The purpose of this study was to assess the performance of ultrasonic (US) vitrectomy devices by quantifying and comparing its impact on extracted vitreous properties to conventional pneumatic blade (PB) cutters using micro-extensional rheology. US vitrectomy is a new technology that offers an alternative to PB cutters used in vitreo-retinal surgeries.Thirty-six porcine vitreous samples were extracted using US and PB cutters. Each sample was kept at 4°C and tested within 24 hours postmortem and 4 hours post-vitrectomy. A recently developed micro-extensional rheology technique is used to infer the relative protein fragment size of extracted vitreous by quantifying the extensional relaxation time.US-extracted vitreous exhibited extensional relaxation times orders of magnitude lower than PB-extracted vitreous . Relaxation time is directly correlated to the fragment size of the collagen fibers in the vitreous. The formation of beads-on-a-string droplets within the PB samples indicates the presence of larger collagen fragments. These droplets were not seen on US samples.This new micro-extensional rheology technique can identify significant differences in physical properties of extracted vitreous. Long relaxation times and beads-on-a-string droplets within the PB vitreous samples indicate larger protein fragments compared to the US samples.Higher fragmentation of vitreous and lower extensional relaxation times may improve retina safety due to a reduction in vitreo-retinal traction resulting from the continuous shear action and aspiration applied by ultrasonic vitrectomy technology. Vitreous is a transparent gel-like substance found between the lens capsule and retina. It is composed of an inflated and highly hydrated collagen matrix with 99 wt.% water, 0.9 wt.% salts, and less than 0.1 wt.% collagen and hyaluronic acid.–Conventional pneumatic blade (PB) cutters use a “guillotine-style” cutter to aspirate vitreous out of the eye . These c––,Rheological study of the vitreous dates back to Lee and colleagues in 1992.Extensional behavior of protein solutions is characterized by the formation of fluid structures where elastic protein interactions dominate the fluid flow.Pig vitreous was used as a tissue analog for human vitreous, extracted from eyes using two different vitreous cutters: a PB cutter  and a US cutter . Freshly harvested, unscalded, enucleated pig eyes were purchased from Sierra Medical Supplies . Preparation of each eye was performed under a surgical microscope . Eyes were acquired within 24 hours postmortem, and the tests were performed within 8 hours of receipt and 4 hours post-vitrectomy to ensure consistency in results. All eyes were stored at 4°C prior to the experiments.The eyes underwent an open-sky pars plana vitrectomy while secured to a custom polystyrene holder. The procedures were conducted using the Bausch & Lomb (B&L) Stellaris PC Vision Enhancement System with hardware and software modifications to provide ultrasonic vitrectomy drive capability (Bausch & Lomb). A trocar was placed temporally 4 mm behind the limbus followed by a temporal corneal incision created with a 2.8 mm keratome blade. Surgical ophthalmic scissors were then used to remove the cornea by cutting through the corneal incision and following the edge of the corneoscleral limbus. Then the lens was squeezed out of the eye in one whole piece and the capsule was removed. Minimal iris manipulation was attempted to avoid contaminating the subsequent vitreous samples with pigment or other tissue fragments. Each probe was introduced via the pre-placed trocar to perform the vitrectomy and collect the undiluted chopped vitreous samples into sterile “BD Vacutainer Glass Serum” tubes  using a vitreous trap technique.The dual-action 23- and 25-gauge PB probes were tested at 100, 300, and 600 mm Hg and at 15,000 cuts per minute (cpm). The 23- and 25-gauge US probes were tested at 60 µm stroke and at 100 and 300 mm Hg vacuum setting. Flowrate is the factor that determines the vitreous fragment size at a given cut rate. Due to the US cutter's open smaller port design and larger inner needle lumen, the flowrate at 300 mm Hg is comparable to the upper limit of the PB cutter achieved at 600 mm Hg . Furthermore, the US cutter is not used in surgeries above 250 mm Hg. Therefore, conducting this experiment with US probes at usable vacuum ranges and comparable port flowrates was found to be adequate. The US cut rate is nominally 38 kHz and 41 kHz for the 23- and 25-gauge needles, respectively. Considering one cycle to be equivalent to one cut, these cutter frequencies correspond to cut rates of more than 2 million cpm for the US cutters.To analyze the extensional rheological properties of small samples, we adapted a simple rheometric method to apply a large strain rate to very small sample volumes.−1 shutter speed captured the evolution of this liquid bridge. The high-speed images were converted to binary in ImageJ, and the evolution of the fluid structure over time was measured using an edge-finding program in Matlab.As the sample dripped from the probe to the rest of the droplet, an elasto-capillary liquid bridge formed between the probe and sample droplet. A high-speed camera  set to 8000 frames per second and 1/10,000 sE) of the elasto-capillary regime was calculated for each trial . Twelve samples were tested after extraction with the US cutter. Twenty-four samples were tested after extraction with the PB cutter see the .The extensional rheology behavior varied dramatically between the US and PB cutter vitreous samples , 4. The Another characteristic difference between the PB and US samples was the beads-on-a-string structures that were only observed with the PB samples see c; 165 msP < 0.001) with up to two orders of magnitude in difference. The inertia-capillary regime of the US samples dominated the flow, and the elasto-capillary liquid bridge existed for only a few image frames in each trial, indicating the protein interactions had little effect on the flow in these samples. The relatively high extensional relaxation time for PB samples culminating in beads-on-a-string structures indicate the elasto-capillary effects of the protein interactions were significant. This is an indication of relatively large protein fragments and high cross-linking of the proteins within the samples.Extensional relaxation times  averagedP = 0.004 and P = 0.018, respectively) and PB 25-gauge samples (P = 0.002). The vacuum pressure did not have a significant impact on the average relaxation time of the PB 23-gauge samples (P = 0.112).Higher vacuum pressure resulted in significantly higher average extensional relaxation time in US 23- and 25-gauge samples (P = 0.032), but not at 100 mm Hg (P = 0.548). For PB samples, the needle gauge did not significantly impact the average extensional relaxation time at any of the three vacuum pressures .Smaller needle gauges exhibited higher average extensional relaxation times for US samples at 300 mm Hg  evaluated the US vitrector by qualitatively examining the histopathological changes in the vitreous after vitrectomy with US and PB vitrectors in pig cadaveric eyes, live swine, and human cadaveric eyes.The effects of vacuum pressure and probe gauge on extensional relaxation time were outweighed by the effect of cutting method (US versus PB). Generally, it is expected that higher pressures, which correspond to higher flow rates,The results of this study support the findings of Pastor-Idoate et al. (2017), that the use of US cutters results in high fragmentation of the vitreous,These results also indicate that many of the limitations identified by Stanga et al. (2017)Finally, the US action may show potential for the development of new tools and techniques. It can be utilized in a variety of geometries to improve ergonomics and promote safety. Because US cutters can effectively liquify the material around it, the results of this study also show promise that the technology can be applied to stiffer biological materials.The goal of this paper was to quantify and assess the effect cutting method has on the rheological properties of extracted vitreous. To do so, we measured the extensional relaxation time, which is an indicator of the relative length of protein polymer fragments contained in a sample. The results presented here indicate that the US cutter chops vitreous into a much smaller fragments, as expected. However, this conclusion was not directly confirmed. If desired, the relative size of the proteins in samples of vitreous extracted using PB and US vitrectors can be directly confirmed using microscopy techniques like those used by Pastor-Idoate et al. (2017).In summary, we have assessed ultrasonic vitrectomy technology and its performance compared to conventional PB vitrectors. By using extensional rheology, we were able to show significant differences in the vitreous samples extracted using the two cutter styles, where typical shear rheology was not likely to do so. When extracted using an ultrasonic cutter, the extensional relaxation times of the vitreous samples were orders of magnitude shorter compared to samples extracted using the pneumatic blade cutter . These results confirm that the US cutter breaks the vitreous collagen into very small fragments compared to pneumatic cutters. This has broad implications for improved ergonomics and safety of the ultrasonic cutter, as well as for the development and improvement of vitrectomy devices and techniques using US technology.


Patients with coronavirus disease 2019 (COVID-19) often develop acute respiratory failure and acute respiratory distress syndrome (ARDS) that requires intensive care unit (ICU) hospitalization and invasive mechanical ventilation, associated with a high mortality rate. In addition, many patients fail early weaning attempts, further increasing ICU length of stay and mortality. COVID-19 related ARDS can be complicated by neurological involvement with mechanisms of direct central nervous system (CNS) infection and with overlapping para-infective mechanisms of the peripheral nervous system (PNS). We aimed to evaluate the possible involvement of the brainstem and PNS in patients with COVID-19 related ARDS and difficulty in weaning from mechanical ventilation. We evaluated electroencephalogram (EEG), brainstem auditory evoked potentials (BAEPs), electroneurography of the four limbs and the phrenic nerve in 10 patients with respiratory insufficiency due to SARS-CoV-2. All were admitted to intensive care unit and were facing prolonged weaning from mechanical ventilation. All ten patients showed a mild diffuse non-specific slowing of brain electrical activity on the EEG. Four patients had an acute motor axonal neuropathy with absent or reduced amplitude phrenic nerve CMAP while four patients showed impairment of the BAEPs. A patient with peripheral nerve impairment suggestive of Guillain-Barré syndrome (GBS) underwent an intravenous immunoglobulin (IVIg) cycle that led to an improvement in the weaning process and progressive motor improvement. The inclusion of a comprehensive neurological evaluation in COVID-19 patients in ICU facilitated the early identification and effective management of Nervous System involvement. Interstitial pneumonia due to SARS-CoV-2 can be complicated by possible neurological involvement with mechanisms of direct CNS infection and/or with para-infective mechanisms of the peripheral nervous system (PNS), shown by some neuropathological findings of COVID-19 patients, as recently reviewed  and increasing in that way complications, morbidity, and mortality. In some cases, there appears to be a discrepancy between the severity of lung involvement and respiratory function. Severe COVID-19 leads to death through multiple mechanisms, including myocardial damage, renal failure, shock, and disseminated intravascular coagulopathy . It has Recent reports describe a Guillain-Barré syndrome related to SARS-CoV-2, characterized mainly by axonal impairment with early involvement of cranial nerves that could lead to severe respiratory failure .Our aim was to consider the possible overlap of central and peripheral nervous system involvement in patients with respiratory insufficiency due to COVID-19 and difficulty in weaning from mechanical ventilation.Miulli Hospital in Acquaviva delle Fonti in the period from 1 March to 30 May 2021, evaluated because they were facing prolonged weaning from mechanical ventilation, despite the improvement in pulmonary conditions. All patients underwent electroencephalogram (EEG), brainstem auditory evoked potentials (BAEPs), electroneurography of the four limbs and the phrenic nerve. A standard 20 min EEG was recorded according to the 10–20 International system of electrode placement. BAEPs were recorded following auditory stimulation by a 100-μs 85-dB ± click applied to one ear, with a (−20 dB) contralateral masking using “white noise.” The recurrence frequency was 11 Hz . Two sets of 2,000 sweeps were averaged. BAEPs were picked up in Cz. The reference electrode was placed at the earlobe ipsilateral to the stimulated ear. Nerve conduction studies were performed according to standardized techniques. Distal motor latency, amplitude and duration of negative peak of compound muscle action potential (CMAP), motor conduction velocity and minimal F-wave latency were measured from different stimulation sites . Sensory studies were performed anti-dramatically in median, ulnar and sural nerves and amplitude of sensory nerve action potential was measured baseline to negative peak. Patients with previous pathology of the central and peripheral nervous system were not considered. At the time of the evaluation, patients were subjected to only mild sedation with dexmedetomidine.We present data about patients with respiratory failure due to SARS-CoV-2 infection admitted to the intensive care unit of the We evaluated 10 patients, 5 male, aged 53–75 years (mean 66.1); the duration of Covid, from the first detection of SARS-CoV-2 RNA in respiratory specimen  until the neurophysiological evaluation, was 6–50 days (mean 25.5). In all subjects, the onset of symptoms was on the same day or the day before arrival in ED and the diagnosis of SARS-CoV-2 infection.The main comorbidities presented by the patients were hypertension (present in the 80% of subjects), obesity (60%), Chronic kidney Disease (20%), diabetes (20%). All ten patients showed a mild diffuse non-specific slowing of brain electrical activity on the EEG. Three patients showed normal BAEPs, while in two patients we found non-evocable responses and in 5 patients increased interpeak III-V wave latency monolaterally . We perfIn 6 patients the neurological evaluation showed no specific or significant findings. In these patients the electroneurographic study showed normal findings or abnormal studies that did not allow a specific electrodiagnostic classification . Four ofWhilst respiratory failure in COVID-19 arises from severe interstitial lung involvement , SARS-CoAnother important aspect that emerged from our evaluation in patients with COVID-19 is the need to consider a Guillain-Barré syndrome associated with severe respiratory impairment. Typically, GBS is a post-infectious condition with symptom onset for 76% of the patients occurring in about 4 weeks after the preceding respiratory or gastrointestinal infection . The parThis study has several limitations. First, the number of cases was very small. Second, it is a retrospective study and some findings such as antiganglioside antibody titres and cerebrospinal fluid analysis were not available. However, it should be considered that these patients were studied in a pandemic context and under the pressure of and exceptional health emergency in a hospital of southern Italy. Therefore, the interpretation of our findings should be made with caution and should be interpreted as hypothesis-generating.Overall, our results suggest that a central, mainly at brainstem level, and peripheral nervous system involvement likely contributes to respiratory failure in COVID-19 patients. The inclusion of a comprehensive neurological evaluation in SARS-CoV-2 patients with clinical and radiological lung amelioration, but difficulty in weaning from mechanical ventilation, facilitated the identification and effective treatment of neurological involvement.The original contributions presented in the study are included in the article/Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Patient's next of kin provided their written informed consent to participate in this study.EV, LG, and NC: conception, design, and drafting article. VR, LD, GP, and LP: acquisition of data. EV and AP: analysis and interpretation of data. FT and VD: study supervision. All authors critically revised the article and reviewed final version of the manuscript and approved it for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


N = 10,603) we report a novel LDL-C association in the GATB region . Meta-analysis with four other African cohorts  provides supporting evidence for the LDL-C association with the GATB/FHIP1A region and identifies a novel triglyceride association signal close to the FHIT gene . Our data enable fine-mapping of several well-known lipid-trait loci including LDLR, PMFBP1 and LPA. The transferability of signals detected in two large global studies (GLGC and PAGE) consistently improves with an increase in the size of the African replication cohort. Polygenic risk score analysis shows increased predictive accuracy for LDL-C levels with the narrowing of genetic distance between the discovery dataset and our cohort. Novel discovery is enhanced with the inclusion of African data.Genetic associations for lipid traits have identified hundreds of variants with clear differences across European, Asian and African studies. Based on a sub-Saharan-African GWAS for lipid traits in the population cross-sectional AWI-Gen cohort ( Genetic associations and polygenic scores for lipid traits have low transferability to African individuals. Here, the authors perform a large sub-Sarahan African lipid GWAS and find that larger datasets and better global representation in discovery GWAS help to bridge this gap. Although modulated by environmental and behavioural factors, the heritability of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and triglycerides (TG) has been estimated at between 0.35 and 0.766. However, trait heritability demonstrates considerable inter-ethnic differences, some of which could be explained by environmental differences such as diet and availability of medications7. Although large representative datasets are sparse, at a population level lipid profiles from African Americans generally reflect lower TG, and higher HDL-C levels compared to populations from Asia and Europe8.Circulating lipid levels and their genetic associations are important indicators of the risk for developing cardiometabolic diseases including stroke, coronary artery disease, hypertension, and are also associated with kidney disease9, SNP arrays enriched for known associated genomic regions (Metabochip)11, exome SNP arrays14, genome-wide SNP arrays16, whole exome sequencing (WES) and whole genome sequencing (WGS)17 approaches to reveal over 400 robustly associated independent loci. Many of the studies included multiple ethnicities mainly representing Americans of European- and African-ancestry, Hispanics17, American Indians18 and Asian populations20. These studies have generally shown that the majority of lipid-trait associations are universal; however, several studies also revealed population- or ancestry-specific lipid-associated loci and allelic heterogeneity21. A recent systematic assessment of the transferability of an European genome-wide association study (GWAS) derived signals across ancestral groups highlighted these differences, especially in Africans, despite the shared genetic architecture for lipid traits22.Genetic associations with lipid levels have been extensively studied using candidate loci23. However, understanding the genomic architecture of lipid traits in Africans is currently dominated by studies on admixed African-ancestry individuals resident in the USA, who represent only a portion of the genetic diversity that exists on the continent. Moreover, these studies have mostly used SNP arrays based on common genetic variation in European and admixed populations25. Only a few studies on lipid traits27 have been solely or predominantly based on sub-Saharan African populations. The modest sample sizes and lack of geographic spread of participants have limited our understanding of the genetics of lipid traits in these populations.There are demonstrable benefits in using ancestrally diverse populations in genetic-association studies. These include refinement of previously associated loci by highlighting different effect sizes across ancestral populations, enhancing the potential to identify functional variants using linkage disequilibrium (LD)-based fine mapping, and the identification of associated variants that are ancestry-specific for either novel or known loci. Arguably, studies in African populations have the potential to contribute much benefit, as these populations have increased diversity, lower LD and higher population structure, but require the use of African-centric arrays and appropriate imputation panels to maximize novel discovery28. However, the predictive ability of these scores not only varies widely between traits but also between GWAS for the same trait due to factors such as sample size and LD architecture30. Moreover, PRS models for several traits have shown that their application to ancestries other than the ones in the original GWAS often leads to a loss in predictive accuracy33. Accordingly, an African population was shown to have the lowest predictive accuracy using a European-derived PRS model for lipid traits22.Genetic associations from a GWAS are often used as a collective set for the generation of polygenic risk score (PRS) models. The application of these models to lipid traits shows an overall high predictability. For example, in a study of 94,674 ancestrally diverse treatment-naive participants from the Kaiser Permanente members, a GWAS using electronic health records revealed novel and sex-specific genetic associations and demonstrated that a 477 SNP PRS could predict age at first use of lipid-lowering drugs with relative accuracyhttps://www.h3abionet.org/h3africa-chip), enriched for common variants in sub-Saharan Africans, to perform GWAS for fasting serum TC, LDL-C, HDL-C, and TG in the AWI-Gen cohort based on 10,603 adults, resident in six study sites across four countries in sub-Saharan Africa35. This was followed by a meta-analysis with previously published summary statistics from four sub-Saharan African cohorts reaching a combined sample size of about 24,000. We then performed an in depth assessment of the replication of previously associated loci (using the summary statistics from the Population Architecture using Genomics and Epidemiology (PAGE)36 and Global Lipid Genetics Consortium (GLGC)15) in AWI-Gen and other sub-Saharan African cohorts. Finally, we examined the predictability of PRS models based on GWAS from European (GLGC)15, multi-ancestry (PAGE)36 and sub-Saharan African populations7, in our cohort.In this study, we report on the use of the H3Africa SNP genotyping array . This dataset was imputed using the African Reference Panel at the Sanger Imputation facility and only SNPs with MAF > 0.01 and Info Score>0.6 were included in the analysis. Stage 2 (building on the outcome of Stage 1) was the meta-analysis of these results with published summary statistics from four cohorts (the Uganda Genome Resource (UGR) study, the Africa-America Diabetes Mellitus (AADM) study, the Durban Diabetes Study (DDS), the Durban Case Control (DCC) study) all of which were included in the Gurdasani et al.7 study , the Stage 2 analysis was restricted to the ~14M SNPs that were present in the AWI-Gen study. This difference, despite the use of the same imputation panel, was due to a more stringent MAF cut-off (MAF > 0.01) compared to that used in Gurdasani et al.7 (MAF > 0.005).The distribution of the four lipid traits  and associated covariates (age and sex) in the six AWI-Gen study sites are summarized in Supplementary Data 36, we defined the number of relevant principal components (PCs) based on a joint plot of the first twenty PCs for representative ethnolinguistic groups  genomic regions that were associated with at least one of the four lipid traits at a genome-wide significance threshold of P-value <5 × 10−8 , the lack of any previous lipid signal in the extended genomic region (+/−500 kb) around this SNP  that also lacked any report of previous association either in GWAS catalog or PhenoScanner43 (Table 39 MAF = 0.015 in African Americans and MAF < 0.001 in non-African populations in the gnomAD database40) which could explain its absence in previous GWASs. Similarly, the lead SNP rs2435386 near the well-knownTDRD15 gene also has no reported associations in the GWAS catalog41. However, a PhenoScanner search shows this SNP to be associated with high cholesterol as well as cholesterol lowering medication. The geographic-region-specific analyses  as a potential causal variant for both LDL-C and TC. The prediction was supported by the other two methods. This stop-gained variant is nearly absent in non-Africans in the 1000 Genomes39 and GnomAD datasets40 and consequently has been detected as associations in only three of the previous lipid-trait GWAS that were either African based7 or included a substantial portion of African ancestry47. rs28362286 was observed as the lead variant for associations with TC and LDL-C in both Stage 1 and Stage 2 GWAS . In addition, we observed a marked reduction of credible set size of the signal near the well-knownLDLR locus in the African dataset . Most of the signals were within or near known lipid-associated genes such as ABCA1, LPL and APOB (Table ABCA1) and rs2126263 (near LPL) were observed as suggestive hits in the Stage 1 GWAS  and PMFBP1 with LDL-C (rs4788609), detected in the Stage 2 GWAS, were also found to be represented by novel lead SNPs.Stage 2 of our GWAS, based on a meta-analysis of the results from the Stage 1 GWAS and summary statistics for four other African cohorts, identified about 15 additional independent genomic regions associated with one or more of the four lipid traits Table . Most ofGATB gene showing LDL-C association in the Stage 1 GWAS was found to be nominally replicated  in the AADM cohort and was close to the genome-wide significance threshold  in the Stage 2 GWAS  gene at the genome-wide significance threshold  on chromosome 19, are also strong candidates for being novel signals. A potential novel signal for TG was observed in an intergenic region near the FHIT gene (rs75064672). The comparison of results from various meta-analysis models demonstrated that most of these associations were significant at the standard genome-wide significance threshold irrespective of whether a fixed, random or binary effect model was considered 15 to the African dataset, we assessed the P-value of the signals from the GLGC study in the Stage 1 GWAS, Stage 2 GWAS and the four African cohorts included in the meta-analysis. To delineate independent loci for evaluation, genome-wide significant signals in the GLGC summary statistics that were within 50 kb of each other were considered as belonging to the same locus. For each locus, if any of the SNPs that were genome-wide significant in the GLGC GWAS showed P-value <5 × 10−4 in an African cohort, the locus was considered to be replicated/transferable. The cut-off was decided on the premise that each of four lipid traits were represented by about 100 independent loci in the GLGC dataset and therefore a minimum correction for 100 tests was necessary . In addition, we also assessed replication at a more stringent level  and a less stringent threshold , the entire analysis was restricted only to the ~14 M SNPs that were present in the AWI-Gen dataset.To evaluate the transferability of signals from the predominantly European-based GLGC Consortium study22 has shown the transferability of signals from GLGC to vary considerably with the strength of the signals; signals with P-value < 10−100 show much higher transferability compared to signals with higher P-values. To assess this further we classified signals into three categories—very strong , strong  and moderate  and studied the levels of transferability in each of these categories separately. The results presented in Fig. P-values at which these replications were observed also decreased consistently with the increase in the size of the replication cohort.A previous study on lipid-trait signals36 showed the same trend along with an overall higher transferability  compared to signals from the GLGC study across all lipid traits. As there were very few signals with P-value < 10−100, we only categorized the signals into strong  and moderate , and once again observed better replication in the former compared to the latter. Although the variation in the level of transferability of signals for the four traits was not consistent across the PAGE and the GLGC study, in both cases we observed the signals for LDL-C and HDL-C to show better transferability compared to the other two traits .A similar analysis of signals from the multi-ethnic but much smaller PAGE consortium studyity Fig.  compared15, PAGE36 and a sub-Saharan African meta-analysis GWAS7 could predict the levels of the four lipid traits in the AWI-Gen cohort participants  and triglycerides  while the multi-ancestry (PAGE) based PRS better predicted HDL-C  and total cholesterol  as illustrated in Fig. Next, we assessed the extent to which PRS derived from GLGCnts Fig. . The PRSAPOE p.Arg176Cys) shows almost two-fold higher MAF in South African cohorts compared to both the East African cohorts and one of the West African cohorts. However, with the exception of the DDS and UGR cohorts the effect sizes for association of this SNP with LDL-C were comparable among other cohorts. The SNP rs4788609 in PMFBP1 has similar MAFs in Eastern, Western and Southern Africa  but very different effect sizes (for LDL-C) in one of the Eastern and Southern African cohorts. Finally, the novel lead SNP rs6845395 for LDL-C shows major variations in both allele frequencies and effect sizes across the cohorts. Overall, the results from the three smaller cohorts DDS, DCC and AWI-Gen East showed the maximum deviation from other studies and also exhibited greater magnitude of standard error estimates  for lipid-trait genetics in sub-Saharan African populations. Before highlighting the key findings we discuss some of the strengths of the AWI-Gen study. These include the use of a single laboratory for measuring lipid biomarker levels for all the samples, as well as the same genotyping array and joint imputation for capturing genotype data which ensured much higher comparability of the data from different parts of the continent. The population cross-sectional nature of the cohort, in contrast to cohorts from disease-based studies such as AADM, DDC and DDS, was also expected to reduce the influence of other conditions on lipid levels. Finally, almost all the AWI-Gen participants were naive to medication for dyslipidemia and other cardiometabolic diseases removing another potential confounder from the study.The lpaucity of GWAS in African populations has been consistently highlighted in recent literature51. This, added to environmental and lifestyle differences within and between geographic regions, as observed in several phenotype studies based on the AWI-Gen cohort53, could limit the ability of modest sample size GWAS based on pan-African datasets to identify associations accurately. We have employed a LMM based approach to conduct association testing on the full dataset with principal components as covariates for the Stage 1 GWAS. Given the modest sample size of our study, we considered this composite or “mega-analysis” to be appropriate as it was expected to have higher statistical power compared to meta-analysis of separate GWAS for the three African regions36. However, the high concordance in the outcomes of the mega- and meta-analyses of AWI-Gen East, West and South African data demonstrates that these two approaches perform comparably in our study. However, further in-depth analysis from other similar cohorts will be required to develop comprehensive guidelines for addressing population structure and heterogeneity in pan-African GWAS based on modest sample sizes.A key requirement for the success of a pan-African GWAS is the identification of an optimal approach to control for population structure. This is driven by the fact that in addition to pronounced genetic differences between Eastern, Western and Southern African populations, genetic differences are common even among participants from the same geographic region. For example, a recent study reported that fine-scale population structure was strong enough to influence association signals among the South African participants from our cohort7. The combined sample size of around 24,000 participants makes this study considerably larger than previous GWASs for lipid traits conducted in sub-Saharan African populations. We restricted the analysis to only those SNPs that were included in the final AWI-Gen dataset and in at least three of the four other cohorts. While this could have limited our potential for discovering novel associations, this approach ensured that the associations were based on a significant proportion of the combined sample set and are thereby relatively robust. Another ongoing discussion given the overall lower LD in African genomes, concerns the optimal P-value cut-off for African GWAS/meta-analysis, especially those using imputed data. Approaches such as using an overall lower threshold of significance (< 5 × 10−9)7 or allele frequency dependent cut-offs have been recommended54. As there is a lack of consensus, we have used the currently accepted genome-wide significance threshold P-value< 5 × 10−8. However, our novel signal (rs6845395) for LDL-C in the Stage 2 GWAS reached a P-value of 3.28 × 10−9.To bolster the power of our GWAS, we performed a meta-analysis of the Stage 1 results with the summary statistics from four African cohorts included in the Gurdasani et al. 2019 studyGATB gene  in the ene Fig. . The nomFHIT from the Stage 2 meta-analysis (rs75064672), reached a P-value < 0.05 in three independent African cohorts suggesting that this is a reliable association signal. Several variants from this gene have been detected to be associated with body composition related traits including body mass index, obesity and lean mass41.We also detected a novel association for TG in the Stage 2 GWAS. The lead TG-associated SNP near 55 showed that these SNPs occur at much lower frequencies in both these cohorts in comparison to AWI-Gen  as well as the trans-ethnic meta-analysis of the GLGC cohort (~1.5 M samples) from the recent GLGC studySENP7 gene. Despite being a globally common SNP, this lead SNP has no previous report for association with any lipid traits; however, a PhenoScanner43 search showed another SNP from the region, rs149851118, to be associated  with “Treatment with lipantil micro 67 mg capsule” in a UK Biobank study. Fenofibrate, the active ingredient of the drug, is widely used to treat abnormal lipid levels, which suggests a possible involvement of the variant/region in modulating lipid metabolism. However, further investigations would be required to establish a functional connection between this genomic region and lipid traits.In addition to signals at the continental level we report a novel West African specific signal for association with TG in the The absence of large-effect novel associations in the meta-analysis probably hints at saturation in the discovery of major lipid traits associated with globally common variants. However, the detection of novel African-specific lead SNPs, potential causal variants and relatively smaller credible sets for some well-known lipid-associated loci highlights the importance of including large African GWAS in fine-mapping of lipid traits.P-value < 5 × 10−322. The relatively modest size of the African cohort (~6400 samples) included in that study could have been a major reason for the low transferability of signals to African GWAS. To test whether the use of larger African replication cohorts could address this lack of transferability, we studied the transferability of signals from two large discovery cohorts (GLGC15 study and PAGE36 study) to various African replication sets . Signals from smaller discovery GWAS, due to higher effect sizes can be expected to be more transferable compared to lower effect signals in larger studies. Consistent with the differences in GWAS sizes, we observed that the signals from the PAGE study showed considerably better transferability compared to the much larger GLGC study. Moreover, the multi-ethnic nature of the PAGE study cohort compared to predominantly European samples in the GLGC study could have contributed to these differences. Nevertheless, for both the discovery cohorts, the transferability was found to increase more or less consistently with the size of the replication cohort and in general the SNPs with stronger signals  from a study showed better transferability in comparison to modest signals from the same study. In line with the previous observation22 that transferability of signals is not uniform across traits and associations for some traits (such as HDL-C) have much higher transferability in comparison to others (such as TG), for both the discovery sets and across all the replication cohorts, we observed that LDL-C and HDL-C showed higher transferability in comparison to TG.To assess the extent to which current genetic knowledge gained from GWAS conducted in European populations could benefit risk prediction in populations from other continents, it is critical to ascertain that the same loci are associated with the trait across such populations. An in-depth study of the transferability of known lipid-trait associations detected in a predominantly European cohort, to GWAS conducted in East Asian and African ancestry populations has shown that of the hundreds of loci associated with lipid traits, only a portion of signals replicate in African populations even at a moderate replication threshold of 33. Several of these studies have demonstrated an overall poor transferability of existing European-based PRS models to African ancestry populations for most of the traits investigated. Allele frequency and LD differences as well as environmental differences have been suggested as the major sources of the low transferability of PRS models to Africans. The study by Kuchenbaekar and colleagues22 highlighted the lack of transferability of European-based PRS models for lipid traits to African populations. The analysis of predictability of PRS based on summary statistics from three different studies  in the AWI-Gen cohort, showed progressively better predictability for signals from genetically closer cohorts for the lipids traits such as LDL-C. It is therefore critical to increase the number of non-European participants in GWAS to enhance polygenic prediction across diverse populations56.The promise of global application of precision medicine approaches will require efficient transferability of polygenic risk score (PRS) models across diverse populations. However, since research is currently largely based on European populations, there is a strong bias for potential beneficiaries. Studies on the transferability/predictability of existing PRS models for various traits from the European-based discovery cohorts to East Asian and African populations are of critical importanceAs factors such as allele frequency and effect size could strongly influence the detection of a signal in a GWAS, we performed an in-depth comparison of effect sizes and MAFs of association signals detected in our study in the Eastern, Western and Southern African subsets of our cohort and four other African cohorts. The inclusion of at least two cohorts from each of three African regions  enabled us to investigate if there are systematic geographic trends in these estimates. Although our results show considerable heterogeneity between African cohorts, we did not see clear geographic differences suggesting that there is probably more intra-region variation compared to inter-region variation in the effect size and frequencies of these SNPs in the cohorts studied. The high level of heterogeneity also indicates that effect sizes for a SNP detected in one particular African cohort might not correspond to or have predictive relevance for other African cohorts, including those from the same geographic region. However, it needs to be highlighted that due to factors such as differences in sample size, age, proportion of participants with type 2 diabetes (as some of these were diabetes-based cohorts and type 2 diabetes might have influenced lipid levels) that exists between them, these cohorts are not completely comparable. The use of more homogenous cohorts across the continent will be required to perform a robust investigation into possible geographic trends.The modest sample size of the AWI-Gen study is a limitation to the power of the GWAS. In addition, pronounced differences in genetic variation, and environmental and lifestyle factors between the AWI-Gen study sites may have impacted the overall statistical power. Despite these challenges, the detection of novel signals and novel lead SNPs for lipid traits in the AWI-Gen cohort and the meta-analysis with other African studies emphasise the promise and potential of African GWAS in enhancing our understanding of the genetic aetiology of lipid traits and dyslipidemia. In addition to demonstrating that the extent of transferability might vary widely between lipid traits and discovery cohorts, our results suggest that the transferability for PRS might be further enhanced with the use of more multi-ethnic based GWAS datasets.This study was approved by the Human Research Ethics Committee  of the University of the Witwatersrand (Wits) (protocol numbers M121029 and M170880). In addition, each research site obtained approval from their local ethics review board prior to commencing any participant-related activities.34 and Ali et al.35.The participants in this study are part of the Africa Wits-INDEPTH partnership for Genomics studies (AWI-Gen) which aims to examine genetic and environmental factors related to cardiometabolic diseases in Africans. It is part of the Human Heredity and Health in Africa Consortium (H3Africa). From 2012 to 2016, ~12,000 participants, primarily between the ages of 40 to 60 years were enrolled across the six AWI-Gen centres, and a further 552 over the age of 60 enrolled at the Agincourt centre. Following community engagement and individual informed consent, data and samples were collected from six study centres in four SSA countries: in South Africa, the MRC/Wits Agincourt Health and Demographic Surveillance System Site (HDSS) (referred to as Agincourt), the Dikgale HDSS of the University of Limpopo, and the Soweto centre which is coordinated by the South African Medical Research Council/Wits Developmental Pathways for Health Research Unit (DPHRU); in Kenya, the African Population and Health Research Center HDSS in Nairobi; in Ghana, the Navrongo HDSS in the Navrongo Health Research Centre; and in Burkina Faso the Nanoro HDSS hosted by the Institut de Recherche en Sciences de la Santé Clinical Research Unit. Participants were given a small compensation to cover their travel and incidentals. Further details are available in Ramsay et al.57. The coefficient of variation of the laboratory measurements for lipids was less than 1.5 %. Each lipid phenotype was checked for extreme outliers and inverse normal transformed prior to genetic-association analysis.Fasting serum lipids were analyzed using a Randox Plus clinical chemistry analyzer  using colorimetric assays and all assays were performed at the DPHRU laboratories in Soweto, Johannesburg. The concentrations for triglycerides (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C) were determined directly from the assay. The concentration of low-density lipoprotein cholesterol (LDL-C) calculated using the Friedewald equation® FastTrackTM Microarray services . The genotype calling was performed using the Illumina pipeline. Pre-imputation quality control (QC), performed using H3ABioNet/H3Agwas pipeline (https://github.com/h3abionet/h3agwas) and involved the removal of SNPs showing missingness greater than 0.05, MAF less than 0.01, and Hardy-Weinberg equilibrium (HWE) P-value less than 0.0001. In addition, duplicates, X chromosome, Y chromosome and mitochondrial SNPs, and SNPs that did not match the GRCh37 references alleles were also removed. Samples which were potential duplicates (PIHAT > 0.9), had a missing SNP genotyping rate greater than 0.05, and showed sex inconsistencies (between recorded and genetic sex) were excluded. Population stratification in this dataset, was assessed using a principal component (PC) analysis, based on a LD pruned subset of SNPs, using the smartPCA program implemented in EIGENSTRAT58. As the genotyping was performed in 4 independent batches, with some overlapping samples, both PC and genotype concordance of these overlapping samples was used to identify possible batch effects. Both the approaches ruled out any serious batch effect in the dataset.Approximately 11,000 samples were genotyped on the 2.3 M SNP H3Africa array at Illuminahttps://imputation.sanger.ac.uk/). Post imputation quality control (QC) involved removal of indels, rare SNPs  < = 0.01) and poorly imputed SNPs (Info score < = 0.6) resulting in the final dataset containing 10,603 participants and 13.98 M SNPs.Imputation was performed using the African Genome Resources reference panel (EAGLE2 + PBWT pipeline) at the Sanger Imputation Server  and imputed data using dosage format, we performed an association analysis using BOLT-LMM implemented in the h3agwas pipeline (github.com/h3abionet/h3agwas/assoc/assoc.nf)59. For each dataset we employed BOLT-LMM37 with leave-one-chromosome-out (LOCO) analysis and 1,000,000 SNPs to build models. Independent SNPs were pruned using PLINK60 (“--indep-pairwise” option with window size of 100 kb by step size of 20 kb and threshold of ld of 0.6 (r2)). For reference LD score, tables were calculated using the LDSC software61 using the 1000 Genomes African Dataset39 and genetic map (build hg19) as described in the LD Score Estimation Tutorial . We reported non-infinitesimal mixed model association test P-values for Stage 1 GWAS. All association tests were based on impute2 dosage. The FUMA online platform38 was used for partitioning associations into locus, lead SNPs and independent SNPs based on the 1000 Genomes African dataset.Using residuals adjusted for age, sex and first 62 implemented in https://github.com/h3abionet/h3agwas/tree/master/meta/meta-assoc.nf. We also performed a meta-analysis of the summary statistics from AWI-Gen data and 4 cohorts AADM, UGR, DDS, DDC included in Gurdasani et al. 2019. We primarily used the Han and Eskin’s Random Effects model (RE2), as it has been suggested to best address the heterogeneity in a cohort like ours, but also recorded P-values derived from the Fixed Effect (FE) Binary Effects model (BE) for comparison. We restricted the analysis to only those SNPs that were included in the final AWI-Gen dataset and in at least three of the four other cohorts. The FUMA online platform38, as mentioned above, was used for classifying the associations.To evaluate the robustness of associations detected in joint analysis of the AWI-Gen dataset, we performed a meta-analysis of summary statistics from the South, East and West African cohort specific analyses using METASOFT (v2.0.1)P-value into Very Strong , Strong  and Moderate  signals. For each locus we recorded the lowest P-value that was observed for any of the significant SNPs in the discovery set in each of the 6 replication sets . If a locus was represented by a SNP with a P-value < 5 × 10−4 it was considered as a replication. We also assessed replication at the nominal P-value cut-off of 0.05 as well as a genome-wide significant threshold. For PAGE study based signals only two categories – Strong  and Moderate were considered .Genomic regions associated with each of the four lipid traits in the GLGC and PAGE consortium studies were identified from the respective summary statistics. For each trait, genome-wide significant SNPs in the discovery set, if also present in the AWI-Gen study, were selected. Individual hits, if separated by less than 50 Kb, were merged into a single locus. Using this approach, for the GLGC study we defined 138, 181, 174, 135 independent regions or loci for LDL-C, HDL-C, TC and TG, respectively. Similarly, for the PAGE study 21, 26, 32 and 17 loci were detected for LDL-C, HDL-C, TC and TG, respectively. The loci from the GLGC study were then partitioned based on lowest 7 summary statistics file . Minor allele frequencies for the AWI-Gen East African, AWI-Gen West African and AWI-Gen Southern African populations were estimated using PLINK60 and effect sizes were obtained from geographic-region-specific GWAS conducted in these populations.Minor allele frequencies and effect sizes  for the four lipid traits in the AADM, UGR, DDS and DDC studies were obtained from the Gurdasani et al.38 was used to annotate, prioritize, visualize and interpret the GWAS results. This included extensive functional annotation of all SNPs matching their chromosome base-pair position, and reference and alternate alleles to databases containing known functional annotations. Genes implicated by the mapping of association signals were further investigated using the GENE2FUNC procedure in FUMA, which provides hypergeometric tests of enrichment of the list of mapped genes in 53 GTEx tissue-specific gene expression sets, 7,246 MSigDB gene sets and chromatin states. Finally, the Multi-marker analysis of genomic annotation  analysis, implemented in FUMA, was performed using summary statistics of our association results as input. These gene-based analyses enabled summarization of SNP associations at the gene level and association of the set of genes to biological pathways.The FUMA online platformhttps://github.com/h3abionet/h3agwas/tree/master/finemapping) that employ Bayesian calculation of posterior probability and/or annotation information to identify potential causal variants. For each region around the Stage 1 GWAS signals, using FINEMAP44, we performed fine-mapping with stepwise conditioning (--cond) and shotgun stochastic search (--sss), providing us with Posterior Inclusion Probabilities (PIP). The PIP for the l th SNP is the posterior probability that this SNP is causal and the Bayes factor quantifies the evidence that the l th SNP is causal with log10 Bayes factors greater than 2 reporting considerable evidence . PAINTOR45 empirically estimated the contribution of each functional annotation to the trait of interest directly from summary association statistics while allowing for multiple causal variants at any risk locus. Additionally, we used CAVIAR-BF46 for fine-mapping using marginal test statistics in the Bayesian framework. In addition, COJO-GCTA63 was used to perform a stepwise model selection procedure to select independently associated SNPs (--cojo-slct); and we also performed association analysis of the included SNPs conditional on the given list of SNPs (--cojo-cond).To perform fine-mapping, we used three software packages included in the h3agwas pipeline  and the AWI-Gen study participants were used as the target dataset. The prediction models were corrected for age, sex and residual population structure using principal components. The R2 (variance) was used to determine the best predictive PRS. The polygenic risk scores were first tested on a third of the AWI-Gen participants (n = 3500) and then the best predictive polygenic risk scores were validated in the remaining two-thirds of the AWI-Gen dataset (n = 7103). Decile plots were used to evaluate the risk stratification of PRS, by comparing the mean difference of the lipid traits of the upper deciles from the lowest decile.The clumping and thresholding (C + T) approach in PRSice2Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1-10Reporting Summary


Fasciola hepatica is a zoonotic neglected disease affecting animals and humans worldwide. Infection occurs upon ingestion of aquatic plants or water contaminated with metacercariae. These release the newly excysted juveniles (FhNEJ) in the host duodenum, where they establish contact with the epithelium and cross the intestinal barrier to reach the peritoneum within 2–3 h after infection. Juveniles crawl up the peritoneum towards the liver, and migrate through the hepatic tissue before reaching their definitive location inside the major biliary ducts, where they mature into adult worms. Fasciolosis is treated with triclabendazole, although resistant isolates of the parasite are increasingly being reported. This, together with the limited efficacy of the assayed vaccines against this infection, poses fasciolosis as a veterinary and human health problem of growing concern. In this context, the study of early host-parasite interactions is of paramount importance for the definition of new targets for the treatment and prevention of fasciolosis. Here, we develop a new in vitro model that replicates the first interaction between FhNEJ and mouse primary small intestinal epithelial cells (MPSIEC). FhNEJ and MPSIEC were co-incubated for 3 h and protein extracts (tegument and soma of FhNEJ and membrane and cytosol of MPSIEC) were subjected to quantitative SWATH-MS proteomics and compared to respective controls  to evaluate protein expression changes in both the parasite and the host. Results show that the interaction between FhNEJ and MPSIEC triggers a rapid protein expression change of FhNEJ in response to the host epithelial barrier, including cathepsins L3 and L4 and several immunoregulatory proteins. Regarding MPSIEC, stimulation with FhNEJ results in alterations in the protein profile related to immunomodulation and cell-cell interactions, together with a drastic reduction in the expression of proteins linked with ribosome function. The molecules identified in this model of early host-parasite interactions could help define new tools against fasciolosis.Fasciolosis caused by the trematode  Fasciola hepatica poses a serious concern for animal and human health worldwide. Treatment and prevention of this disease is challenging due to the appearance of parasites that are resistant to the current treatment and the suboptimal efficacy of assayed vaccines. In this context, the importance of early interactions with the host for parasite migration and infection progression prompted us to develop and study new models of interaction between F. hepatica juveniles and their host. Here, an in vitro model coupled with a quantitative proteomics approach was developed to evaluate the changes in F. hepatica newly excysted juveniles right after their contact with intestinal epithelial cells, which represent the first line of host cells that the parasite encounters upon host infection. Additionally, this model has been used to decipher potential changes elicited by the parasite in the host intestine. A proteomic study of parasites and host cells upon co-incubation revealed specific changes in the repertoire of proteins from both host and parasite following their interaction. These proteins could be of importance for parasite migration and survival inside the host, and give new important insights to understand the host-parasite molecular crosstalk in early fasciolosis.Fasciolosis caused by  In vitro models to dissect the early infection stages of parasitic infections constitute a valuable tool that has been successfully implemented for the study of different endoparasites of medical and veterinary interest ).Up and down-regulated proteins in FhNEJ and MPSIEC were identified by database search and bioinformatic analysis , cathepand 24 h , but onland 24 h . Our resollagens . Provideollagens  in orderollagens . Togetheollagens .Interestingly, some proteins traditionally associated with the nucleus such as histones H4 and H2B and histone deacetylase were also up-regulated in the FhNEJ detergent-soluble extract enriched with tegument after co-incubation with host intestinal epithelial cells. These proteins have important functions in chromosomic DNA organization  and theyF. hepatica GST  has shown anti-inflammatory properties [F. gigantica CABP4 stimulated IL-2 and IFN-γ Th1-type cytokines and reduced IL-10 (Th2) expression levels by activated goat monocytes [Potentially linked to immune regulation, we found glutathione-S-transferase (GST) and the calcium-binding EF-hand protein 4 (CABP4) to be overexpressed in this protein fraction of FhNEJ upon incubation with MPSIEC. operties , and F. onocytes . As obseonocytes ), these F. hepatica infection. This could be the case of the above-mentioned cathepsins. In fact, the results obtained in this study showed a down-regulation in the expression of two isoforms of cathepsin B after contact of FhNEJ with MPSIEC. FhCB peptidases appear to be highly expressed by FhNEJ and down-regulated in adult flukes [F. hepatica in correlation to parasite development and migration through host tissues.Down-regulated proteins in the detergent-soluble extract enriched with tegument of the parasite include components of the muscle contraction machinery  and proteins involved in metabolite transport  along with other multi-purpose proteins . Noteworthy, some biological processes with up-regulated proteins (i.e. proteolysis or antioxidant response) also exhibit down-regulated representatives, and might therefore be finely regulated during the early phases of t flukes , 57, altin vitro [Interestingly, the downregulated protein with the highest Log2FC in this FhNEJ extract, also corresponding to the fifth most downregulated protein in somatic extracts of FhNEJ upon MPSIEC incubation, is CD59, a complement regulatory protein that protects cells from the destructive action of the complement system. Homologue CD59 proteins have been previously described to be expressed and secreted by FhNEJ in vitro , 58, togin vitro , 38, 58,Schistosoma mansoni or Opistorchis viverrini have elicited promising results as vaccine candidates against schistosomiasis and opisthorchiasis, respectively [In the somatic FhNEJ extract, the main GO terms in the BP category were proteolysis, carbohydrate metabolic process, protein folding and microtubule-based process , and traectively , 62. HowDown-regulated proteins in the soma of FhNEJ included proteins also down-regulated in their tegument . Additionally, legumain like and leucyl aminopeptidase, superoxide dismutase, transketolase and pyruvate dehydrogenase, representing functions similar to those down-regulated in the tegument of FhNEJ, were also found as down-regulated in the somatic extracts of FhNEJ.F. hepatica excretory/secretory products and that these enzymes have a key role in detoxifying both endogenous and exogenous toxins arising from the oxidative host defence response [Regarding antioxidant enzymes, our data revealed that the expression of four isoforms of one of their main representatives, GST, is up-regulated within the FhNEJ somatic and tegument extracts after co-culture with MPSIEC. It has been described that GSTs account for 4% of response . For tharesponse . Howeverresponse ,65.The comparison of the results of the present analysis with that performed before by our group by using the iTRAQ methodology after 24 h co-incubation of FhNEJ and MPSIEC , shows tOn the other hand, muscle contraction and cytoskeleton reorganization processes seem to be more represented at 24h pi, as at this time several related proteins are found to be up-regulated while they are not found at 3h pi, including dynein, cadherin, myophilin and catenin alpha. Several metabolic enzymes could be also differentially expressed at both times pi. The landscape at 3h pi is mainly aerobic , while at 24h pi it seems to evolve to an anaerobic profile with a greater abundance of glycolytic enzymes . Interestingly, histone isoforms are found as up-regulated proteins at both times pi, with histone H4 present at both times, while histone H2B is found at 3h and histone H2A at 24h. In summary, comparison of the results obtained here at 3h pi obtained by SWATH and the previous results at 24 h pi obtained by iTRAQ, show that proteolysis and its regulation, muscle contraction and metabolism could be differentially regulated in FhNEJ upon MPSIEC co-incubation at different times pi. Nevertheless, this should be further explored in more comparable studies, since the methods used to check up- and down-regulated proteins in FhNEJ after co-culture with host MPSIEC between the two above-mentioned studies are different regarding the proteomic and statistical approaches.In vertebrate animals, the small intestine is the organ with the largest surface area in contact with the outside environment. Although its main function is the absorption of water and nutrients from the diet, it is also the entry point for a multitude of pathogens, including foodborne parasites . As a maOur proteomics approach showed a considerable number of altered processes that were identified in the extracts of MPSIEC upon interaction with FhNEJ. In particular, some of the main modified BPs in MPSIEC cytosolic extracts were regulation of catalytic activity, actin cytoskeleton organisation, response to inorganic substance and vesicle-mediated transport , whereasOver-expressed proteins in the cytosol of mouse cells were involved in functions such as vesicle transport  and lipid biosynthesis  along with proteins involved in the response to stress (Hspa1b protein) or viruses (interferon-induced transmembrane protein 3 (IFITM3)). Among down-regulated proteins, there was an abundance of ribosomal constituents together with cytoskeleton-related proteins. Moreover, our results revealed that the expression of pro-inflammatory mediators (prostaglandin E synthase 3 (PTGES3)) and peptides with antimicrobial activity (Lysophospholipase 1) was also decreased.Up-regulated proteins within the MPSIEC membrane extracts included nuclear lamin components , chromatin-interacting proteins  and enzymes involved in energy metabolism . Down-regulation of proteins related to carbohydrate catabolism ) or response to pathogens (phospholipase A-2-activating protein (PLAA), annexin A2) was observed in this extract.Taken together, these results suggest a potential modulation of MPSIEC immune responses by FhNEJ via the regulation of important host defence mediators. These mechanisms could have a crucial role not only in relation to the overall success of parasite invasion but also in the outcome of concomitant infections. Particularly, phospholipase A-2 plays a key role in the initiation, propagation and resolution of inflammation at the intestinal level. This enzyme is considered one of the main components of the innate immune system of the intestinal epithelial barrier against invading microbes due to its bacteriolytic properties . LikewisThe stimulation of MPISEC with FhNEJ also resulted in the upregulation of IFITM3, an interferon-induced protein that is also constitutively expressed by virtually all human and mouse cell types. IFITM3 restricts cellular entry of enveloped viruses, including SARS coronaviruses , by eithNotwithstanding, and although a number of proteins with potential immune-related functions have been identified, further experiments aimed at testing the ability of MPSIEC exposed to FhNEJ to respond to specific Th1-inducing ligands should be performed in the future to clarify the immunomodulatory capacity of the FhNEJ in this model.F. hepatica infection  and soma (B) extracts of FhNEJ are shown. The size and colour of each circle represents the Nodescore of each GO term, and the spacing between circles refers to the similarity between the terms represented.(JPG)Click here for additional data file.S2 FigThe MF GO terms corresponding to up- and down-regulated proteins in the cytosol (A) and membrane (B) extracts of MPSIEC are shown. The size and colour of each circle represents the Nodescore of each GO term, and the spacing between circles refers to the similarity between the terms represented.(JPG)Click here for additional data file.S1 TableF. hepatica sequences available in this database (downloaded on 25 February 2020), whereas the remaining data were obtained during the Blast2GO annotation pipeline described previously.Uniprot accession codes were assigned as the match with the highest score in a BLAST search against the (XLSX)Click here for additional data file.S2 TableData were obtained during the Blast2GO annotation pipeline described previously.(XLSX)Click here for additional data file.S1 VideoIn the video, a single FhNEJ can be observed crawling over the MPSIEC monolayer with a characteristic piston movement.(MOV)Click here for additional data file.


Minimum clinically important difference (MCID) is a measure that defines the minimum amount of change in an objective score of a clinical test that must be reached for that change to be clinically noticeable. We aimed to find the MCID for patients with predominantly negative symptoms of schizophrenia at its earliest occurrence.1); as the difference between the PANSS-FSNS change associated with the first instance and the PANSS-FSNS changes associated with the last recorded clinically unchanged status across all visits (MCID2); with the effect size approach (MCID3); as the Youden Index based cut-off value between no clinical change and minimal improvement (MCID4); as the relative likelihood of minimal improvement (MCID5).Data of a 26-week long, double-blind study with 454 patients  treated with cariprazine 4.5 mg/d or risperidone 4 mg/d were analyzed. The Clinical Global Impression—Improvement scale was used to quantify minimum improvement (CGI-I = 3) and no clinical change (CGI-I = 4) on the PANSS-FSNS, and the MCID was estimated with the following methods: as the mean PANSS-FSNS changes corresponding to the first instance of minimal improvement across all visits  and a 1.5-point (7.3%) decrease from baseline severity on the PANSS-FSNS. Greater values were required for the MCID at later evaluation times. The cut-off between minimum improvement and no clinical change defined by the Youden Index was a−3-point (15%) change in the PANSS-FSNS. The effect size approach indicated the 1.5-point difference between minimally improved and unchanged patients to be a medium effect (ES = 0.6).The MCIDApplying different methods led to different results, ranging between 7.3 and 18.5% improvement from the baseline for the MCID at its earliest occurrence in patients with predominantly negative symptoms of schizophrenia. The efficacy of various treatment interventions can be assessed in clinical trials by testing for statistical significance, yet a statistically significant change on a symptom scale score does not necessarily indicate a clinically relevant improvement . Thus, vCalculations of the MCID are usually divided into two groups: anchor- and distribution-based approaches. In anchor-based methods, an objective outcome measure of change is linked to a clinically meaningful external anchor, largely corresponding to patient perception , 6, 7 orIn schizophrenia, the Positive and Negative Syndrome Scale (PANSS) measuring positive, negative, and general psychopathology, is the gold-standard instrument for assessing symptom severity . The cliDepending on the diagnostic criteria applied, negative symptoms of schizophrenia are present in 5–60% of patients with schizophrenia . These sFinding the minimum clinically important change for negative symptoms may help physicians in better assessing treatment results as well as fostering the development of new instruments. In this paper, we further analyse the MCID in negative symptoms of schizophrenia, hypothesizing that as patients get better by taking their medication, more extensive changes in the PANSS-FSNS are needed to be considered clinically relevant. The previous estimation by Leucht et al. took all PANSS-FSNS changes associated with minimal improvement into account, regardless of their timepoint  meaning Data were analyzed from a large randomized, double-blind clinical trial treating patients with schizophrenia with predominantly negative symptoms; the study's methods and results have been previously published . The stuMale and female patients with schizophrenia and predominantly negative symptoms, between 18 and 65 years of age, who were diagnosed with schizophrenia (as defined by DSM-IV-TR) and an onset of illness ≥2 years prior, were included in the study. Patients also needed to be in stable condition for at least 6 months with no hospitalisations. For study inclusion, patients must have presented with predominantly negative symptoms for ≥6 months, a PANSS-FSNS ≥24, and a score ≥4 on at least 2 of the following 3 PANSS negative symptom items: blunted affect, passive/apathetic social withdrawal, and lack of spontaneity and flow of conversation. The presence of a current DSM-IV-TR axis I disorder other than schizophrenia, and an unstable condition , a PANSS factor score for positive symptoms (PANSS-FSPS) >19, or a PANSS-FSPS score increase of ≥25% during study lead-in were grounds for study exclusion. Other clinical exclusion criteria included substance abuse/dependence, treatment with clozapine during the 12 months before the study, a history of non-response to an adequate trial of risperidone for a psychotic episode, or treatment with risperidone within 6 weeks of screening .The clinician-rated CGI-I scale was used to quantify minimum improvement (CGI-I = 3) and no clinical change (CGI-I = 4) on the PANSS-FSNS. To demonstrate any meaningful results by linking an objective scale measuring symptom severity to a subjective scale that estimates the clinical state correlation between the two scales must be demonstrated in the target population. This was done by Leucht et al., who performed the correlation analysis in the same population on which this work is based .According to the definition of the MCID, a within-subject design was applied to estimate the difference between no clinical change and minimal improvement. Our observation was that PANSS-FSNS changes corresponding to minimal improvement get higher and higher over time, and thus, in order to capture the minimum clinically important difference, the MCID should be calculated on the basis of PANSS-FSNS changes associated with the earliest instance of minimal improvement. The MCID was estimated with the following methods.1: The mean PANSS-FSNS changes corresponding to the first instance of minimal improvement (CGI-I = 3) across all visits. In other words, MCID1 is based on the original definition by Jaeschke et al.  and the PANSS-FSNS changes associated with the last recorded clinically unchanged status (CGI-I = 4) across all visits. Thus, MCID2 represents the mean score method of Redelmeier and Lorig  analysis with improvement and visit as fixed effects. The subject was used as random effect in the model. To avoid losing cases with minimal improvement at the first visit after baseline (Week 1), zero PANSS-FSNS change was imputed for the baseline visit (where no improvement can be present by definition).3: the effect size approach, based on the standardized response mean difference, a widely used distribution-based method to estimate the MCID, where MCID is the mean difference between the last unchanged (CGI-I = 4) and the first minimally improved (CGI-I = 3) PANSS-FSNS values divided by the pooled standard deviation (SD) of the two. It formally corresponds to the effect size calculation, making it possible to interpret the MCID in terms of the effect sizes  based on the cut-off value between no clinical change (CGI-I = 4) and minimal improvement (CGI = 3). A logistic regression model, with CGI-I as dependent and PANSS-FSNS as independent variables as well as baseline PANSS-FSNS as a covariate, was fitted to the data. To examine the accuracy of predicting improvement based on the PANSS-FSNS change, a receiver operating characteristics (ROC) curve was derived. The strategy used in the ROC analysis was to maximize both sensitivity and specificity, and the MCID was estimated as a cut-off value corresponding to the maximal Youden's index  of being in improved vs. unchanged state at a certain degree of FSNS decrease. This estimation is based on the logistic regression model as above and expresses the strength of the predictive power of unity change in FSNS for clinical improvement.MCIDp = 0.0022; effect size = 0.31]. Also, for the secondary efficacy parameter, least squares mean change from baseline to endpoint in PSP total score, was greater for cariprazine than risperidone . In the parameters controlling for secondary negative symptoms, least squares mean changes from baseline for PANSS-FSPS, CDSS total score, and movement scales were small and similar for cariprazine and risperidone.The primary results of the study were previously published : change A total of 454 patients from the intent-to-treat population with at least one post-baseline PANSS assessment were pooled for this analysis from both the cariprazine and risperidone treatment groups; the mean factor score on the PANSS-FSNS at baseline was 27.6, with points decreasing to 19.0 points at week 26.Minimal improvement on the clinical global impression scale (CGI-I = 3) was associated with PANSS-FSNS changes ranging from −2.5 points (Week 1) to −7.1 points (Week 26), consistent with our hypothesis of the MCID to be smaller at its earliest occurrence .The mean PANSS-FSNS change from baseline corresponding to the first occurrence of minimal improvement (CGI-I = 3) and the last recorded unchanged status across all visits were −3.8 and −2.3 points, respectively. The statistical analysis of the two arithmetic PANSS-FSNS means showed a significant difference .Based on the observed PANSS-FSNS changes the standardized effect size was determined according to the following formula:  − (Mean PANSS-FSNS Change from baseline at the last recorded unchanged status)/pooled SD. Our computation resulted in a standardized effect size for the improvement with a value of 0.6 .The ROC curve indicated statistically robust predictive values of PANSS-FSNS changes, as the model fitted to our data (Model) was highly significantly different from the reference line (ROC1) . Based op < 0.05) favoring CGI-I = 3 vs. 4, The logistic regression analysis described above yielded an estimated OR of 2.07  for a 2-point decrease in the PANSS-FSNS factor score during the study. Thus, this analysis indicates that such an improvement  in the PANSS-FSNS factor score more than doubles the likelihood of achieving minimal clinical improvement in the study.The odds ratio (OR) indicates the strength of association between the decrease in FSNS and the improved state based on CGI-I. For the −1.7 shift in FSNS obtained above as estimated MCID based on the Least Squares Mean Difference (LSMD), the OR is 1.86  as being associated with a 27% decrease in the PANSS-FSNS for patients with predominantly negative symptoms of schizophrenia  which maIt is important to note that the five different approaches that we adopted in the current investigation to characterize MCID complement each other and delineate the MCID from various vantage points. The distribution-free approaches characterize change over time in terms of the original units on the scale of interest (PANSS-FSNS), either as a baseline-end point difference associated with the minimal improvement on the CGI-I (score = 3) (MCID1) or the difference between unchanged and minimally improved PANSS-FSNS values (MCID2). The first one of the distribution-based methods that we adopted (MCID3) expresses the difference between unchanged and minimally improved PANSS-FSNS values  in terms of standard deviation  units. The additional two distribution-based approaches employ logistic regression modeling in order to predict the minimally improved status on the basis of the PANSS-FSNS change over time. They express MCID either as measures of the ROC Curve  or an OR (MCID5).Applying these approaches, we confirmed that over time, more and more prominent symptom changes were needed to achieve minimal clinical improvement. In view of this finding, we conclude that the absolute minimum clinically meaningful difference should be considered at the earliest instance, since this approach provides the highest assay sensitivity to detect clinically important changes of symptom severity over time . We note that the PANSS-FSNS of the patients whose clinical status remained the same slightly decreased as well, although to a much smaller extent. This slight symptom reduction of the clinically not improving patients may be attributable to unspecific changes, such as the regression to the mean effect, a phenomenon often seen when applying strict inclusion criteria for the patients regarding their symptom severity . The pre3, i.e., statistical effect size in terms of standardized mean difference = 0.6); and highly significant predictive power for the PANSS-FSNS scale for differentiating Minimally Improved from Clinically Unchanged status in terms of ROC measures  and the odds ratio .Overall, applying various underlying concepts to calculate the MCID, different methods led to different estimates with respect to the PANSS-FSNS change that need to be considered as minimally clinically significant. Our estimates from the anchor-based analyses, ranging from 7.3 to 18.5% for patients with predominant negative symptoms of schizophrenia, were below the estimates reported from studies that relied on more heterogeneous patient populations and did not take the time effect into consideration. However, it is important to bear in mind that the distribution-based statistical approaches showed a marked separation , analysis, and interpretation of data and decided to submit it for publication.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The recent coronavirus disease (COVID-19) pandemic mainly affects the respiratory system; however, several oral and maxillofacial post-COVID-19 complications have also been observed. This series reports the growing number of osteonecrosis cases associated with post-COVID-19 patients.This is a retrospective, multi-center case series that reports cases with maxillary osteonecrosis after various periods of SARS-CoV-2 infection in the period between January and August 2021 based on the PROCESS guidelines.Twelve cases were reported with post-COVID-19 manifestation of spontaneous osteonecrosis of the maxillary jaw. Five patients were hospitalized during COVID-19 management and all of the twelve cases had at least one systematic Co-morbidity, and undertake corticosteroids prescription based on the COVID-19 disease treatment protocol. The mean onset of osteonecrosis symptoms appearance was 5.5 ± 2.43 weeks calculated from the day of the negative PCR test. The management was successfully done through surgical debridement and pre and post-operative antibiotics. No anti-fungal medications were prescribed as the fungal culture and the histopathological report were negative.Post-COVID-related osteonecrosis of the jaw (PC-RONJ) could be now considered as one of the potential post-COVID-19 oral and maxillofacial complications that occurs unprovokedly and mainly in the maxilla.The online version contains supplementary material available at 10.1186/s12879-022-07518-9. Although many patients recover from COVID-19, it is important to keep in mind that there may be complications after recovery. One such complication in the maxillofacial region is Avascular Necrosis (AVN), which may lead to deteriorating outcomes and significant tissue loss -Scan) to delineate the extent of the affection. The CT image also showed the affection of the maxillary sinus, and whether it was obliterated or clear. Any provoking stimulus in the post-COVID infection was reported, including recent dental treatment, any type of trauma, or wearing a removable partial denture Fig. . A microManagement of the patient comorbidities was performed before the start of the surgical phase. All patients signed informed consent before the start of the treatment protocol. All of the reported subjects were hospitalized and prepared for general anesthesia, where surgical debridement was performed. The necrotic maxilla was exposed via a sulcular incision, and the margins between the vital bone and the sequestrum were detected. Separation and debridement of the dead bone were performed, followed by smoothing of any sharp bony edges, copious saline irrigation, and water-tight suturing. The specimen was sent for histopathology analysis. The patients were put under culture-specific postoperative antibiotic coverage for a minimum of 3 weeks based on the clinical pharmacist’s recommendation.The study was based on the declaration of Helsinki recommendations. All of the investigated patients have signed an informed consent before embarking in this study after clarifying the study purpose, risks, and benefits  weeks, calculated from the day of the negative PCR test. No precipitating event or provoking factor were reported in the period prior to the onset of the osteonecrosis symptoms in any of the twelve reported patients.Regarding the clinical manifestations, a mobile dentoalveolar maxillary segment was observed in nine patients, a pus-oozing fistula in eight patients, palatal swelling in three patients, and exposed necrotic bone with mucosal ulceration in four patient Fig. .Fig. 2VaEnterococcus spp, Neisseria spp, and Staphylococci were isolated respectively.The fungal culture and microscopic examination came out to be negative in all of the reported cases, indicating the absence of any fungal infestation or growth. Bacterial growth was detected from the bacterial-culture swab of all of the reported cases. A stained smear of gram negative, ampicillin and sulbactam resistant bacilli was isolated in nine swabs. In the remaining three swabs, A common surgical protocol was proposed to all of the study patients, where a surgical debridement was performed, followed by culture-based antibiotic therapy for a minimum period of 3 weeks. No antifungal agent was prescribed in any of the reported subjects. All of the patients were followed for a minimum of 2 months, during which, no recurrence or propagation of the inflammatory processes was reported. A confirmatory C-reactive protein (CRP) blood test revealed normal values in all cases.A recurring histopathological picture was reported in all cases. Examination of Hematoxylin and Eosin-Stained (H&E) sections demonstrated bony trabeculae with numerous necrotic fragments and empty osteocytic lacunae. The bone marrow showed heavy infiltration of inflammatory cells, mainly composed of lymphocytes, histiocytes, plasma cells, and dense sheets of polymorphous and osteoclasts giant cells. This chronically inflamed fibrous tissue was found along with areas of hemorrhage and dystrophic calcification Fig. . No fungMany factors have the potential to contribute to the induction and propagation of osteonecrosis of the jaw related to post COVID-19 infection. The main factor is the virus itself, which causes ACE-2 downregulation and subsequent hyperinflammatory state, with elevated cytokines and immune dysregulation, in addition to microvascular thromboses and the hypercoagulability state , 4. The This is a report of twelve SARS-CoV-2 infection-survived cases with manifestations of maxillary jaw osteonecrosis, which developed after the infectious course of the disease over various periods. None of the patients had fungal infection, confirmed by negative preoperative fungal swab, negative histopathological reports, and the treatment consisted of surgical debridement and antibiotics without any antifungal medications. The mean time until the diagnosis of osteonecrosis was 5.5 ± 2.43 weeks after recovery from SARS-CoV-2 infection. This time frame is in accordance with Boymuradov et al., who reported a minimal elapsed period of 2 months after SARS-CoV-2 infection to develop the symptoms of osteonecrosis, and a maximum symptom-free period of 8 months .All patients in this study received corticosteroids for the treatment of COVID-19. Recent studies on COVID-19 recovered patients have shown the increased risk of developing osteonecrosis with the use of glucocorticoids , 17. DalAll of the included patients have additional comorbidities; where all of them were diabetic. Several reports have linked the association between COVID-19 in patients with diabetes with the occurrence of necrosis of the jaws and other bones , 20, 21.Interestingly, all of the reported cases which considered to have a potential relationship between COVID-19 and osteonecrosis of the jaw were exclusively found in the maxilla. This may be attributed to the close proximity to the nasal mucosa and maxillary sinus. Furthermore, the high expression of ACE-2 receptors in the nasal and oral mucosa epithelial cells may act as a vital risk factor for the occurrence of decreased blood supply , 6. It iBagaria (2021), in an attempt to explain those peculiar orthopedic post-COVID manifestations, postulated that aggressive SARS-CoV-2 infection depletes immune defense mechanism, which leaves the patient vulnerable to infection episodes. This phenomenon is called ‘Consumption Immuno-compromisation’. In his report, Bagaria (2021) reported the occurrence of two cases of spontaneous osteonecrosis of the long bone and two cases of acute primary septic joint, along with a high rate of postoperative infection . All of Several authors have reported the spontaneous occurrence of jawbone necrosis without any precipitating events or predisposing factors , 24. KhoTo the best of our knowledge, this is the first series of reported cases with maxillary osteonecrosis following SARS-CoV-2 infection. This article is limited by its observational pilot nature and limited sample size. The exact pathogenesis of jaw necrosis, and whether the virus contributing effect alone can initiate pathological vascular necrosis without the ramifying effect of steroid therapy or patients comorbidity are topics for future research. Furthermore; the decrease in blood supply to the affected hard and soft tissues of the maxilla could not be evaluated in this study. A prophylactic approach should be presented for the early detection of bone osteonecrosis by the assessment of the vasculature of the maxillary hard and soft tissues in suspected patients.Finally, several recurring risk factors must be built before considering a patient to have PC-RONJ. A recent SARS-CoV-2 infection with concurrent cumulative corticosteroid prescription, systematic co-morbid conditions, no history of current or previous radiotherapy, antiresorptive, or antiangiogenic agents intake all of which are considered as precipitating factors for the occurrence of un-provoked jawbone osteonecrosis.A practical guidelines is called for the prevention, diagnosis, and management of jaw osteonecrosis in patients with recent SARS-CoV-2 infection. With the increasing rate of COVID-19, the general dentists and oral and maxillofacial surgeons must be best prepared and positioned for the early identification and prevention of PC-RONJ to avoid permeant deformity. There is a need for communication between oral and maxillofacial surgeons and physicians to reduce the severity and the vast degree of avoidable morbidity that may occur as a consequence of an unjustified corticosteroid prescription, where they can modify the COVID-19 treatment protocol. A specialized follow-up plan for the early detection of PC-RONJ should be proposed for patients at risk of developing osteonecrosis, as the spontaneous nature of this condition occurs with silent impact.Jaw osteonecrosis occurrence in patients recovered from SARS-COV-2 infection is of a potential concern. Medically compromised patients and those who are prescribed corticosteroids are considered as significant risk factors. Future studies are required to confirm whether the virus itself or the other factors lead to jaw osteonecrosis.Additional file 1. A video document demonstrating a mobile dento-alveolar segment.