However, we observe a large defect in successful expression of proviral genes at early times after infection by an acetylation-deficient IN mutant that cannot be explained by delayed integration dynamics. We demonstrate that the difference between the expression of proviruses integrated by an acetylation mutant and WT IN is likely not due to altered integration site distribution but rather directly due to a lower rate of transcription. Further, the effect of the IN mutation on proviral gene expression is independent of the Tat protein or the LTR promoter. At early times after integration when the transcription defect is observed, the LTRs of proviruses integrated by the mutant IN have altered histone modifications as well as reduced IN protein occupancy. Over time as the transcription defect in the mutant virus diminishes, histone modifications on the WT and mutant proviral LTRs reach comparable levels. These results highlight an unexpected role for the IN protein in regulating proviral transcription at early times post-integration.The central function of the retroviral integrase protein (IN) is to catalyze the integration of viral DNA into the host genome to form the provirus. The IN protein has also been reported to play a role in a number of other processes throughout the retroviral life cycle such as reverse transcription, nuclear import and particle morphogenesis. Studies have shown that HIV-1 IN is subject to multiple post-translational modifications (PTMs) including acetylation, phosphorylation and SUMOylation. However, the importance of these modifications during infection has been contentious. In this study we attempt to clarify the role of acetylation of HIV-1 IN during the retroviral life cycle. We show that conservative mutation of the known acetylated lysine residues has only a modest effect on reverse transcription and proviral integration efficiency  A key step of the retrovirus life cycle is the insertion of the viral DNA genome into the host cell genome, a process called integration. The process of integration is solely catalyzed by the virally encoded integrase (IN) protein. IN has been reported to influence a number of other viral processes such as reverse transcription, nuclear import and particle morphogenesis. The HIV-1 IN protein is known to be heavily post-translationally modified. In light of the known effect of post-translational modifications on the function of the orthologous proteins of certain retrotransposons, we were motivated to ask how post-translational modifications of HIV-1 IN may regulate its various functions. In this study, we examined the consequences of mutations preventing the acetylation of the IN protein on the retroviral life cycle. Surprisingly, we saw that mutations blocking IN acetylation had only modest effects on viral DNA integration. Instead, we uncovered a novel function for HIV-1 IN in regulating proviral transcription at early times after infection. Our data suggests that IN may be retained on proviral DNA at early times after integration and promote proviral gene expression by altering chromatin modifications at the viral transcriptional promoter. A key component of a successful retroviral infection is the integration of the proviral genome into the host cell genome. The irreversible process of retroviral integration is imperative for maintenance of infection as it establishes a stable template for proviral transcription and thus continued viral replication. Integration also establishes the provirus as an inextricable component of the host genome, which is a major barrier to treatment and cure of retroviral infections.The process of integration is catalyzed by the virally encoded integrase (IN) protein. After the viral RNA genome is reverse transcribed to a double-stranded DNA, IN specifically recognizes and binds the ends of the linear viral DNA, consisting of long terminal repeats (LTRs). Integration is a concerted reaction that consists of two catalytic steps–end processing and joining. During end processing, a dinucleotide is cleaved from the 3’ strand at either LTR end exposing a 3’-hydroxyl group that subsequently attacks the host target DNA during the joining process. This leaves a gapped DNA with viral overhangs that must subsequently be repaired by host enzymes .In addition to the main catalytic function of the IN protein, it has long been known that this protein plays additional roles in the viral life cycle . MutatioSuccessful viral integration is important for persistence of the virus, but is also required for efficient transcription of proviral genes ,9. Gene Post-integration transcription of the HIV-1 provirus is regulated by availability of host transcription machinery, and the virally encoded Tat protein, and is reflected in the chromatin state of the viral promoter . HIV-1 pAfter the HIV-1 DNA is integrated into the host cell genome, it is also subject to the same chromatin-based regulatory mechanisms that modulate host gene expression . TranscrEvidence suggests that the site of HIV integration in the human genome plays an additional role in determining proviral transcriptional activity . RetroviThis mechanism of integrase-mediated targeting is seen in other retroviruses as well as retrotransposons . Murine in vitro [in vivo has been more contentious. Initial studies reported an extreme defect in integration in vivo in the absence of IN acetylation [The HIV-1 IN protein is also known to be post-translationally modified but little evidence to date suggests that these post-translational modifications (PTMs) similarly regulate integration site selection . The besin vitro . The efftylation . Howevertylation .In this study, we conservatively mutate the four known acetylated lysine residues in the CTD of HIV-1 IN. We show that ablating acetylation of the HIV-1 IN protein has only modest effects on early steps of the retroviral life cycle such as reverse transcription and integration. However, we observe a severe defect in transcription of proviral genes at early times after infection by a virus expressing an acetylation-deficient IN mutant. The observed transcriptional defect present in proviruses integrated by an acetylation-deficient mutant IN cannot be explained by delayed integration dynamics in the mutant and is also not confined to the context of Tat-dependent transcription or even the LTR promoter. Mapping the genomic locations of proviruses integrated by either the acetylation mutant or WT IN reveals that these differences are not due to altered integration site selection but rather to a lower rate of transcription. The poor transcription is accompanied by the presence of altered histone modifications present on the viral LTR. We observe significantly less of the active chromatin mark of H3 acetylation on the LTRs of proviruses formed by an acetylation-deficient IN. The transcription defect in the acetylation-deficient mutant virus diminishes over time and the presence of active chromatin associated histone modifications correspondingly returns to wild-type levels. Further, these mutations in IN decrease the occupancy of the IN protein on the proviral DNA suggesting that IN may normally be retained on the viral DNA after integration and plays an active role in establishing a permissive chromatin environment for transcription. To our knowledge this is the first evidence that the IN protein can influence proviral transcription post-integration and raises the possibility that PTMs of IN, such as acetylation, may coordinate this function.HIV-1 integrase has four lysine residues in the C-terminal domain known to be acetylated–K258, K264, K266 and K273 ,42. We cHeLa cells were infected with equal quantities of fresh virus and successful transduction was quantified two days post-infection using a luciferase reporter assay . MutatioPrevious reports, while controversial, also described reductions in reporter gene expression from acetylation-deficient IN mutants, and attributed this difference to integration defects . To deteResidues in the CTD of IN have also previously been identified as class II mutants that are defective for viral RNA binding and particle morphogenesis, and as a consequence the virions are defective in early steps of infection such as reverse transcription and nuclear import . To detetat shown). To account for any differences in levels of integrated proviral DNA, the viral mRNA levels were normalized to viral DNA content as measured by qPCR. We observed a highly significant decrease in global viral mRNA levels from proviruses formed by the acetylation deficient QA mutant IN . To ensure that our introduced mutations were not uniquely affecting tat message, we also quantified steady state levels of other viral transcripts , the luciferase reporter assays suggested a large defect in successful retroviral transduction. We therefore tested the effect of the QA mutation on the next step of the viral life cycle–proviral transcription. We measured steady state mRNA levels of various viral genes at 48 hours after infection in HeLa cells  pulse-labeling experiment. This allows us to more directly measure differences in newly synthesized proviral transcripts. Cells were infected with QA mutant and WT virus preparations, and at 48 hours post-infection were incubated for 4 hours with EU to label nascent transcripts. Total cellular RNA was harvested and labeled RNA was subsequently isolated. Viral-specific RNA was quantified by qRT-PCR using primers against various transcripts and normalized to total viral DNA. We observed a substantial 80-fold average decrease in nascent viral transcripts produced from proviruses integrated by QA mutant IN vs. WT IN regardless of the viral message quantified  mutant IN we monitored detectable proviral integration over a 14-day time course using an Alu-gag nested qPCR approach . Consisttat mRNA splicing or expression, then a deficit of Tat protein would cause a dramatic decrease in proviral transcription regardless of the mutation status of the IN protein.The pNL4.3 viral vector utilized to read out the effects of IN mutations on the level of transduction generates virion particles with mutant IN protein, and thus monitors the function of that protein during the early steps of infection. But in addition, the provirus formed by infection carries the same mutations, and thus changes in the level of reporter gene expression, in principle, could be mediated by effects on viral mRNA processing or regulation. This is not implausible, because many of the IN mutations are located in regions of the viral genome utilized for splicing, generating Tat and Rev proteins, and HIV-1 proviral transcription relies heavily on the presence of sufficient Tat protein . If mutagag-pol allowing us to separate out the mutation from the integrated viral DNA sequence.To determine if the effect of the acetylation-deficient mutant IN on transcription is genuinely due to the mutant IN protein or to defects in splicing dynamics, we made use of a reporter gene driven by a Tat-independent, CMV promoter. Virus was made by co-expressing a plasmid expressing HIV-1 Gag-Pol protein with either the WT or QA mutant IN sequences, along with a minimal transducing viral genome carrying a CMV-driven ZsGreen reporter gene. This allows us to assay reporter gene expression from a provirus that is integrated by WT or QA mutant IN but whose expression is independent of Tat protein levels and even independent of the proviral LTR promoter. Further, the mutant IN will only be present in the form of the incoming protein, as the reporter encodes no At 2 days post-infection, we performed flow cytometry on infected HeLa cells to quantify ZsGreen expression. The expression from the proviral ZsGreen reporter gene integrated by the acetylation-deficient QA mutant IN was dramatically decreased relative to proviruses integrated by WT IN. In cells infected with virus possessing a QA mutant IN, there were substantially fewer ZsGreen positive cells , 10-foldThe fact that integration by the QA mutant IN still induces a transcriptional defect of this CMV-driven ZsGreen reporter strongly suggests that the observed phenotype is not a product of perturbation to Tat levels. However, the integrated minimal viral genome in this experiment still contains LTR sequences that could somehow mediate this phenotype. To further remove any effect of the LTR promoter, we repeated this experiment using a self inactivating (SIN) viral genome carrying a GFP reporter gene expressed from a human PGK promoter. Unlike the previous CMV-driven reporter, the integrated SIN-GFP proviral genome has no functional LTR. Even in this context we still observe a strong defect in viral gene expression at two days post-infection despite comparable levels of integrated DNA .These data demonstrate that the large effects of the introduced QA IN mutation on proviral expression are attributable to the mutant IN protein itself and are not Tat-dependent or confined to the context of the LTR promoter. These experiments exclude the possibility that the observed transcriptional defect is a byproduct of perturbations of viral regulatory mRNA processing or changes in the levels of Tat protein. The effect is seen with multiple vectors utilizing varying promoters, and in multiple cell lines.The data presented thus far indicate that proviruses integrated by an acetylation-deficient mutant IN have a significantly lower rate of transcription. This effect is not due to decreased frequency of integration and is independent of the Tat protein or the viral LTR promoter. If the mutations ablating IN acetylation are not affecting IN catalytic activity, another plausible explanation is that these mutations may be affecting integration site targeting. If acetylation deficient IN alters integration site selection, then transcription from these mutant proviruses could be reduced compared to WT proviruses due to chromosomal position effects. WT IN normally binds LEDGF and targets integration to active gene regions . The mutWe collected genomic DNA from independent infections in HeLa cells and amplified the viral–host genome junctions using nested rounds of PCR. Subsequent high throughput sequencing of the viral-host junctions allowed us to map viral integration sites in the human genome and to compare locations of integrations with various genomic features such as annotated genes, transcription start sites and CpG islands as well as the pre-infection genomic locations of common proteins or histone modifications as deduced from ChIP-seq data. Three distinct NGS runs were performed from independent infections to account for biological variation. Integration frequency near features was determined for each run and is presented as an average of the repeat experiments. Differences in integration site distribution of proviruses formed by WT or QA IN were gauged for significance relative to each other as well as relative to a matched random control (MRC) using a paired t-test .We observed that the WT and QA mutant proviruses have a similar distribution of integration sites. Both mutant and WT proviruses integrated with equivalent frequencies near RefSeq genes, CpG islands, DNase hypersensitive regions and RNA polymerase II binding sites . The disMapping of integration sites in infected cells allowed us to determine preferences for integration near the location of specific pre-infection histone modifications but does not report the histone modifications on the viral LTR itself. To look directly at the histone modifications present on the viral DNA we performed chromatin immunoprecipitation with antibodies specific for total or acetylated histone H3 followed by qPCR with LTR specific primers. At early points after infection (2 dpi), the WT proviral DNA was heavily loaded with acetylated histone H3 (H3K27ac), a chromatin mark known to be associated with active chromatin. Significantly less of the QA mutant proviral LTR than the WT LTR was associated with acetylated histone H3 , p<0.05.The IN protein could affect the local chromatin environment on the viral LTR in a number of ways. First, IN is known to interact with and recruit many host cell factors that aid in the process of integration and it seems plausible that the introduced mutation may disrupt IN-host factor interactions that could lead to changes in chromatin. Second, the QA mutation in IN may affect binding or retention of the IN protein on the viral DNA itself after integration and thereby fail to recruit necessary machinery for active transcription.To differentiate between these possibilities we first immunoprecipitated either WT or QA mutant IN proteins and performed a mass-spectrometry screen of the associated proteins to identify differentially binding host factors (data not shown). The repertoire of host factors that precipitated with both IN proteins was nearly identical, with no single host factor detected with significantly differential binding. The result made it unlikely that differential host factor interaction could cause such a large discrepancy in proviral expression levels.In vitro studies on acetylation-deficient IN have shown that acetylation of IN promotes viral DNA binding [Recently, it has been shown that the PFV intasome is stabilized on the provirus after strand transfer . It is n binding . Thus, win vivo, we infected HeLa cells with mutant and WT virus preparations, and performed chromatin immunoprecipitation (ChIP) of genomic DNA using antibodies against IN, and scored for the recovery of viral DNA sequences by qPCR. We found that the IN protein was indeed associated with viral DNA in these assays. Importantly, we detected significantly less viral DNA bound by the acetylation-deficient QA IN as compared to WT  of the IN protein is the least conserved portion of the protein. Despite this, avian leukosis virus (ALV) has six lysines in the CTD, three of which are in sequences that can be aligned with sequences in the HIV-1 IN  cells with ALV vectors encoding either the WT or K6R mutant IN with VSV-G envelope protein. At 2 days post-infection, luciferase reporter expression was assayed and was found to be indistinguishable between WT and mutant viruses . Viral Din vivo. Our mutant vectors show at most a 2–3 fold decrease in integration frequency as compared to the WT control, which appears to be largely a consequence of decreased viral DNA levels available for integration  vector and thus has no LTR promoter present after integration. In both cases, the proviral reporter gene was expressed significantly less efficiently when integrated by an acetylation-deficient mutant IN protein, indicating that the effect we see is independent of both Tat and the viral LTR promoter . The replication-defective pNL4.3 luciferase reporter construct lacks envelope and expresses firefly luciferase from the gag-pol was used to generate virus preparations with various reporter constructs. Two different reporter constructs were used to separate effects of Tat dependency and the LTR promoter on proviral transcription. The pLVX-IRES-ZsGreen plasmid is a minimal lentiviral expression vector consisting of HIV-1 packaging and LTR sequences and an internal CMV-promoter driven ZsGreen reporter gene (Takara Bio). Second, a self inactivating (SIN) reporter construct, pRRLSIN.cPPT.PGK-GFP.WPRE (Addgene 12252), was used to deliver a GFP reporter gene driven by a PGK promoter.The packaging construct pCMV delta R8.91 expressing HIV-1 ALV viral constructs were based on the single-round infection vector pRIAS which lacks envelope and contains a luciferase reporter gene ,62.Conservative point mutations in the integrase coding sequence were introduced into all plasmids using PCR site-directed mutagenesis (See All DNA transfections were performed using Lipofectamine 3000 (Life Technologies) following the manufacturer’s protocol.gag-pol with WT or mutant IN sequence was co-transfected with the appropriate reporter construct–either pLVX-IRES-ZsGreen or pRRLSIN.cPPT.PGK-GFP.WPRE–and plasmid pMD2.G expressing VSV-G envelope. Pseudotyped ALV was produced in chick embryonic fibroblast (CEF) cells by co-transfection of the pRIAS viral vector and pMD2.G.To generate pseudotyped HIV-1 virus for infections, the viral vector pNL4.3.Luc.R-E- (or derivative mutant vector) was co-transfected with a plasmid encoding VSV-G (pMD2.G) in HEK293T cells. To package lentiviral reporters, pCMVdeltaR8.91 encoding HIV-1 Viral supernatants were collected 48 hours post-transfection and treated with RQ1 DNase (Promega) to remove residual plasmid DNA. Viruses were used immediately after collection and diluted 3-fold with cell culture medium for infections. Virus quantities were normalized prior to infection by quantification of viral RNA genome content by qRT-PCR.Successful viral transduction was assayed using either a luciferase assay or flow cytometry depending on the reporter construct used. Luciferase activity was assayed using the Promega Luciferase Assay System (Cat# E4550). ZsGreen or GFP positive cells and mean fluorescence intensity (MFI) were measured by flow cytometry using an automated cell analyzer .-ΔΔCt method and the results were first normalized for total DNA input by a host housekeeping gene and then expressed as fold change relative to the WT control.To assay viral DNAs formed after acute infections, total cell DNA was collected from infected cells using the Qiagen DNeasy Blood and Tissue kit. Subsequent quantitative PCR was performed on genomic DNA using FastStart Universal SYBR Green Mastermix (Bio-Rad) according to manufacturer’s protocol on ABI 7500 Fast Real Time PCR System. Total viral DNA was quantified using primers complementary to the luciferase reporter sequence. Late reverse transcription products were assayed with primers in the LTR. 2-LTR circles were quantified as previously published and normalized to a housekeeping gene . Integra-ΔΔCt method for quantification was used to score levels of integrated DNA, and expressed relative to the last collected time point as a measure of the final levels produced by the single-round vectors. Finally, as a more direct comparison, the ratio of integration quantified in cells infected with the mutant QA IN virus relative to WT virus was calculated.To monitor integration timing dynamics, DNA was collected from infected HeLa cells at 2, 4, 7, 10 and 14 days post-infection. Integration frequency was measured by Alu-gag nested PCR. The 2A minimum of three biological replicates were performed for each assay. Biological replicates consist of completely independent virus preparations and cells, infections, and assays. DNA preparations and qPCR assays were done independently. Within each biological replicate there are technical duplicates for consistency. A single factor ANOVA analysis was used to confirm significant changes within each experiment . When appropriate, this analysis was followed by pairwise comparisons using a two-tailed t-test assuming unequal variance in Prism.Steady state RNA levels were measured by collecting total RNA from infected cells at designated times post-infection using a standard Trizol protocol. Reverse transcription was subsequently performed using random hexamer primers and Maxima H Reverse Transcriptase (Thermo Fisher).5-ethynyl uridine (EU) labeling was performed using the Click-It Nascent RNA Capture Kit according to manufacturer’s protocol (Thermo Fisher). Briefly, two days post-infection, cells were pulse-labeled by incubation for 4 hours in medium containing 0.5 mM EU ribonucleotide homologs containing a reactive alkyne group. Total RNA was collected, biotin azide was attached to the incorporated EU ribonucleotides and the RNA was subsequently recovered by binding with and elution from streptavidin beads. Viral RNA was then quantified by qRT-PCR.tat mRNA, luciferase mRNA, or envelope mRNA as well as gag mRNA and all LTR containing sequences . When appropriate, this analysis was followed by pairwise comparisons using a two-tailed t-test assuming unequal variance in Prism.Sequencing libraries of viral-host DNA junctions were prepared as described previously ,66. SpecViral-host DNA junction reads were demultiplexed by unique dual barcodes and filtered to exclude reads not containing an initial viral LTR sequence at the host junction using a custom python script . Reads fUsing Bowtie2, sequences were aligned end-to-end using a seed length of 28 nucleotides, with a maximum of 2 mismatches permitted in the seed. Alignments for reads were suppressed if more than one reportable alignment existed to prevent multiple mapping and ensure that reads corresponded to unique integration sites. Multi-mapping reads were written to a separate FASTA file and subsequently mapped to the RepeatMasker genome to analyze integrations into repeat sequences .Genomic coordinates of RefSeq genes, transcription start sites, DNase hypersensitivity regions and CpG islands were extracted from the hg38 genome assembly via the UCSC Genome Browser. Genomic locations of RNA polymerase II binding and histone modifications was extracted from ENCODE data sets . Genomic coordinates of “super enhancers” in HeLa cells were extracted from the SEA 3.0 database . A matchTo gauge the statistical significance of differences in integration patterns between WT IN and acetylation deficient (QA) IN we used a paired t-test of three independent replicate data sets for each condition . A one-sInfected HeLa cells were crosslinked using 1% formaldehyde for 10 minutes at 37°C. Cells were subsequently lysed with ChIP lysis buffer . Chromatin was sheared by sonication with a Branson 450 Digital Sonifier to an average size of 500–1000 nucleotides in length. 25 ug of chromatin as measured by spectrometer was mixed with 2 μg of various specific antibodies and incubated overnight at 4°C. For immunoprecipitation of H3K27ac, a ChIP-validated polyclonal mouse antibody was used (EMD Millipore). Integrase IP was performed with either a mouse monoclonal antibody  or polyclonal antibodies (IN-9379). ChIP with histone H3 (abcam ab10799) and control mouse IgG (abcam 37355) antibodies were performed in parallel as a positive and negative control respectively. Post-incubation, the chromatin-antibody mixture was mixed with Protein A and Protein G beads and incubated with rotation for 2 hours at 4°C. Beads were then washed extensively as described . CrossliThe monoclonal antibody used for HIV-1 IN immunoprecipitation was validated for comparable binding to WT and acetylation deficient QA mutant IN by Western blot. The WT or mutant IN open reading frame was cloned into a pJET mammalian expression vector and HA-tagged at the C-terminal end. Equal quantities of plasmid were transfected into HEK293T cells. Transfected cells were lysed and used for Western blot with antibodies targeting the HA-tag or IN.S1 Figtat mRNA levels by RT-qPCR. All measurements were done in parallel. Data shown is average of a minimum of three independent biological replicates +/- SEs. Statistical significance was gauged by paired t-test.Viruses produced from pNL4.3R-E- viral vector with either a WT IN or QA mutant IN sequence were used to infect either (A) HeLa, (B) HEK293T, (C) CEM or (D) Jurkat cells. All infected cells were collected at two days post-infection. From each independent experiment, cells were collected to measure luciferase activity, and genomic DNA and total RNA was isolated in parallel. From genomic DNA, total reverse transcription (RT) products as well as integrated provirus were quantified by qPCR. Transcription was roughly quantified by measurement of steady state (TIF)Click here for additional data file.S2 Figgag-pol. VSV-G pseudotyped virions were produced by co-transfecting the plasmid expressing either WT or QA mutant gag-pol along with a minimal self-inactivating (SIN) viral construct carrying a GFP reporter gene driven by a human PGK promoter. Infected HeLa cells were collected two days post-infection. (A) Representative flow cytometry data of one independent experiment is shown. (B) Average percent of GFP positive cells and (C) mean fluorescence intensity (MFI) after infection with viruses carrying WT or QA mutant IN. Data shown is average +/- SEs of three independent experiments run in duplicate.The QA mutation in the CTD of IN was introduced into pCMV-delta-R8.9 vector expressing only HIV-1 (TIF)Click here for additional data file.S3 FigHeLa cells infected with virus expressing either a WT or QA mutant IN were collected at 24 hours post-infection. Quantity of viral DNA bound to IN protein was estimated via chromatin immunoprecipitation (ChIP) using a polyclonal antibody against the IN protein followed by qPCR using LTR-specific primers. Data shown is average of two independent biological replicate experiments run in duplicate +/- SEs.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TablePaired two-tailed t-test of three independent biological replicate NGS experiments.(DOCX)Click here for additional data file.S3 TableMutated base is highlighted in red.(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


High degree atrioventricular block (HDAVB) is a rare complication of non-ST-elevation myocardial infarction (NSTEMI) and often requires pacemaker implantation. We describe a case of reversal of HDAVB in a patient with dual atrioventricular node blood supply following coronary revascularization and potential mechanisms of such complications. As the prevalence of coronary artery disease in the general population increases, so does the incidence of non-ST-segment elevation myocardial infarction (NSTEMI) . An uncoA 47-year-old female, with a past medical history of hypothyroidism and no significant cardiac history, was admitted with complaints of atypical chest pain and generalized weakness. An initial electrocardiogram (EKG) was performed that revealed 1st-degree atrioventricular (AV) block with a ventricular rate of 42 beats per minute. Physical examination was unremarkable, and labs were significant for a Troponin-I level of 4.89 ng/ml. Emergent cardiac catheterization revealed complete occlusion of the right coronary artery (RCA) Figure . Two houThe resolution of high degree AV block following RCA revascularization has been previously described, but this is the first case of HDAVB resolution in the presence of co-dominant coronary circulation in the setting of NSTEMI .HDAVB is a form of advanced second-degree heart block. It is differentiated from complete heart block by the conduction of some atrial impulses to the ventricles and absence of complete AV dissociation, whereas in former, there is no conduction of atrial impulses and there is complete AV dissociation. In an analysis of nationwide inpatient database, 0.6% of patients admitted with NSTEMI developed HDAVB, and a third of those patients required permanent pacemaker placement . OverallIn our case, the patient presented with 1st degree AV block which quickly progressed to HDAVB and the patient required emergent percutaneous intervention. Left heart catheterization demonstrated variant coronary anatomy seen in only 2% of patients, where the AV nodal branch originates from both the RCA and LCA . The retWe report first case of the patient with dual atrioventricular nodal supply presenting with NSTEMI complicated by high degree AV block. Rapid identification and early coronary revascularization of this abnormality may lead to reversal of conduction block and establishing 1:1 AV conduction thus avoiding the need for pacemaker insertion. Our case supports the ischemia-related mechanism of such complications and emphasizes the role of early intervention in order to improve outcomes.


X-ray ptychography is a rapidly developing coherent diffraction imaging technique that provides nanoscale resolution on extended field-of-view. However, the requirement of coherence and the scanning mechanism limit the throughput of ptychographic imaging. In this paper, we propose X-ray ptychography using multiple illuminations instead of single illumination in conventional ptychography. Multiple locations of the sample are simultaneously imaged by spatially separated X-ray beams, therefore, the obtained field-of-view in one scan can be enlarged by a factor equal to the number of illuminations. We have demonstrated this technique experimentally using two X-ray beams focused by a house-made Fresnel zone plate array. Two areas of the object and corresponding double illuminations were successfully reconstructed from diffraction patterns acquired in one scan, with image quality similar with those obtained by conventional single-beam ptychography in sequence. Multi-beam ptychography approach increases the imaging speed, providing an efficient way for high-resolution imaging of large extended specimens. It has become popular in many research areas  because quantitative information can be obtained on both sample and illumination, with a high-spatial resolution not limited by optics but rather by the maximum angle of scattered signals. Recent advances in technique and algorithm developments also made ptychography more robust to deal with many problems, such as partial coherence18, scanning errors20, and beam variation21.X-ray ptychography is an emerging coherent lensless imaging technique that can produce a high-resolution image of an extended sample22, however, it contradicts the coherence requirement in ptychography due to the decrease of spatial coherence. Recent efforts were made on reconstruction algorithms to deal with partial coherence so that one can use a source with higher flux. For example, the development of the mixed-state method greatly relaxes the coherent requirement of ptychography and enables the use of partial coherence illumination to significantly reduce the exposure time by delivering higher flux on the sample23. In practical scans, a continuous motion scheme 26 has been recently implemented in ptychography to speed up imaging by avoiding mechanical overheads, while the decoherence effect caused by the continuous motion is solved in the reconstruction.Ptychography doesn’t put a limit on the imaging field-of-view (FOV), but the increase of FOV comes at the cost of long scanning time and large data volume. To reduce the experiment time, one can use a higher-flux illumination achieved by opening the source aperture. This method is effectively used in some incoherent imaging systems such as scanning transmission X-ray microscopy (STXM)27 or spatially28 separated) or mutually coherent beams29. In the second case, the interference of exit surface waves from two separated sources makes reconstruction complicated, and careful processing on the data is required to filter out the interference terms29.Imaging with multiple illuminations while maintaining enough coherence degree on each illumination is also a novel and effective approach for high-throughput imaging since separated sample sites with each illumination are imaged simultaneously. This idea was firstly demonstrated in visible light domain using either mutually incoherent beams  array, and used for ptychography scanning. The imaged FOV was doubled in this demonstration while leaving the scan time and acquired data volume unchanged. We also incorporated the partial coherent illumination and the fly-scan technique to the multi-beam approach to further increase the ptychographic throughput. The transmission function of both sample and probe for two illumination beams were reconstructed simultaneously, and the two sample regions were successfully separated and had similar image quality compared with the conventional single beam ptychography.In this paper, we proposed and demonstrated this multi-beam approach for X-ray ptychography, which was completed independently from another similar workO(r) is scanned by multiple illuminations mth illumination, the complex-valued exit wave from the object at the jth scan point can be expressed asr is the coordinate on object plane and jth . X, y encoder positions from the sample translation stages were used as the initial positions for ptychography reconstructions and position correction algorithm32 was implemented during the reconstruction process. The far-field diffraction patterns were collected by a Dectris Eiger 500K detector (75 We demonstrated this technique on a fluorescence microscope at beamline 2-ID-D at the Advanced Photon Source (APS) in Argonne National Laboratory. Ptychography experiment was performed at 8.8 keV photon energy defined by a double-crystal Si111) monochromator. Gold Fresnel zone plate arrays with 2 (row) 11 monoch33. In the reconstruction, both probe functions used an array size of 128 34) on different areas but on the same window acquired by a high-resolution ptychography instrument-the Velociprobe33. Therefore, the line patterns are real and are presumably due to the defects from the fabrication process, displaying the parallelogram (or trapezoid) filling scheme of electron beam lithography35.In the first experimental demonstration, two zone plates with a horizontal spacing of 1 mm as shown in the inset of Fig. entclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minima25 was calculated for the reconstructed region marked by the green box in Fig. To access the quality and the spatial resolution of the double-beam ptychography method, the power spectral density (PSD)30 or additional constraints  in the reconstruction algorithm may be helpful for separating different imaged regions.The two sample regions shown in Fig. For the proof-of-concept experiment, a large separation of 1 mm between two zone plates was used in the above scans. To further increase the scan efficiency using multiple beams, one would consider decreasing the distance between the zone plates so that a higher number of beams can be used in one scan. To decrease the beam distance, we rotated the FZP array by 9029, or developing algorithms to deal with the partial coherence.Further decrease in the beam separation is possible based on the above result with In addition to the data acquisition time, the reconstruction time is also an important factor that influences the ptychographic imaging efficiency. For the same FOV, the reconstruction time of double-beam ptychography is about the same with the time needed for two sequential single-beam ptychography reconstructions, while preserving the same image quality. The reconstruction speed for multi-beam ptychography can be further improved by algorithm development, such as implementing parallelization for multiple beams during the reconstruction process, thus further increasing the ptychography imaging efficiency.The work reported here demonstrates a multi-beam ptychography technique using zone plate focused beams. A set of focusing optics including a FZP array, central stops and OSA array was developed to focus the coming X-rays into multiple compact illuminations which were mutually incoherent. In this proof-of-concept experiment, two illuminations from the FZP array were used under several conditions to show the robustness of multiple-beam ptychography. All the objects and probes corresponding to multiple beams were successfully reconstructed, with imaging quality comparable with conventional single-beam ptychography. In some cases, multiple-beam ptychography works better in position correction because of more information constraints. Since different regions of the sample are illuminated simultaneously with multi-beams in one ptychographic scan, the imaged FOV is proportionally increased by a factor equal to the number of beams, while having a small data volume compared to sequential single-beam scans on the same FOV. Even though only two beams were demonstrated for this multiple-beam technique, more zone plates can be introduced by optimizing the beam separation and the transverse coherence length to further make use of the available X-ray flux to improve ptychography throughput.Supplementary Figures.


The COVID-19 outbreak has had a major impact on clinical microbiology laboratories in the past several months. This commentary covers current issues and challenges for the laboratory diagnosis of infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In the preanalytical stage, collecting the proper respiratory tract specimen at the right time from the right anatomic site is essential for a prompt and accurate molecular diagnosis of COVID-19. Appropriate measures are required to keep laboratory staff safe while producing reliable test results. The COVID-19 outbreak has had a major impact on clinical microbiology laboratories in the past several months. This commentary covers current issues and challenges for the laboratory diagnosis of infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In the preanalytical stage, collecting the proper respiratory tract specimen at the right time from the right anatomic site is essential for a prompt and accurate molecular diagnosis of COVID-19. Appropriate measures are required to keep laboratory staff safe while producing reliable test results. In the analytic stage, real-time reverse transcription-PCR (RT-PCR) assays remain the molecular test of choice for the etiologic diagnosis of SARS-CoV-2 infection while antibody-based techniques are being introduced as supplemental tools. In the postanalytical stage, testing results should be carefully interpreted using both molecular and serological findings. Finally, random-access, integrated devices available at the point of care with scalable capacities will facilitate the rapid and accurate diagnosis and monitoring of SARS-CoV-2 infections and greatly assist in the control of this outbreak. The identification by U.S. public health officials of presumptive COVID-19 cases believed to be due to community transmission of this infection brings into sharp focus the importance of the laboratory diagnosis of infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 15. The cu6–(i) Initial respiratory tract specimen collection for diagnosis and screening of patients with COVID-19 pneumonia. Within 5 to 6 days of the onset of symptoms, patients with COVID-19 have demonstrated high viral loads in their upper and lower respiratory tracts (–n = 398) were used much more frequently than nasal swabs (n = 8) in China during the COVID-19 outbreak; however, the SARS-CoV-2 RNA was detected in only 32% of OP swabs, which was significantly lower than the level in nasal swabs (63%)  . While cIn order to properly obtain an NP swab specimen, the swab must be inserted deeply into the nasal cavity. Patients will likely flinch, but that means the swab has hit the target. Swabs should be kept in place for 10 s while being twirled three times. Swabs should have flocked nontoxic synthetic fibers, such as polyester, as well as synthetic nylon handles . Collect19–After collection, swabs should be placed in viral  transport medium for rapid transportation to the clinical microbiology laboratory, ideally under refrigerated conditions . It shou(ii) Late detection and monitoring of patients with severe COVID-19 pneumonia. Ideally, sputum sampling or bronchoalveolar lavage should be used for collecting lower respiratory tract specimens as they have yielded the highest viral loads for the diagnosis of COVID-19  Safety measures for specimen processing for PCR processing and testing. Processing of respiratory specimens should be done in a class II biological safety cabinet (–http://www.chinamerlin.com/en/index.php?p=products_show&id=166&s_id=&c_id=68&lanmu=2). Because this test is a reverse transcription method, the saliva/swabs used to collect the clinical specimens should be quickly added to lysis buffer to disinfect the specimen as well as to stop degradation of the coronavirus RNA  , 38, cob(i) Assay selection. Immunoassays have been developed for rapid detection of SARS-CoV-2 antigens or antibodies. These rapid point-of-care immunoassays are generally lateral flow assays, but high-throughput immunoanalyzer versions are also in development for population-level screening. Such lateral flow assays have been developed for detecting antigens such as the SARS-CoV-2 virus or for detecting antibodies (IgM and IgG) against COVID-19.Rapid antigen lateral flow assays would theoretically provide the advantage of a fast time to result and low-cost detection of SARS-CoV-2 but are likely to suffer from poor sensitivity early in infection, based on the experience with this method for influenza (Flu) viruses –44. Mono–Serology measures the host response to infection and is an indirect measure of infection that is best utilized retrospectively. Serological methods are rapidly being developed and have proven to be useful in confirming past COVID-19 . Serolog(ii) Assay selection for molecular detection of SARS-CoV-2. Random-amplification deep-sequencing methods played a major role in the initial identification of SARS-CoV-2  Target selection for real-time RT-PCR assays. A real-time RT-PCR method is recommended for molecular testing .In the United States, regulatory issues have complicated the development and implementation of laboratory-developed molecular tests for the diagnosis of COVID-19. On 29 February 2020, the FDA issued new guidance for laboratories to be able to develop and implement COVID-19 molecular diagnostic tests prior to obtaining EUA. Laboratories are required to submit an EAU to the FDA within 15 business days after validation. Moreover, the validation must include the specimen types  that are to be used clinically. Although these new regulatory burdens did not prohibit the development of molecular laboratory testing for the diagnosis of COVID-19, they did create a lot of extra work. At the time of writing, the U.S. FDA had granted quite a few EUAs ((i) Interpretation of molecular results. In the United States, initially if both of two targets in the CDC assay (nucleocapsid proteins N1 and N2) test positive, a case is considered to be laboratory confirmed (TC) value of less than 40 is defined as a positive test, while a TC value of 40 or more is defined as a negative test. A TC value of <40 for only one of the two nucleocapsid protein (N1 and N2) is defined as indeterminant and requires confirmation by retesting  Test of cure and test of infectivity. Monitoring patients with resolution of COVID-19 pneumonia may also be important in terms of when they should be released from isolation and discharged. If discharged patients are still shedding viable coronavirus, they are likely to infect other people  Serology of COVID-19. Members of the coronavirus family have four structural proteins: the spike (S), membrane (M]), envelope (E), and nucleocapsid (N) proteins. Two of these proteins appear to be important antigenic sites for the development of serological assays to detect COVID-19. Serological methods have focused on detecting serum antibodies against S proteins from the coronavirus spike  produced during COVID-19 have been developed . SeroconThe ongoing, unprecedented outbreak of COVID-19 globally has emphasized the importance of the laboratory diagnosis of human coronavirus infections in order to limit the spread as well as to appropriately treat those patients who have a serious infection. This commentary has addressed current issues regarding such testing for SARS-CoV-2. For example, an NP rather than OP swab is recommended for early diagnosis or screening because it provides higher diagnostic yields, is better tolerated by the patient, and is safer for the operator. An NP swab can be combined with an OP swab to increase sensitivity but requires twice the number of swabs. Should the NP swabs become scarce, self-collected saliva or nasal washes could be used as an alternative specimen type for epidemiological screening and for the “worried well,” who are asymptomatic persons with no exposure history who wish to be tested just to be sure they are not infected. NP swabs would then be reserved for hospitalized patients; those who test negative may need deep sputum or BAL fluid samples collected. The importance of repeated testing or the use of bronchoscopy in patients with severe illness should the first screening test be negative must be understood. The role of rectal swabs in testing patients with late infection or as a test of infectivity/cure is currently not well studied but needs urgent attention. Equally unappreciated is the need for broad screening/testing with molecular testing and/or serological testing in order to determine the true mortality rate as well as other epidemiological markers. Finally, the importance of rapid development of integrated, random-access, point-of-care molecular devices for the accurate diagnosis of SARS-CoV-2 infections cannot be overemphasized. These short-turnaround-time (STAT) tests will be very important for real-time patient management and infection control decisions, especially when other less infectious forms of pneumonia are present and respiratory isolate resources are scarce. These assays are safe, simple, and fast and can be used in local clinics and hospitals that already have the needed instruments and that are responsible for identifying and treating such patients.


Mass drug administration (MDA) of ivermectin for onchocerciasis has been disrupted by the coronavirus disease 2019 (COVID-19) pandemic. Mathematical modelling can help predict how missed/delayed MDA will affect short-term epidemiological trends and elimination prospects by 2030.Onchocerca volvulus microfilarial prevalence and intensity for different treatment histories and transmission settings, assuming no interruption, a 1-y (2020) interruption or a 2-y (2020–2021) interruption. Biannual MDA or increased coverage upon MDA resumption are investigated as remedial strategies.Two onchocerciasis transmission models (EPIONCHO-IBM and ONCHOSIM) are used to simulate microfilarial prevalence trends, elimination probabilities and age profiles of Programmes with shorter MDA histories and settings with high pre-intervention endemicity will be the most affected. Biannual MDA is more effective than increasing coverage for mitigating COVID-19’s impact on MDA. Programmes that had already switched to biannual MDA should be minimally affected. In high-transmission settings with short treatment history, a 2-y interruption could lead to increased microfilarial load in children (EPIONCHO-IBM) and adults (ONCHOSIM).Programmes with shorter  treatment histories should be prioritised for remedial biannual MDA. Increases in microfilarial load could have short- and long-term morbidity and mortality repercussions. These results can guide decision-making to mitigate the impact of COVID-19 on onchocerciasis elimination. These disruptions are expected to be particularly pronounced in low- and middle-income countries due to already underresourced healthcare systems. On 1 April 2020, the World Health Organization (WHO) advised that mass drug administration (MDA) and epidemiological surveys for neglected tropical diseases (NTDs) tackled by preventive chemotherapy and transmission control (PCT) should be postponed.4 However, comparability is limited due to the stark differences in the scale and manifestations of Ebola and COVID-19. There are concerns that delaying MDA might increase onchocerciasis morbidity and undermine progress made towards the 2030 elimination of transmission (EOT) goals proposed in the recently launched WHO 2021–2030 Roadmap for NTDs.5 It is therefore important to quantify where (in terms of transmission setting and treatment history) the impact of postponements of ivermectin MDA for onchocerciasis will be most pronounced and to identify the most effective mitigation strategies to help affected programmes get back on track. This will allow better planning and prioritisation of ivermectin distribution/treatment upon safe MDA resumption.Although so-called lockdowns and delayed MDA might be effective in temporally reducing the transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, the implications for both short- and long-term onchocerciasis transmission are less clear. The impact of the Ebola outbreaks in West Africa (2013–2016) on morbidity due to other diseases (such as tuberculosis and human immunodeficiency virus) can provide some insight into the implications of withdrawing routine public health services.7 Deadlines for drug orders , as required by the Mectizan Donation Program , might also be problematic. In addition to these challenges, programmes will have to be adaptive in the face of unforeseen setbacks that may emerge as MDA recommences, remedial strategies (such as increased MDA coverage and/or frequency) are attempted and as the COVID-19 pandemic progresses.Delayed ivermectin MDA, or reduced treatment coverage, could result not only from population-wide lockdowns to reduce COVID-19 transmission and the resulting redirection or disruption of health services, but also from shortages in drug availability (due to slower production and supply chains or exceeded drug shelf-life by the time MDA recommences).8 Delays in treating children who would otherwise receive ivermectin when turning 5 y of age could result in higher microfilarial loads experienced early in life, which may impact health outcomes in later years.9Mathematical models of onchocerciasis transmission provide a useful predictive tool for understanding the impacts of ivermectin MDA interruptions on the short-term (increases in transmission intensity) and long-term (elimination prospects), as well as the potential benefit of remedial mitigation strategies to help programmes get back on track. Although the resurgence of helminth transmission is typically slower than that of viral, bacterial and protozoan infections (due to differences in life history), increases in infection prevalence and intensity may increase onchocerciasis-associated morbidity.10 and provided preliminary but less comprehensive results for a report to the WHO on the impact of COVID-19 on NTD programmes.11In this article we use the EPIONCHO-IBM and ONCHOSIM transmission models to quantify where (in terms of transmission setting and treatment history) the impact of interruptions to ivermectin MDA for onchocerciasis will be most pronounced, allowing better planning and prioritisation of ivermectin distribution/treatment upon resumption, and investigate how mitigation strategies based on increased frequency  of increased coverage can help affected programmes to get back on track. Both models contributed insights to inform the WHO 2021–2030 NTD Roadmap12 and ONCHOSIM13 to address seven questions concerning the effect of MDA disruptions due to COVID-19: (i) What is the impact of delaying treatment for 1 or 2 y on microfilarial prevalence trends and the probability of elimination by 2030?; (ii) Which pre-intervention endemicity levels are particularly vulnerable?; (iii) How much more vulnerable are programmes with shorter  treatment histories (those with fewer years of MDA) than those with longer ones?; (iv) Which age groups will be most affected?; (v) Are remedial strategies based on increasing treatment frequency  or increasing treatment coverage during the early stages of MDA resumption useful for mitigating any setbacks?; (vi) How will programmes that had already switched to biannual MDA be impacted?; and (vii) What will be the effect on (West African) countries whose NTD programmes had already been disrupted by the Ebola outbreak in 2014?We used two individual-based stochastic transmission models, namely EPIONCHO-IBM2 additional MDA (either an extra round or increased coverage within one round) is delivered. Hence ‘remedial’ biannual MDA can be interpreted as ‘delayed treatment’ if a round of annual MDA is not delivered when planned but given in addition to the planned round in the following  year. Although, for simplicity, modelling results are presented as if MDA were delivered at the beginning of each year, what is important is the duration between two consecutive MDA rounds and whether MDA is delivered with increased frequency or coverage when treatment can recommence.In the context of a 1-y interruption or 1 y of missed MDA, the terms ‘interruption’ or ‘missed’ are used to describe a situation in which the MDA round planned to take place 12 months after a successful round pre-COVID-19 is not delivered . ‘Remedial’ MDA implies that once MDA can safely resume,14 to advocate and adhere to using good practice for policy-relevant modelling. We followed the five principles of the Neglected Tropical Disease Modelling Consortium15 and the African Programme for Onchocerciasis Control .16We simulated a range of scenarios reflecting pre-control endemicity and historical treatment durations in the former Onchocerciasis Control Programme in West Africa , specifically areas in the western extension in which ivermectin MDA was implemented without vector control,Onchocerca volvulus microfilarial prevalence of 20–85% (i.e. from hypoendemic to highly hyperendemic settings), although the APOC prioritised treatment only in areas with microfilarial prevalence ≥40%.For the sake of completeness, we considered pre-intervention onchocerciasis endemicity levels given by a baseline Annual ivermectin MDA programmes were simulated with start years 2000, 2003, 2006, 2009, 2012, 2014, 2017 and 2020, assuming that 65% of the total population is treated per round . The level of systematic non-participation was assumed to be 5%.17 where the main vector is Simulium damnosum sensu stricto (for which both models are parameterised). These pre-existing biannual programmes were assumed to have started in 1994 with annual MDA at 25% coverage  until 2012, when biannual treatment with 75% coverage of the total population  began.17 A pre-intervention 50% microfilarial prevalence was motivated by the baseline values for Adjumani-Moyo16 and Kitgum18 in the Madi-Mid North focus.We also modelled scenarios for pre-existing biannual programmes preceded by low-coverage annual MDA motivated by the situation in the Madi-Mid North focus in Uganda,We modelled settings previously affected by the Ebola outbreak in western Africa in 2014, which were broadly motivated by the treatment histories in Sierra Leone, Liberia and Guinea.We projected microfilarial dynamics and estimated elimination probabilities assuming either a 1-y (2020) or 2-y (2020 and 2021) interruption to ivermectin MDA. Additionally, we tested various assumptions of delays in achieving pre-COVID-19 coverage upon treatment resumption, e.g. missing MDA in 2020 but gradually increasing coverage from 30% in 2021 to 50% in 2022 to 65% in 2023–2030. Levels of systematic non-participation were set at 5%.We modelled the effectiveness of increasing frequency  or therapeutic coverage  as strategies for mitigating the impact of missed treatment rounds (either in 2020 or in 2020 and 2021). For a selection of the scenarios described above, we investigated the capacity for remedial treatment to revert the microfilaridermia trends and restore elimination probabilities (with MDA ceasing in 2030) to those predicted without interruption. Typically MDP only approves ivermectin donations for biannual treatment if programmes can demonstrate sufficiently high coverage in preceding years. Therefore we assumed that remedial biannual MDA followed only after a year of annual 65% coverage once programmes restart.We present three model outputs to understand the implications of interruptions to MDA: temporal trends in microfilarial prevalence (%) to understand the short-term implications, probabilities of achieving elimination by 2030 to understand the long-term implications and age profiles for microfilarial prevalence (%) and intensity (microfilariae/skin snip) to identify the most affected age groups.Figure Although EPIONCHO-IBM generally predicts lower elimination probabilities than ONCHOSIM, the models agree qualitatively on the impact of MDA interruptions due to COVID-19. Both models predict that interruptions to MDA will reduce the prospects of onchocerciasis elimination by 2030 if no mitigation strategies are implemented. Both models also predict that programmes with shorter  treatment histories will be more vulnerable to MDA disruptions than those that have distributed MDA for longer, particularly if treatment is delayed for 2 y . We illustrate these results after a 2-y interruption (2020–2021) when MDA initially resumes at low annual coverage 30%, 2022), returns to annual 65% coverage (2023) and then either increases to a 6-month frequency for 2 consecutive years 2024–2025) at 65% coverage , a 1-y interruption did not result in pronounced microfilarial resurgence according to either model Figure . For thiEPIONCHO-IBM predicts that the impact of a 1-y interruption due to COVID-19 in 2020 on microfilarial prevalence dynamics in West African programmes that were affected by the Ebola outbreak in 2014 will depend (like in other scenarios) on the transmission setting Figure . When asEPIONCHO-IBM and ONCHOSIM predict noticeably different age (and sex) patterns of microfilarial prevalence and intensity after three rounds of annual ivermectin MDA, with or without a 2-y (2020–2021) interruption  Figure .1 These disruptions to ivermectin MDA created concern in the NTD community regarding potential short-term increases in transmission and impacts on longer-term elimination prospects for onchocerciasis, particularly in the context of the EOT goals proposed by the WHO in its 2021–2030 NTD Roadmap.5 In response to these concerns, we used two stochastic onchocerciasis transmission models, EPIONCHO-IBM and ONCHOSIM, to investigate the impact of (1- and 2-y) interruptions to MDA in a variety of settings motivated by the epidemiological situation in endemic areas in Africa.While in the grip of the COVID-19 pandemic in April 2020, the WHO recommended suspending all epidemiological surveys and MDA activities for NTDs to help curtail the transmission and spread of the SARS-CoV-2 virus.Generally the models suggest that programmes with short treatment histories of pre-existing annual MDA (late-start programmes) will be the most vulnerable to a 2-y (2020–2021) interruption. Programmes with longer treatment histories (early-start programmes) could also be adversely affected if initial endemicity levels indicate intense (hyperendemic) transmission. The influence of baseline microfilarial prevalence was more pronounced in EPIONCHO-IBM for both early- and late-start MDA programmes than in ONCHOSIM. Although for late-start programmes a 1-y interruption to MDA impacted the elimination probabilities and microfilarial prevalence in 2030, the effect would be more tolerable than for a 2-y interruption; early-start programmes with lower pre-intervention endemicities were mostly unaffected.2 Therefore the implementation of mitigation strategies in the models allowed for a gradual scaling-up of therapeutic coverage once MDA resumes. Both models predict that remedial biannual MDA  would be more effective at controlling increases in infection and decreases in elimination probabilities than remedial high (75%) coverage of annual MDA .In July 2020, the WHO issued guidelines for the resumption of MDA provided it can be delivered ‘safely’ following a case-by-case risk–benefit assessment, with due consideration of a health system's capacity to conduct such modified activities effectively in the context of the ongoing COVID-19 pandemic.17 A 1-y (rather than a 2-y) interruption was explored for these settings because of their pre-COVID-19 strong programmatic performance.17 It was also assumed that these programmes would be able to resume biannual MDA at the same high coverage they had recorded before the pandemic given their existing structure and commitment to achieving EOT.Both models also predicted that pre-existing biannual MDA programmes—which had achieved a high treatment coverage for nearly a decade (in our simulations 75% pre-COVID-19 coverage from 2012 inclusive to reflect a 90% coverage of eligible population)—would not be adversely affected by a 1-y interruption. This was despite assuming a slow start of annual MDA at low coverage  to capture initial difficulties in implementing treatment.Some West African countries  had already had their ivermectin MDA programmes interrupted in 2014 due to the Ebola epidemic. We therefore considered this situation in addition to a 1-y COVID-19 interruption in 2020. Both models predicted that in meso-endemic settings , these two separate 1-y interruptions would not greatly impact a programmes’ performance, provided that coverage levels can catch up reasonably quickly following resumption of MDA (50% in 2021 and 65% in 2022 onwards). In hyperendemic and highly hyperendemic settings  the impact would be more pronounced (particularly according to EPIONCHO-IBM).22 children may reach their fifth birthday (when they become eligible for ivermectin treatment) with a sizeable microfilarial load. This has been shown to be a significant risk factor for the development of epilepsy later in life,9 ocular lesions23 and could also contribute to increasing the relative risk of mortality, which for a given microfilarial load is higher in children than in adults.24 In the Maridi villages, Ov-16 serology (a marker of exposure to infection) revealed a 20%  seroprevalence in 3- to 6-year-olds.22 Following a 2-y MDA interruption, children may not receive treatment until their seventh birthday, leading to a further microfilarial load build-up. Our modelling results indicate that in late-start (e.g. 2017) and high initial endemicity programmes , there might be substantial increases in infection intensity in children <10 y of age, particularly according to EPIONCHO-IBM. In addition, both models predict that in these settings, and following a 2-y MDA interruption, there will also be increases in infection prevalence and intensity in older age groups (moderate in EPIONCHO-IBM but pronounced in ONCHOSIM). Although here we do not explicitly model morbidity, this increase could lead to exacerbation of other onchocerciasis-associated sequelae, such as troublesome itch and acute ocular lesions (due to microfilaria in the anterior eye chamber),25 according to epidemiological8 and modelling studies linking infection and disease.27 These results are mostly determined by the assumed patterns of age- and sex-dependent exposure to infection, which differ markedly between the two models.28Although programmes measure progress in terms of reducing mean infection prevalence and intensity at the population level, favourable infection trends may mask heterogeneities among different population subgroups. In areas of intense transmission and only recent MDA implementation, which could be exemplified by the situation of the Maridi Dam in South Sudan,29 In addition, the aforementioned differences in the assumed age- and sex-specific exposure patterns, and associated age and sex profiles of infection, results in ONCHOSIM predicting a lower microfilarial intensity in children <5 y of age, in EPIONCHO-IBM there exists a larger reservoir of infection in untreated children, which also contributes to lower elimination probabilities.Although the models agree qualitatively on the role of treatment history in the outcome of interruptions to ivermectin MDA, and the differences between remedial strategies in mitigating the ensuing setbacks, ONCHOSIM predicts higher elimination probabilities than EPIONCHO-IBM. This is partly because EPIONCHO-IBM assumes strong regulatory processes operating on parasite establishment within humans that are relaxed as transmission declines during interventions, increasing the stability and resilience of the host–parasite system; these processes are not assumed in ONCHOSIM.An assumption made in most of the modelled scenarios is that when MDA resumes, coverage will initially be lower than pre-pandemic levels and could take several years to recover. However, the usual drug distribution modality, by which members of the community present at a focal point to receive ivermectin, could be replaced by door-to-door drug distribution . Depending on local circumstances, some programmes may achieve well-documented and high coverage levels, although the latter would also rely on well-stocked supply chains.2We did not consider the potential use of moxidectin and/or vector control as alternative remedial strategies to mitigate the impact of the current pandemic. This is because, while these approaches are very promising,Both EPIONCHO-IBM and ONCHOSIM indicate that ivermectin MDA programmes with shorter  treatment histories will be most vulnerable to MDA interruptions caused by the COVID-19 pandemic, particularly if treatment cannot be resumed safely in 2021. This impact may be noticeable in local infection resurgence in higher-endemicity settings and may reduce the probability of achieving EOT by 2030. Programmes with longer treatment histories of annual MDA that have achieved and maintained coverage of 65% are predicted to be less affected  by interruptions of ivermectin MDA, particularly if high initial endemicity indicates highly propitious transmission conditions. Young children have the potential to be negatively affected by increased levels of transmission resulting from missed MDA rounds, particularly for a 2-y interruption in highly endemic  settings recently incorporated into ivermectin MDA programmes, depending on local age-exposure patterns.The impact of COVID-19 on progress towards the WHO 2021–2030 goals is best ameliorated by implementing biannual MDA as soon as pre-COVID-19 levels of therapeutic coverage are restored . This mitigation strategy is indicated as more effective than increasing annual MDA coverage (to 75%).33 so alternative modalities of MDA delivery can be put in place safely and effectively to resume MDA and regain pre-COVID-19 levels of coverage where possible . Additionally, in August 2020, the United States Agency for International Development (USAID) released guidance on how best to implement MDA in the context of COVID-19.34 To complement the WHO and USAID documentation, we offer a decision-making tree to summarise our results and recommendations, which we hope will help programme managers to navigate the landscape of ivermectin MDA for onchocerciasis during the ongoing pandemic (Figure MDA programmes should promptly conduct the risk–benefit evaluations indicated by the WHO,c Figure .traa193_Supplemental_FileClick here for additional data file.


This study investigated a rare ultrasonographically detected thyroid petal-like calcification and its relationship with thyroid carcinoma and biological behavior. We described the clinical and ultrasonographical features of thyroid nodules with petal-like calcifications in 18 patients undergoing thyroid surgery and cervical lymph node dissection. All of the thyroid nodules with petal-like calcifications were papillary thyroid carcinomas (PTCs). Of the 18 patients, 13 (72.2%) had cervical central lymph node metastasis, and five (27.8%) had cervical lateral lymph node metastasis. Petal-like calcifications occurred in malignant thyroid nodules with a high incidence of lymph node metastasis, which may be a specific ultrasonographic feature associated with the aggressive biological behavior of PTC. Calcifications are commonly detected by ultrasonographic images in thyroid nodules and could be classified into various patterns –6. Taki A total of 18 patients with 18 nodules with petal-like calcifications that were detected with preoperative conventional ultrasound (US) and contrasted-enhanced ultrasound (CEUS) and underwent postoperative histopathologic analysis following resected hemithyroidectomy or total thyroidectomy from December 2016 to November 2019 were enrolled in this prospective study. Of the 18 patients, five underwent cervical central and lateral lymph node dissection, and 13 only underwent central lymph node dissection.The thyroid nodules were imaged with a Siemens Acuson S3000 US scanner equipped with a 9L4 linear array transducer  and/or an 18L6 linear array transducer (6–18 MHz). Petal-like calcifications appeared as scattered hyperechogenic spots <2 mm in diameter around solid thyroid nodules with the appearance of flower petals and had a cystic-like dark area ahead of each hyperechogenic spot. The cystic dark area and hyperechogenic spot constituted black-and-white foci . The thyCEUS was performed using contrast pulsed sequencing technology with a low mechanical index following intravenous injection of SonoVue microbubbles . CEUS videos were recorded for at least 60 s with dedicated software . With respect to the surrounding thyroid parenchyma enhancement, the time-intensity curves (TICs) of the thyroid nodules with regions of interest (ROIs) were acquired, and CEUS features were classified , 9, inclThe histopathological results obtained after surgery were used as the only reference standard for the final diagnoses of the thyroid nodules. Patients were staged according to the eighth edition of the American Joint Committee on Cancer (AJCC)/Tumor Lymph Node Metastasis (TNM) staging system –13.In this study, a total of 18 patients with 18 nodules with petal-like calcifications were summarized for their clinical, ultrasonographic and pathologic characteristics. The clinical characteristics of the 18 patients with petal-like calcifications were summarized in The ultrasonographic characteristics of the thyroid nodules with petal-like calcifications are outlined in The pathologic characteristics of all the patients are summarized in High-resolution US is recommended for preoperative screening of malignant thyroid nodules from benign nodules and evaluating cervical lymph node metastasis –16. PrevIn the present study, we collected 18 thyroid nodules with petal-like calcifications, which appeared as many scattered hyperechogenic spots around solid thyroid nodules with the appearance of flower petals, with a cystic-like dark area ahead of each hyperechogenic spot. The histopathologic result from all the patients was PTC, and 13 (72.2%) patients had cervical lymph node metastasis; however, previous studies have indicated that only 60–75% of thyroid nodules with microcalcification were PTC , 5, whicAccording to the Kwak TI-RADS classification, five US suspicious features  were used to categorize the thyroid nodules, with TI-RADS scores of 3 (no suspicious US features), 4a (one suspicious US feature), 4b (two suspicious US features), 4c (three or four suspicious US features), and 5 (five suspicious US features) . In the This study had many limitations. Firstly, all of the nodules with petal-like calcifications identified in our study ended up being PTC. So, data on whether benign thyroid nodules display such petal-like calcifications is lacking in our study. The sample size of our study population was small. Second, an unavoidable selection bias existed due to only patients who underwent surgery included in this study. A large-scale study is needed in the future to evaluate the association of petal-like calcifications with their corresponding pathogenetic characteristic.In conclusion, we first reported a special pattern of calcification in thyroid nodules, petal-like calcifications, which only occurred in malignant nodules with a high incidence of lymph node metastasis. This kind of calcification appears as numerous scattered hyperechogenic spots around solid thyroid nodules with the appearance of flower petals, with a cystic-like dark area ahead of each hyperechogenic spot in ultrasonographic images, which is called black-and-white foci. In addition, the majority of these nodules had ill-defined margins, marked hypoechoic or hypoechoic echogenicity, capsule contact with protrusion, a hyper- or isoenhancement type, a centripetal perfusion pattern, a PI index ≥1 and an AUC index ≥1. Therefore, petal-like calcifications in thyroid nodules may be a specific ultrasonographic feature associated with the aggressive biological behavior of PTC.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The studies involving human participants were reviewed and approved by the ethics committee of the Second Xiangya Hospital of Central South University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.CN contributed to the conception and design of the work. QP and CN participated to data analysis and manuscript writing. QZ and SC participated to data collection and patient follow-up.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.


This work presents a comprehensive study on the effects of the Friction-based Injection Clinching Joining (F-ICJ) process on the microstructure and local properties of the stake head. The manuscript evaluates the consequences on the quasi-static mechanical performance of hybrid joints of amorphous polyetherimide (PEI) with aluminium AA6082. Through an overlay of microhardness map on a cross-polarized transmitted-light optical microscopy (CP-TLOM) image, two lower-strength microstructural zones in the PEI stake head were observed: a plastically-deformed zone (PDZ) and a thermo-mechanically-affected zone (PTMAZ). When compared to the base material, PDZ and PTMAZ have a reduction of 12%–16% and 8%–12%, respectively, in local mechanical properties. The reduced local strength was associated with distinct volumes of loosely packed PEI chains with unsteady chain conformation and thus larger free volume in the affected regions. The mechanical strength reduction is reversible through physical aging by thermal annealing the joints, which additionally shows that process-induced thermomechanical degradation of PEI by chain scission, as evidenced by size exclusion chromatography (SEC) analysis, does not appear to affect local mechanical strength. An evaluation of typical loading regimes of staked joints in lap shear (average ultimate force of 1419 ± 43 N) and cross tensile (average ultimate force of 430 ± 44 N) testing indicates that the process-induced changes of PEI do not compromise the global mechanical performance of such a structure. These findings provide a better understanding of the relationships between processing, microstructure, and properties for further F-ICJ process optimization. Lightweight design has been established as one of the most successful strategies for the reduction of emissions in transport industry. By applying the right material in the right place, it is possible to obtain a multi-material structure with optimized weight and strength. This approach has driven research on several new joining methods which are potentially able to assemble these advanced polymer-metal hybrid structures. Several staking processes have been developed in recent years. Threaded Hole Friction Spot Welding (THFSW), based on filling of the pre-threaded metallic hole by melting and re-solidifying polymer, was used to join AA5052 aluminum to short-carbon-fiber-reinforced polypropylene (PP-SCF) composite . The incHowever, the application of new technologies in demanding industries such as aircraft and automotive requires a deep comprehension of several aspects of the joints. Manufacturing processes often causes alterations to the microstructure of materials, which in turn affects the final properties and performance of the joint. Therefore, not only the mechanical performance and damage tolerance are important investigation subjects, but also the effects of the process on local material properties. This understanding is essential for clarifying the influence of the process on joint properties to target joint efficiency.Although much of the research in this topic has been performed with metallic materials, there is interest on the behavior of polymer and polymer composites affected by various joining processes. Simōes and Rodrigues  used traThe effect of these microstructural changes on local properties of the joined materials can be further investigated by indentation testing (microhardness)  and physThis work describes the process-related changes observed in an amorphous engineering thermoplastic  when joined to a metal  by a friction-based staking process (F-ICJ ). The naFriction-based Injection Clinching Joining (F-ICJ) has been recently explored as an alternative advanced staking process for new lightweight structures by Abibe et al. ,18,19. TF-ICJ polymer stake heads are flush to the surface of the metal part b. A therThe basic process steps for the F-ICJ welding technique are shown in The fundamentals of the F-ICJ process have been described in ,19. JoinPolymer parts with a stud were machined from 6.35 mm thick extruded polyetherimide plates , as showed schematically in The pre-assembled parts were joined by F-ICJ using an automated gantry system  equipped with a high-speed friction welding machine . The system operates with rotational speeds ranging from 6000 to 21,000 rpm and axial forces of up to 24 kN. A torque sensor  was used to obtain the materials’ torque response. The unified system allows signal monitoring from rotational speed, axial force, spindle displacement, and torque. The specimens were cleaned with pressurized air (PEI) and acetone (AA6082) prior to joining. They were clamped in a standard sample holder to avoid slippage during joining. Microstructural analysis of PEI was carried out with a Leica DM IRM optical microscope . Reflected light optical microscopy (RLOM) and transmitted light optical microscopy (TLOM) were used for general microstructural and material flow observation. Samples were prepared by cutting the specimens 1 mm from their center and embedding them in low-temperature epoxy resin. Embedded samples were ground and polished for RLOM analyses according to the standard materiallographic procedure. For TLOM analyses, thin sections of 1 mm thickness were cut from embedded samples and subsequently had both sides polished.Qualitative evaluation of residual stresses in the PEI part after joining was performed using transmitted optical microscopy with crossed polarizers (CP-TLOM) on 1 mm thick section samples. The light source was a standard microscope filament lamp producing a continuous white light spectrum. The observed image produces a colored fringe pattern, in which each isochromatic corresponds to a local stress level caused by F-ICJ. A grayscale image of this pattern can qualitatively indicate the stress levels. In this study, the zero-order fringes were identified in color images as the non-stressed regions, then the grayscale images were used to interpret the local stresses and its dependence on the F-ICJ process parameters. Typical colored and grayscale fringe patterns and zero-order fringe identification of the PEI base material are showed in Local mechanical properties of PEI were measured on embedded and polished cross sections of joints. Zwick ZHV  equipment was used with an indentation load of 0.495 N over 15 s, and distance between indentations of 200 µm. The testing procedures are based on the ASTM E384 .−1. The analyses were performed using 200 µL of PEI/TCB solution at 150 °C and flow rate of 1 mL min−1. The calibration curve was built using monodisperse polystyrene standards with molecular weights between 845 and 1,900,000 g mol−1.Changes in molecular weight distribution (MWD) of PEI were evaluated by size exclusion chromatography (SEC) with a HT-GPC equipment  using HT-806 M columns coupled to a refractive index detector. Samples of PEI were removed with a scalpel from the PTMAZ of each specimen, and dissolved in trichlorobenzene (TCB) in a heated bath at 150 °C for 10 min at concentration of 2 mg LThe energy input provided by each set of F-ICJ process parameters was calculated to establish a correlation with physical–chemical changes in the PEI. Mechanical work is commonly used to estimate energy input in friction welding processes ,26. EquaBoth constants A typical F-ICJ joint results from the effect of heating and deformation imposed by rotational and axial movement of the tool in contact with the polymeric stud. Its microstructure is highly influenced by heat input, which in turn depends on the tool geometry and process parameters. The cross-section of such a PEI-aluminum F-ICJ joint is shown in The use of transmitted-light optical microscopy (TLOM) through a thin section of an F-ICJ joint makes it possible to observe the microstructural features and discontinuities in the polymer. A dark line across the diameter of the stake shaft delineates a polymer-polymer interface b. The voTo better visualize the microstructural zones and understand their local properties, further characterization methods were performed. The right-hand side of Beneath the shear layer boundary of the PTMAZ (dotted line in g)) during cooling or at room temperature. Plastic deformation by yielding is described as conformational changes of the chains, leading to increased free volume in amorphous polymers [PEI undergoes strain softening under compression in the 7%–13% strain range , as showpolymers ,29,30,31polymers ,9.Identification of the shape and limits of the PDZ can be additionally supported by the birefringence pattern with crossed polarizers. Birefringence patterns are associated with the residual stresses in a material through the stress-optic law (Equation (2) ).(2)σreasticity  can be uasticity ,36, whicicity [δ . In thision (2)) .material  where noIt can be also observed in g are seen, because this volume is above the Tg of polymer during processing. Competing phenomena may act in this volume to change its microhardness: topological changes affecting internal order, and molecular weight changes [Following the polymer–polymer interface line in  changes . Conform changes ,9,39. Re changes . By cont changes . A combig of PEI (215 °C), and it cools down rapidly (≈35 °C·s−1) during consolidation [g in a glassy state [Chain entanglement density in PEI at the stake head of F-ICJ joints is related to the available energy for chain diffusion at the end of the friction phase. During the friction phase, the PTMAZ reaches temperatures (up to 385 °C) far above the Tlidation . At the sy state ; this dosy state ,32. Thissy state . Either n and Mw) and increasing of the polydispersity (Mw/Mn) towards higher energy input levels can be observed. This trend is an indication that the temperature and shear rate imposed by the F-ICJ process are high enough to cause thermomechanical degradation of PEI through chain scission [To investigate if a certain level of degradation is present, physical–chemical properties related to chain length were studied. The hypothesis of PEI degradation caused by F-ICJ stated in the previous sections is proven in scission . Previouscission  showed tIn the previous discussion, it has been shown that microstructural changes in PEI joined by F-ICJ decrease local strength in the PTMAZ and PDZ  and creaA cross-section of the joint showing the indentation positions prior to and after thermal aging are showed in The microhardness distribution for the as-joined PEI shows clear boundaries for the typical low-strength PTMAZ and PDZ of an F-ICJ joint. After annealing no microstructural zones can be distinguished. A region of lower strength is observed below the keyhole, corresponding to non-process-related cracks which develop during annealing d. AveragIt is of interest to correlate significant microstructural changes in a joint with the expected mechanical behavior of the structure in service. The mechanical behavior of staked joints is commonly tested through lap-shear and cross-tensile configurations. These tests simulate typical stresses in rivet-like assemblies.Lap shear tests are carried out in overlap specimens as depicted in A correlation can be established with the as-joined microstructure of an F-ICJ joint. At the stake base, where the final crack grows, there were no microstructural changes due to the joining process. This region’s resistance to failure can mainly be improved by geometrical design. On the other hand, the stake head region is the PTMAZ, with lower local mechanical strength. The stake head crack grows through this volume, and the crack growth is influenced by its properties and features. For instance, extreme levels of thermal–mechanical degradation may reduce PTMAZ strength to a point where the crack growth is facilitated. The presence of pores c allows Cross-tensile tests use an overlap specimen as shown schematically in Typically, the lower strength and degradation of the PTMAZ will not influence the reliability of assemblies suffering loads as from cross tensile test. The base plate bending failure is a function of the base material’s properties and mechanical design of the polymeric part. However, in some cases foreign particles in the PTMAZ have been shown to nucleate cracks at the stake head during cross tensile tests . The groThe joining of polyetherimide (PEI) amorphous engineering thermoplastic to aluminum alloy through the new F-ICJ staking joining process was presented. A comprehensive study on the effects of processing on the microstructure and local properties was carried out through light optical microscopy, microhardness testing, and size exclusion chromatography techniques.An analysis through qualitative transmitted-light optical microscopy combined with quantitative microhardness testing allowed to identify and clearly delimitate two microstructural zones in the stake head of PEI: a thermo-mechanically-affected zone (PTMAZ) and a plastically-deformed zone (PDZ).g of PEI (215 °C), and quickly cooled (≈35 °C s−1) afterwards, presented an 8%–12% reduction in the microhardness values compared to the base material (BM), as well as a few volumetric defects. This zone was characterized by a distinct birefringence pattern, as revealed by cross-polarized transmitted-light optical microscopy (CP-TLOM) analysis, resulting from thermomechanically-induced residual stresses. Furthermore, thermomechanical degradation of PEI by chain scission was identified through size exclusion chromatography (SEC) analysis.The PTMAZ, a polymer layer below the keyhole that was molten and sheared by the action the stirring tool at temperatures up to 385 °C well above the TThe PDZ, a polymer volume beneath the PTMAZ boundary that underwent strain softening as a consequence of developed compressive stresses resulting from F-ICJ, showed a 12%–16% reduction in the microhardness values and a different birefringence pattern. The boundary between the PDZ and the base material (BM) was characterized by the difference in the number of fringes presented in the CP-TLOM image.A post-joining annealing treatment eliminated residual stresses in the PTMAZ and PDZ, as a consequence of physical ageing of PEI. This helped to identify the nature of the above-mentioned microstructural local changes as distinct volumes of loosely packed PEI chains with unsteady chain conformation and thus larger free volume, which in turn reduced microhardness values. Although thermomechanical degradation of PEI on the staked head was evidenced by SEC, it seems not to contribute to the reduction in joint global mechanical strength.The consequences of the microstructural changes and thermal degradation of PEI on the global mechanical properties of staked joints were evaluated in terms of typical mechanical loading in lap shear (average ultimate force of 1419 ± 43 N) and cross tensile (average ultimate force of 430 ± 44 N) testing. Neither of the loading situations rely largely on the PDZ and PTMAZ, therefore the process-induced local strength reduction and PEI degradation by chain scission in the stake head do not compromise global mechanical properties of staked PEI-aluminum joints.These findings extend the understanding of the relationships between processing, microstructure, and properties, as well as provide the basis for further F-ICJ process optimization.