve the effect of traditional motor imagery tasks and promotes more effective connections with different brain regions to better integrate motor-cortex functions for aiding the development of more effective MI-BCI therapy. Clinical and Translational Impact Statement The proposed bcMI-BCI provides more effective connections with different brain areas and integrates motor-cortex functions to promote motor imagery rehabilitation for patients’ impairment.Motor imagery-based brain-computer interface (MI-BCI) currently represents a new trend in rehabilitation. However, individual differences in the responsive frequency bands and a poor understanding of the communication between the ipsilesional motor areas and other regions limit the use of MI-BCI therapy.  I.Recent studies have reached a consensus on the feasibility of motor imagery-based brain-computer interface (MI-BCI) for stroke rehabilitation applications, especially in upper limb rehabilitation A major challenge that limits the use of MI-BCI therapy in stroke patients is the use of channel-based information to discriminate MI patterns. This is because the responsive frequency bands of MI-BCI are not consistent between subjects and within subjects Although MI-BCI can improve the movement function of the ipsilesional motor cortex, brain connectivity analysis can provide more clear evidence to support the notion that performing the unilateral motor imagery tasks benefits the connections between the ipsilesional motor region and other brain areas.In this study, we designed a bimanual coordination MI-BCI (bcMI-BCI) that incorporates the BAT concept to study brain connectivity when performing unilateral motor imagery tasks. In addition, we compare bcMI-BCI with another two conditions: traditional signal static hand and single hand with rotation MI-BCI for the differences in brain connectivity, analyzed by transfer entropy (TE) This study will compare three different motor imagery tasks: (a) Traditional MI (tMI) (b) Rotation Movement MI (rmMI) and, (c) Bimanual Coordination MI (bcMI). For the results of this study, we would approve the effect of bcMI and rmMI by comparing with tMI using machine learning algorithms: common spatial pattern (CSP) for feature extraction and linear discriminant analysis (LDA) for MI classification. Furthermore, this study aims to explore brain connectivity while performing ipsilateral motor imagery tasks in tMI, rmMI, and bcMI. We hypothesized that brain connectivity analysis would reveal that the proposed novel rehabilitation approach of bimanual and rotated motor imagery effectively reorganizes neurons for motor function and reconnects other brain regions to achieve complete hand coordination function.II.A.Fourteen healthy adults were recruited to participate the experiments . All subjects are right-handed with no mental or medical illnesses and no previous experience with MI-BCI. Before the experiment, they signed a consent form indicating that they have a clear understanding of the experimental protocol, which was approved by the Research Ethics Committee for Human Subject Protection, National Chiao Tung University.B.Three types of motor imagery tasks as  were desDuring the MI experiment, all participants sat in an electrically shielded cabin watching a 24” monitor from a 1-m distance. As shown in C.EEG data were acquired with a 32 Ag/AgCl electrode cap referenced to the average of left and right mastoids and a SynAmps amplifier  digitized at a 1000-Hz sampling rate. The contact impedance between all electrodes and the skin was maintained at D.Since the mu  ERD and ERS are the main index for MI-BCI E.After EEG signal preprocessing, a typical signal processing pipeline is used for feature extraction and classification approaches for MI-BCI verification and applications. Currently, common spatial pattern (CSP) is one of the most common feature extraction methods used in MI BCI Two windows from the multivariate signal are represented as Subsequently, linear discriminant analysis (LDA) is applied to identify the EEG signals in each sub-band spectrum F.Brain connectivity analysis, particularly functional and effective connectivity, represents one of the most promising approaches to investigate the brain operation of MI and improve MI-BCI performance TE is a model-free measure based on information theory and can be used to calculate the amount of directed information transmission between two systems The conditional probability If two different processes were not dependent, there is no transfer of information, and it would cause TE indicates directional information flow and was inherently asymmetric; thus,The estimation of parameters and the calculation of TE were performed using TRENTOOL (Version 2.0.4) In this study, the estimation of effective connectivity between the selected channels have been conducted over various MI conditions and different EEG frequency bands, alpha (8-13Hz), beta (14-30Hz), and gamma (31-50Hz) bands, for both left- and right-hand motor imagery. The TE mean values and standard deviations of any two of the selected channels’ connectivity pairs (source to sink) across all subjects have been calculated for each MI task under three frequency bands when they performed left- and right-hand imagery.G.The TE values of the MI stage in three EEG frequency bands were normalized by subtracting the mean baseline (fixation stage) for the motor imagery of both hands under each MI condition. Therefore, in this study, the normalized TE values are relative-TE values, which represent the changes with respect to the baseline period. Nine electrodes  were selected to investigate brain connectivity during the motor imagery stage of the three MI conditions. For further analysis, subjects’ TE values of the connectivity pairs in each MI task were averaged and tested using the Wilcoxon signed rank test with an FDR correction (p<0.05). Given the complexity of the MI task, two comparisons  were taken in this study to test whether the rotation movement and bilateral coordination can evoke more brain interaction when performing single hand motor imagery tasks.III.A.B.To confirm whether MI-BCI performance is influenced by the rotation movement (rmMI) and both hands representation (bcMI) designed in this study, the CSP and LDA were applied to test the classification accuracy of these three MI conditions.The optimal value for the parameter ‘m’ in the CSP algorithm was determined by testing values in the range of 1 to 8 to maximize the classification accuracy. For each value of m, after the CSP projection, 10-fold cross validation was applied to data 100 times. The test results for all subject under the three MI conditions were averaged and these results are shown in After extracting the EEG features by CSP, LDA was applied to perform the classification for all the three MI conditions. Here, 10-fold cross validation was applied 100 times. The above accuracy results of LDA classification with CSP filters among three MI tasks indicate that the newly proposed MI conditions can also evoke similar responses as compared to the traditional MI condition as all participants were able to correctly perform the motor imagery under all conditions without any prior experience.C.In The pairwise connectivity measures that achieve statistical significance (FDR-adjusted p<0.05) during tMI and rmMI conditions are shown in As shown in In IV.In this study, novel MI-BCI, bimanual and rotated motor imagery is proposed to explore brain connectivity when performing ipsilateral motor imagery tasks. The basic MI phenomena were supported in all three conditions based on the ERD changes in the contralateral alpha frequency band, and the results were classified by CSP and LDA.The subjects performed the ipsilateral motor imagery task with bilateral or rotation visual feedback. Based on these MI criteria, the bcMI-BCI can evoke increased brain connectivity, especially when performing the left-hand imagery task.As shown in (1)mutual communication occurs between the prefrontal area (F3 and F4), which indicates that the nonimagined hand  and imagined hand  interact with each other for motor preparation and bilateral arm coordination after receiving visual feedback;(2)communication occurs from the parietal region to bilateral frontal area (Pz to F3 and F4), which indicates that rotation of the hand-bike kept providing the spatial location information of each hand for motor planning. Because the neural patterns are influenced by the function of handedness, less connectivity in right hand condition could be due to all participants are the same right-handed and can image more efficiently Based on our experimental results, this study supports the following conclusions:In summary, this study demonstrates the dynamic connectivity when using bcMI and could be used as the index to investigate the reorganization and reconnection of motor cortex with the MI rehabilitation approach.V.Although the current study validated the brain connectivity while executing bcMI to investigate the reorganization and reconnection of the motor cortex with the MI rehabilitation approach, the present demonstration still has some limitations.First, the connectivity of bimanual and rotated motor condition could be still influenced by the methodological limitation. Therefore, the weighted effect of rotation movement or solely the bimanual component should be further investigated with bimanual static imagery.Second, although the study has yielded consistent results across the subjects participating in the experiment, more testing samples and comparison tests were still needed to confirm effectiveness and robustness.Third, our findings exhibit the ordinary people’s brain connectivity of bcMI as the index; however, for further MI rehabilitation, the future work of the study should collect more data from the patient subjects, such as the stroke patients, for validation.


Patients with Schizophrenia Spectrum Disorders (SSD) often lead unhealthy lifestyles with higher prevalence of obesity and unfavourable cardiometabolic parameters with less life expectancy and often worse quality of life compared with general population.Evaluate the effectiveness of a combined intervention of exercise and psychoeducation in 48 SSD outpatients with metabolic syndrome (MetS), treated with second-generation antipsychotics and also aimed to explore if the effect persisted in a long-term follow-up of 24 months.The intervention included a 12-week aerobic exercise program and a session of lifestyle psychoeducation. Effectiveness was measured in terms a wide range of outcomes involving physical and psychological health, functioning, quality of life, physical activity and changes in motivation to exercise in the context of the self-determination theory.The active intervention group showed benefits after Bonferroni correction over clinical global impression, identified motivation to exercise and changes of physical activity pattern. Maintenance of effects after 24 months of follow-up was observed for identified regulation to exercise and also for negative symptoms of psychosis (Table). Table. Effects assigned-group/time-over *p≤0.05 **p≤0.01.A combined intervention on SSD outpatients with MetS showed effectiveness over several clinical parameters and functioning. Therefore, should be considered an essential part of the integral treatment in mental health services for SSD patients.No significant relationships.