Elasticity prevents fatigue of tissues that are extensively and repeatedly deformed. Resilin is a resilient and elastic extracellular protein matrix in joints and hinges of insects. For its mechanical properties, Resilin is extensively analysed and applied in biomaterial and biomedical sciences. However, there is only indirect evidence for Resilin distribution and function in an insect. Commonly, the presence of dityrosines that covalently link Resilin protein monomers (Pro-Resilin), which are responsible for its mechanical properties and fluoresce upon UV excitation, has been considered to reflect Resilin incidence.Drosophila body, some of which were not described before. Interestingly, the amounts of dityrosines are not proportional to the amounts of Resilin in different areas of the fly body, arguing that the mechanical properties of Resilin matrices vary according to their need. For a functional analysis of Resilin matrices, applying the RNA interference and Crispr/Cas9 techniques, we generated flies with reduced or eliminated Resilin function, respectively. We find that these flies are flightless but capable of locomotion and viable suggesting that other proteins may partially compensate for Resilin function. Indeed, localizations of the potentially elastic protein Cpr56F and Resilin occasionally coincide.Using a GFP-tagged Resilin version, we directly identify Resilin in pliable regions of the Thus, Resilin-matrices are composite in the way that varying amounts of different elastic proteins and dityrosinylation define material properties. Understanding the biology of Resilin will have an impact on Resilin-based biomaterial and biomedical sciences. Drosophila melanogaster, besides the full-length Pro-Resilin isoform with the R&R-2 domain (620 aa), an isoform lacking this domain (575 aa) is present through alternative splicing. Outside the R&R-2 motif, Pro-Resilin sequences in different insects vary considerably. In principle, these sequences are characterised by stretches of non-conserved repetitive blocks that account for the elasticity of Pro-Resilins [Elasticity and resilience are essential for the integrity of tissues that function through repeated and extensive deformation. In vertebrates, for instance, the elastic extracellular polymeric protein Elastin in the lung and blood vessels ensures their functionality after numerous times of use , 2. The Resilins , 16–18.D. melanogaster fruit flies and second to behaviourally characterise flies without Pro-Resilin through genetic manipulation.Presence of Resilin in different body parts argues that matrix composition may vary to accommodate the respective mechanical need. For instance, in adhesive tarsal setae, a fluorescence gradient implying a Pro-Resilin concentration gradient has been reported , while iD. melanogaster, we used transgenic flies expressing a C-terminally GFP-tagged Pro-Resilin (Pro-Resilin-GFP) under the control of the endogenous promoter  between Pro-Resilin monomers that confer auto-fluorescence when excited with UV light , 8, 24. D. melanogaster body, no convincing DT signal was detected that did not merge with a Pro-Resilin-GFP signal  using the UAS/Gal4 expression system. RNAi significantly reduced pro-resilin expression  were unable to fly  phenotype suggests increased fatigue of the Pro-Resilin containing patches. Thus, both flapping and holding of the wing depend on Pro-Resilin. To analyse to what extent wing posture failure affects wing function other than for flight, we filmed pro-resilinRNAi flies during courtship and mating. Wild-type males performed a courtship dance among others moving their wings  and an isoform with a disrupted R&R domain. Mutations in this exon cause a phenotype indistinguishable from the phenotype caused by mutations around the start codon . Of note, we also observe that pro-resilinCC-RR flies are able to climb up the wall of the well suggesting that the tarsi are normal.Additional file 24: Movie S9. Flies homozygous for the pro-resilinCC-ATG allele move freely in a polystyrene multi-well plate with a lid. They keep their wings laterally (hdw phenotype). Of note, we also observe that pro-resilinCC-ATG flies are able to climb up the wall of the well suggesting that the tarsi are normal.pro-resilincc-ATG flies were incubated with the dye Eosin Y in a penetration assay [pro-resilin deficient flies . A more sophisticated experiment supplying food with different densities is needed to further study this issue. To analyse the biological role of Resilin in the spermathecal ducts of females, we assessed fertility. For this purpose, we mated wild-type male flies with pro-resilin mutant females for 4 h (to avoid double mating). Fertility of these females (n = 31) was not reduced significantly  compared to wild-type females (n = 14). We conclude that under laboratory conditions the function of Resilin in female reproduction, i.e. spermathecal ducts is dispensable.In a series of assays, we studied the biological role of Pro-Resilin in the adult fly beyond flight and courtship (see above). To test whether Resilin in the tracheal openings is needed for the gate function of these structures, on assay , 28. Eospro-resilin is not essential for survival at least in the laboratory or that a Resilin-like protein compensates for the reduction or elimination of Pro-Resilin function. A candidate for this redundant function is Cpr56F, a cuticular R&R-domain protein related to Resilin as proposed by Ardell and Andersen and Karouzou et al. [cpr56F expression is not lethal, and the respective flies do not display any obvious phenotype  is detected in the tracheal endings, the spermatheca ducts, at the wing articulations, at or near to limb joints and the proboscis. Those body parts used for locomotion and movement  have already been described extensively to require Resilin in other insect species , 11. In  muscles .D. melanogaster, we did not detect any Pro-Resilin or DT signal associated with wing veins. Hence, flight mechanics may correlate with wing size and are different in different species.Besides the information on Resilin distribution in the fly body, it is interesting to note where Resilin is absent. In many insect species including honeybees and dragonflies, DT, i.e. Resilin has been reported to occur at the margins of wing veins –37. In DResilin patches in and adjacent to the joints are relatively smaller than those regions described in the legs of jumping insects where they serve energy storage and release (e.g. ). Like iThe spiracles at the tracheal endings control gas exchange and water loss by respiration . A repeaThe largest body area that harbours Resilin is the proboscis. In lepidopteran species, recoiling of the proboscis seems to require Resilin in the dorsal half of the proboscis wall . In the D. melanogaster adult body, in conjunction with non-Resilin containing cuticle parts, Resilin contributes small matrices to skeletal structures, organs and tissues that are subject to repeated movement, flexion or torsion. Its function in these structures remains to be studied in biomechanical experiments.Taken together, in the Pro-Resilin is composed of two types of repeat motifs, named A and B  that flapro-resilin expression had ceased. This tentatively suggests that DT formation occurs within the extracellular space when Pro-Resilin is already incorporated in the cuticle. This is consistent with classical work published by Neville and Kristensen [DT formation activity seems not to be only under spatial but also under temporal control. In our experiments, we found that the DT signal intensified after eclosion when istensen , 42. Of Variable region-specific cross-linking degree in Resilin matrices may inspire the analysis of cross-linking degree of tropoelastins in elastin matrices . Indeed,omim.org).As a flexible, resilient and adhesive element, Resilin is a multi-functional matrix in the cuticle of insects. In spite of this, we show that flies without Pro-Resilin, a key component of the Resilin matrix, are viable under laboratory conditions. In mice, deletions of the gene coding for the elastic protein Elastin lead to perinatal lethality . In humaD. melanogaster is probably due to the presence of the potentially elastic protein Cpr56F that may compensate for Pro-Resilin deficiency. Cooperation or redundancy between Pro-Resilin and Cpr56F needs, of course, further, thorough analyses including the generation of double mutant clones in the adult body. Redundancy of elastic Resilin-forming proteins seems to be prevalent in insects. In the bed bug Cimex lectularius, 13 Pro-Resilin candidates have been described [Anopheles gambiae putative Pro-Resilin protein AgamCPR152 (317 residues) is shorter than the D. melanogaster Pro-Resilin protein (620 residues) [A. gambiae genome. We propose that the Resilin matrix may be composed of more than one elastic protein.Non-lethality in escribed . The AnoD. melanogaster, of at least two proteins that are combined in different ratios to accommodate the respective specific function of the cuticle.In summary, our work supports the notion that the concept of “Resilin” involves more than one dityrosinylated protein. In line with a recent hypothesis , our resThis finding will have an impact on the use of Resilin and the entire resilome in biomaterial sciences including micro-robotics –49. As aFlies were kept in vials with standard cornmeal-based food at 18 or 25 °C. For embryo and larva collection, flies were kept in cages on apple-juice plates garnished with fresh baker’s yeast at 25 °C. Mutations were maintained over balancer chromosomes carrying insertions of GFP expressing marker genes (Dfd-YFP or Kr-GFP). This allowed us to identify homozygous non-GFP embryos or larvae as mutants under a standard fluorescence stereo-microscope (Leica).For the feeding experiments, we chose to apply the CAFE assay . In briepro-resilin (41.9 kb) or cpr56F (33.2 kb) genomic DNA and the gfp-coding region fused to the 3′ end of pro-resilin or cpr56F were obtained from Source Bioscience (https://www.sourcebioscience.com) .The transposon plasmids with either the resilin 4.9 kb or pro-resilin coding region with the coding region of Venus downstream of the UAS promoter, the pro-resilin coding region was cloned into the pTWV transposon plasmid obtained from the Drosophila Genomic Resource Center (DGRC). Transgenic flies (w1118) with this UAS-pro-resilin-venus construct were generated by BestGene. For pro-resilin-venus expression, we used the ubiquitous da-Gal4 driver.To recombine the pro-resilin), 13296 and 110265 (both against cpr56F) from the Vienna Drosophila Resource Center (VDRC) were expressed using the ubiquitous da-Gal4 driver.For RNA interference (RNAi), the hairpin RNA constructs 106773  developed at the Wisconsin University. For pro-resilin, we designed two gDNA g4 (GACGCTGCTCATGGCAATGGTGG) and g5 (GGGCATTCAGAGATGCGCGACGG) that are complementary to sequences in exon 1 and exon 2, respectively. For cpr56F, one gDNA (GAGGCAGCCAGGCACACGAGC) was designed. According to the protocol at the flyCRISPR homepage, complementary oligos with appropriate overhangs were annealed and cloned into the pCFD3-dU6/3 vector at the BbsI site. After amplification in bacteria, these vectors were injected separately into Cas9 expressing embryos . Optical sections were generated with the normal confocal mode yielding 28 z-stack images that were subsequently processed with the Fiji software. Two different modes of validation were implemented in order to possibly detect reasonable DT signal differences between wild-type and hdw flies. In the first approach, we tested how often the signal intensity threshold of 4,000,000 that was determined initially was reached in wild-type control and pro-resilin deficient flies (hdw). The positive signal incidence in the wild-type control was set to 100%; the positive signal incidence in hdw flies is given as a fraction thereof. In the second approach, the intensity values of wild-type control and pro-resilin deficient fly were directly compared statistically; these data are shown for the area and the intensity excited at 355 nm in Fig. t test.For quantitative data generation, signal intensities were measured and compared using intensity data determined with the Fiji software. To obtain a DT-specific signal for Resilin detection, the interfering background signal excited by the 405 nm light source had to be eliminated. This background signal emanates partly from DT, partly from the outermost cuticle layer called envelope , 51. ForA Nikon AZ100 zoom microscope operated with the NIS Elements D software was used for Nomarski (DIC) imaging of wing articulations shown in Additional file n = 20) in a well plate was recorded by a Leica EZ4HD with an in-built camera  fixed with Clean Glass adhesive from Duro on a needle with a diameter of 127 μm was filmed with an u3 cmos 14000KPA microscope eyepiece camera  were incubated with Eosin Y  at 25 °C for 20 min, washed with tap water and deposited on a slide for microscopy and imaging (Leica EZ4HD with an in-built camera).Eosin Y staining was conducted according to the recently published protocol . In briewww.inkscape.org), Leica LAS X, ZenBlue, Adobe Illustrator and Photoshop CS6 and GIMP-2.10 software. For the images shown in Figs. Figures were prepared and assembled using the Microsoft PowerPoint and Excel, Inkscape , RNA was first isolated from flies using the RNEasy kit (Qiagen) and used for cDNA synthesis . qPCR was performed according to the Sybr-Green method  on a QuantStudio 5 cycler (Thermo Fisher Scientific). Data were analysed with the in-built software (Quantstudio5). Expression of idelines . ExperimAdditional file 1: Figure S1. Upper scheme: The transposon harbouring the genomic region of the pro-resilin gene (orange) fused with the ORF of sGFP (green) at its 3′ end before the stop codon encompasses 27,324 bps of upstream and 10,204 bps of downstream sequences. Lower scheme: The transposon harbouring the genomic region of the cpr56F gene (orange) fused with the ORF of sGFP (green) at its 3′ end before the stop codon encompasses 13,674 bps of upstream and 15,487 bps of downstream sequences.Additional file 2: Figure S2. Real-time quantitative PCR analysis indicates that pro-resilin (res) is not expressed before the pupal stage. The expression of the house-keeping gene Rps20 was used to normalize the expression data. In addition, we used a primer pair that does not amplify any DNA in the fly transcriptome as a negative control (neg). The expression value for this amplification was set to 1. The amplification levels of pro-resilin (fold change with respect to the negative control) were identical to the amplification levels of this primers in embryonic and larval samples. In pupae, pro-resilin expression is clearly induced. Bars indicates standard deviation.Additional file 3: Figure S3. A–C: Leg and adjacent thoracic wall of a D. melanogaster. Blue labels “l + number” (l leg) specify the various Resilin patches  found; see Additional file D. melanogaster. Blue labels “w + number”  specify the various Resilin patches  found. Upcal upper calyptere; Sc, R, MA, CuA1, A1 are wing veins, and P1–5 are wing articulation sclerites  using the standard terminologies.Additional file 4: Figure S4. In the tracheal endings (sp) and the bristle sockets , very weak DT signals (A and C) overlap with the Pro-Resilin-GFP signal (A and B). The asterisk (*) marks auto-fluorescence of internal tissues after dissection. Images were generated on a Zeiss LSM880 confocal microscope. The excitation (ex) and emission (em) wavelengths are indicated in the images that were obtained by the normal confocal mode. Details of the settings are described in the Methods section.Additional file 5: Figure S5. Pro-Resilin-GFP in spermatheca. (A) In the spermathecal ducts of freshly eclosed females, we observe a weak Pro-Resilin-GFP signal (black arrows). (B) This signal becomes stronger in one-day-old flies and (C) well visible in seven-day-old females. (D) The spermathecal ducts of freshly eclosed, (E) one-day-old or (F) seven-day-old females with reduced pro-resilin expression (pro-resilinRNAi) appear to be normal. The GFP signal (red) was merged with the bright-field image. A Leica DMi8 microscope was used for imaging. Details of the settings are described in the Methods section.Additional file 6: Figure S6. The morphology of the wing articulation region may depend on Resilin. The wild-type wing (w) articulation region in the thorax is framed by the anepisternum (x) and the anepimeron (*). It is composed of three sclerites (1–3) and the pleural wing process. The sclerites 1 and 2 are separated by the flexible vertical cleft (arrow). In the wild-type wing articulation regions, we spotted a triangular structure (triangle) that is missing in the pro-resilinCC-ATG fly. The wing articulation elements were named according to a reproduction of the thorax at flybase (http://flybase.org/reports/FBim0000793). Images were recorded on a Nikon AZ100 zoom microscope applying Nomarski microscopy.Additional file 7: Figure S7. Pro-Resilin-GFP expression  normalizes the hdw phenotype of pro-resilinRNAi flies. The fly has been illuminated additionally with green light  and UV light . All colours have been merged with a bright-field (BF) image with the aim to show a plastic image of the fly taken with a Leica DMI8 microscope. Details of the settings are described in the Methods section. The image was digitally manipulated without changing the ratios of the intensities of the different signals.Additional file 8: Figure S8. Schematic representation of the pro-resilin gene and its products. (A) The full-length Pro-Resilin protein has 620 residues. It has three functional domains. Following the signal peptide  in the N-terminus, there are a number of tandem repeats , followed by an R&R-2 chitin-binding domain, which precedes an array of tandem repeats . A shorter isoform lacks the R&R-2 domain and has 575 residues (not shown). Deletion of 3 bases in the second exon coding for the R&R-2 domain results in the transversion of the M365 to an I and the deletion of R366 as shown above the full-length Pro-Resilin protein (Crispr/Cas92). Deletion of the cytosine 35 and adenosine 36 of the ORF causes a frameshift of the ORF resulting in a premature stop codon after 22 residues (Pro-ResilinCC-ATG). Information on the mutations at the DNA level is presented in Additional file pro-resilin gene is composed of three exons separated by two introns. Exon 1 and 3 can be spliced together omitting exon 2 that encodes the R&R-2 domain. The recognition sites of hairpin RNAs against pro-resilin expression are indicated below the scheme. (C) The GFP coding region is inserted in frame into the 5′ end of the pro-resilin gene just before the stop codon, thereby disrupting the recognition site of hairpin RNAs.Additional file 9: Figure S9. Schematic representation of the cpr56F gene and its product. (A) The cpr56F gene has three exons and two introns. (B) The GFP coding region is inserted in frame into the 5′ end of the cpr56F gene just before the stop codon, thereby disrupting the recognition site of one hairpin RNA (KK), without affecting the other one (GD). (C) Cpr56F (217 residues), is composed of a signal peptide  and two repeats (grey) separated by an R&R-2 domain (black). We generated two Crispr/Cas9 induced cpr56F mutant alleles. One, cpr5612 gives rise to a protein with a deletion of three amino acids in its signal peptide. The other allele, cpr562, is characterised by the deletion of cytosine 30 in the ORF resulting in a frameshift. The respective protein has 88 amino acids including 78 residues absent in the normal Cpr56 protein (light grey region). Information on the mutations at the DNA level is presented in Additional file Additional file 10: Figure S10. (A) The pro-resilin locus, genomic organisation and mutations. The two bases CA (green box) in the first exon (magenta letters) are deleted in the pro-resilinCC-ATG allele. This causes a frameshift and a premature stop codon resulting in a truncated protein as shown in Additional file pro-resilinCC-RR allele. By this deletion that affects two consecutive codons, the amino acid M365 is changed to an I and the amino acid R366 is deleted as shown in Additional file cpr56F locus, genomic organisation and mutations. The nine bases CGTGTCCCT (underlined green box) in the second exon (magenta letters) are deleted in the cpr56F12 allele. This causes a deletion of the three amino acids LVC in the signal peptide of the resulting protein as shown in Additional file 2458 (framed box) in the second exon (magenta letters) is deleted in the cpr56F2 allele. This deletion causes a frameshift resulting in an aberrant protein sequence of 78 amino acids after L10 and a premature stop codon as shown in Additional file Additional file 11: Figure S11. (A-A″) Pro-Resilin-GFP is predominantly detected in or close to the leg joints  and pro-resilincc-ATG (D) samples, the DT signal is not strongly reduced. Differences in intensity are, however, apparent upon software-based anaylses  that, by contrast, penetrates the tracheal system of parched22 flies (triangles), which have been described to have open spiracles [piracles . Images Additional file 13: Figure S13. Reduction of Cpr56F does not cause any visible phenotype. (A) When resting, the wild-type fly holds its wings at the back.  RNAi against cpr56F does not have any effect on wing posture. (C) Some flies with reduced cpr56F expression have crippled wings. (D) cpr56F is highly expressed in pupae. Its expression drops in eclosed flies. cpr56F expression is not strongly reduced by RNAi. According to the overlapping confidence intervals (CI), the differences in expression levels are not highly significant. Images were generated with a Leica EZ4HD with an in-built camera.Additional file 14: Figure S14. Cpr56F-GFP is detected in the head region of the embryo and larva. (A) The head skeleton and the tip of the head of the ready-to-hatch embryo contain Cpr56F-GFP. (B) This signal persists during larval stages. Images were generated with a Leica M205FA fluorescence binocular. Details of the respective settings are described in the Methods section. Labelling: lsg... larval salivary gland, ci... cibarium, lb...  labellum.Additional file 15: Figure S15. The proximal edge of the marginal cell (mc) in the wing of Drosophila hydei contains dityrosine (A). In the green channel, this signal is missing (B). Cpr56F-GFP is expressed at this position (C), whereas Pro-Resilin-GFP is not . Here, we used an Axio Observer Z1 microscope for imaging. The excitation (ex) and emission (em) wavelengths are indicated in the images. Details of the respective settings are described in the Methods section.Additional file 25: Text. Extended description and discussion of Resilin patches in the fly leg.


In many low‐ and middle‐income countries, HIV viral load (VL) testing occurs at centralized laboratories and time‐to‐result‐delivery is lengthy, preventing timely monitoring of HIV treatment adherence. Near point‐of‐care (POC) devices, which are placed within health facility laboratories rather than clinics themselves (i.e. “true” POC), can offer VL in conjunction with centralized laboratories to expedite clinical decision making and improve outcomes, especially for patients at high risk of treatment failure. We assessed impacts of near‐POC VL testing on result receipt and clinical action in public sector programmes in Cameroon, Democratic Republic of Congo, Kenya, Malawi, Senegal, Tanzania and Zimbabwe.Routine health data were collected retrospectively after introducing near‐POC VL testing at 57 public sector health facilities . Where possible, key indicators were compared to data from patients receiving centralized laboratory testing using hazard ratios and the Somers’ D test.Data were collected from 6795 tests conducted on near‐POC and 17614 tests on centralized laboratory‐based platforms. Thirty‐one percent (2062/6694) of near‐POC tests were conducted for high‐risk populations: pregnant and breastfeeding women, children and those with suspected failure. Compared to conventional testing, near‐POC improved the median time from sample collection to return of results to patient  and to clinical action for individuals with an elevated HIV VL . Near‐POC VL results were two times more likely to be returned to the patient within 90 days compared to centralized tests . Thirty‐seven percent (340/925) of patients with an elevated near‐POC HIV VL result had documented clinical follow‐up actions within 30 days compared to 7% (167/2276) for centralized testing.Near‐POC VL testing enabled rapid test result delivery for high‐risk populations and led to significant improvements in the timeliness of patient result receipt compared to centralized testing. While there was some improvement in time‐to‐clinical action with near‐POC VL testing, major gaps remained. Strengthening of systems supporting the utilization of results for patient management are needed to truly capitalize on the benefits of decentralized testing. CliniciNear point‐of‐care (near‐POC) HIV VL technologies are simpler to operate and do not require the infrastructure of a full laboratory. Near‐POC devices are placed within health facility laboratories rather than directly within the clinic where the patient is being seen (“true” POC). This technology theoretically may enable decentralization of testing to lower levels of health facilities, thus eliminating the need for sample transportation for many patients, and can deliver test results within hours of sample collection. As a result, near‐POC VL testing has the potential to address some of the current gaps in VL monitoring including improving and expediting result return to both clinicians and patients and clinical utilization . TherefoTo date, there is limited research on how best to implement near‐POC testing as part of a national HIV VL network or testing strategy in LMICs, the impact of near‐POC VL on patient care, or the management of specific patient groups that could benefit most from near‐POC VL testing , 12, 13.22.1Delivery of testing services and clinical outcomes was assessed after the introduction of near‐POC VL at 57 public sector health facilities as part of service delivery programmes in seven countries: Cameroon, Democratic Republic of Congo (DRC), Kenya, Malawi, Senegal, Tanzania and Zimbabwe. In Senegal and Zimbabwe, some data were collected as part of ongoing studies with information that could be used for this analysis. Data were collected from May 2017‐October 2019 (country‐dependent). Descriptions of the assessment structure by country and study design can be found in Table National HIV programmes had completed their own processes to select health facilities for near‐POC VL implementation based on criteria such as current device availability, volume of patients on antiretroviral therapy (ART) per facility, history of stockouts and current device utilization rate. For this analysis, 3 to 10 facilities with near‐POC devices were randomly selected within each country. The assessment period ranged from three to nine months per country, depending on logistical factors. Data were retrospectively collected for all patients on ART receiving near‐POC VL testing during the assessment period. Where possible, key indicators were compared to data from all patients on ART receiving VL testing through the centralized laboratory in the three‐ to nine‐month period before near‐POC VL testing was implemented  or to data from patients receiving centralized laboratory testing at the same facilities during the same period . In Cameroon and DRC, no comparison data were available on centralized testing because there was previously no centralized HIV VL programme.2.2The 2016 WHO treatment guidelines recommend that patients receive a first HIV VL test at six months post‐ART initiation, a second at 12 months post‐ART initiation and annual tests thereafter [A single Cepheid GeneXpert  or Abbott m‐PIMA  device was available for near‐POC VL testing at the health facilities included in this assessment. Rollout of near‐POC testing included training existing health facility staff on operational procedures for testing, clinical utilization of rapid test results, results documentation, device maintenance, supply chain and waste management and quality control.Near‐POC VL samples were only collected from patients receiving care at the selected study facilities. In most countries, all patients receiving VL technically were eligible for near‐POC VL testing. However, prioritization of patients at high risk of having viraemia  or at high risk of transmission (pregnant and breastfeeding women) occurred in most contexts. Ultimately, testing decisions were made by individual clinics/healthcare workers. In Kenya, near‐POC VL testing was only made available for paediatric and adolescent populations and pregnant and breastfeeding women.Near‐POC VL devices were placed in the onsite laboratories in the health facilities and were operated by laboratory staff. Centralized VL tests were sent to a district hospital or a National Referral Lab for testing. Whole blood samples were collected by clinic or laboratory staff using venipuncture and were processed for testing per manufacturer’s instruction.2.3Trained data collectors retrospectively extracted data from the health facilities included in this assessment. Data included demographic information, reason for VL test (i.e. routine monitoring or suspected failure), results, clinical action taken, if any, among patients with an elevated VL, and timing of each step in the cascade from sample collection to clinical action for each patient tested using national and facility‐specific laboratory and clinic registers and patient charts. Electronic data collection using SurveyCTO  forms on tablets were used. Data collection for both near‐POC VL and centralized VL testing outcomes allowed for a follow‐up period of 90 days to allow for complete documentation of testing and treatment outcomes in facility registers. In Senegal, only 30 days of follow‐up were possible due to funding/logistical constraints.All samples collected within the study periods were included. Only tests with valid results  were included in the main analyses. The primary outcomes were: clinic receipt of test results during follow‐up (documented in clinic records); patient receipt of test results during follow‐up (documented in clinic records); turnaround times between sample collection and receipt of test results by the clinic and patient; and turnaround time to clinical action for patients with a documented elevated VL result. Turnaround time was defined as the number of days between sample collection and the outcome , for those with an outcome during follow‐up. Clinical action was defined as either a documented enhanced adherence counselling after a first elevated HIV VL (≥1000 copies/mL) or a switch to second‐line regimen upon a second elevated VL. Shorter timeframes were also assessed for these indicators . Patients with missing dates for steps in the cascade of care were considered as failing to complete that step during analysis. In countries without centralized testing comparison data, only descriptive analyses were presented, including percentages and medians with interquartile ranges (IQR).streg command in Stata was utilized, accounting for facility‐level clustering using shared frailties. Models were adjusted for study design, sex, age, test reason, viral load suppression and country. Kaplan–Meier curves were constructed to visually compare groups. Statistical analysis was performed using StataSE 15 .Continuous outcomes were compared using the Somers’ D test, accounting for facility‐level clustering . Adjuste2.4Approval for this study was obtained from a local Institutional Review Board (IRB) in each country where required ; Malawi: National Health Sciences Research Committee #1666/Chesapeake IRB Pro00021907; Tanzania: National Institute for Medical Research NIMR/HQ/R.8a/Vol. IX/3188; Zimbabwe‐2: Medical Research Council of Zimbabwe MRCZ/A/2470; and United States: Advarra Institutional Review Board #Pro00030414). Written informed consent was waived by all IRBs as all activities conducted were considered standard and routine practice and datasets did not include personal identifiers.3Across all countries, 6795 near‐POC VL tests and 17614 centralized VL tests were conducted, of which 31% versus 6% were conducted for high‐risk populations respectively Table . Of the The median turnaround time from sample collection to clinic receipt of results was 1 day (IQR: 0 to 1) for near‐POC VL compared to 35 days (IQR: 20 to 48) for centralized VL testing  Table .The median turnaround time from VL sample collection to patient receipt of results was six days 1 to 33) for near‐POC compared to 68 days (54 to 87) for centralized testing . Near‐POC results that were available at the clinic on the same day as sample collection were almost twice as likely to be returned to the patient within the 90 days of follow‐up .Time to patient receipt also varied according to VL result. For near‐POC VL tests, patients with an elevated VL had a median time to result receipt of two days (IQR: 1 to 15) compared to 21 days (IQR: 1 to 47) for suppressed patients. Similar trends were observed for centralized laboratory testing with a median time of 53 days (IQR: 29 to 65) versus 83 days (IQR: 59 to 87) for patients with elevated VL compared to suppressed respectively.The turnaround time from sample collection to clinical action for patients with elevated VL was three days 1 to 25) for near‐POC compared to 49 days (29 to 70) for centralized testing . Overall, only 37% of patients with elevated VL had received a clinical action within 30 days of sample collection for near‐POC compared to 7% for centralized testing . With near‐POC, when results were available at the clinic on the same day as sample collection, patients were 1.45 times more likely to have a clinical action (95% CI: 1.15 to 1.82). For both pregnant and breastfeeding women and children (under age 15 years) with elevated VL, use of near‐POC VL was associated with a significantly higher rate of clinical action within 90 days than centralized testing   of near‐POC test results were available at the clinic on the same day. Therefore, the provision of near‐POC VL testing alone is not enough and more efforts are needed to ensure that timely results translate into improved clinical decision making for patients.We did observe a significant impact of near‐POC VL on clinical action within high‐risk groups. Pregnant and breastfeeding women as well as children with elevated VL were significantly more likely to receive a clinical action when tested using near‐POC compared to centralized testing, suggesting that these high‐risk groups would benefit substantially from same day result delivery.It is critical to ensure timely result delivery to all patients, including those with suppressed VL, as data has shown that knowing one’s VL improves retention . While tOur findings of improved outcomes with near‐POC are consistent with the limited previously published research available. In a recent trial in South Africa, reduced turnaround times to result receipt by patient and appropriate switch to second‐line therapy with near‐POC was also observed . HoweverTo close gaps in result utilization and improved patient outcomes, a number of systems gaps within programmes were identified for improvement, including result documentation at facilities, patient result communication and follow‐up and clinical acumen and capacity to make switches to second‐line therapy. In many of the health facilities across country settings, we noted poor documentation of patient receipt of results and more importantly, a lack of documentation of clinical actions taken for patients with elevated VL. Poor documentation of results makes it difficult for clinicians to efficiently identify which patients have not yet received their results or started adherence counselling or other follow‐up procedures. Further emphasis on the importance of maintaining accurate records of patient receipt of results and clinical actions taken will improve programme organization and the ability to flag patients who still need follow‐up. Feedback from programme staff indicated that many health facilities did not take proactive measures to return results to patients promptly. This was seen particularly in Kenya and Tanzania, where the time to patient receipt of results was substantially longer than in other settings, even for near‐POC VL. This translates to missed opportunities to provide timely clinical care for patients with an elevated VL, regardless of testing platform. Stronger patient communication and follow‐up are needed. This may take the form of automated SMS notifications or phone calls from the health facility when results are available. These approaches have been shown in numerous studies to be effective in retaining patients in care , 20, 21.This analysis has a number of limitations. First, this work was not designed as a robust study, nor was study design consistent, with the pre/post and cross‐sectional study designs utilized. Three of the studies had no comparison to centralized testing and therefore analyses were entirely descriptive in nature. The government in each country selected facilities to receive near‐POC devices, and this selection process may have introduced bias into study results. There is the potential that other factors changing over time at facilities influenced findings, and/or that differences in patients prioritized for near‐POC VL testing versus centralized testing influenced the outcomes observed, but sensitivity analyses limited to high‐risk groups found similar differences between near‐POC and centralized testing for all outcomes. Senegal only had 30 days of follow‐up, which most likely underestimates their results in comparison to the other countries. Every country also collected data for a different number of months, which could bias comparisons. The data quality in this assessment was limited by what was available in routinely collected register and patient record data available at health facilities. Some of the secondary analyses had relatively small sample sizes. The clinical action‐outcome could have been affected as staff received clinical training in preparation for near‐POC introduction, but not before in the centralized testing arms. We were not able to explore different clinical actions, transmission, qualitative patient impacts or long‐term follow‐up as outcomes. Finally, implementation practices varied across settings, although this can also be viewed as a strength of this analysis, which, along with the large number of patients tested overall across 57 public sector facilities in seven countries, suggests that findings may be generalizable to implementation in other settings in sub‐Saharan Africa.5In this large analysis across seven countries, near‐POC VL was associated with improvements in result availability at the clinic, as well as turnaround times to result availability and clinical action. High‐risk populations were prioritized for near‐POC VL and were more likely to receive clinical action. However, in many cases, test results did not get back to the patient and/or were not acted upon by clinicians. To truly capitalize on VL implementation and especially near‐POC VL, mentoring activities and support for strong facility data systems and patient follow‐up procedures are needed to improve outcomes for high‐risk patients as well as for routine patient management, ultimately reducing morbidity and mortality.The authors declare no competing interests.SK, LV, JAS, TP, ND and CEB designed the study and wrote the study protocol. MW, SK, CA, CB, BK, TM, JM, JN and MRR led study implementation. JJ and SK led the data analysis. CEB wrote the original draft of the manuscript. All authors critically reviewed the analysis and manuscript and approved the final version.Supplementary MaterialClick here for additional data file.


Video consultations are increasingly seen as a possible replacement for face-to-face consultations. Direct physical examination of the patient is impossible; however, a limited examination may be undertaken via video . Little is currently known about what such video examinations involve.This study aimed to explore the opportunities and challenges of remote physical examination of patients with heart failure using video-mediated communication technology.We conducted a microanalysis of video examinations using conversation analysis (CA), an established approach for studying the details of communication and interaction. In all, seven video consultations (using FaceTime) between patients with heart failure and their community-based specialist nurses were video recorded with consent. We used CA to identify the challenges of remote physical examination over video and the verbal and nonverbal communication strategies used to address them.Apart from a general visual overview, remote physical examination in patients with heart failure was restricted to assessing fluid retention (by the patient or relative feeling for leg edema), blood pressure with pulse rate and rhythm (using a self-inflating blood pressure monitor incorporating an irregular heartbeat indicator and put on by the patient or relative), and oxygen saturation (using a finger clip device). In all seven cases, one or more of these examinations were accomplished via video, generating accurate biometric data for assessment by the clinician. However, video examinations proved challenging for all involved. Participants  needed to collaboratively negotiate three recurrent challenges: (1) adequate design of instructions to guide video examinations (with nurses required to explain tasks using lay language and to check instructions were followed), (2) accommodation of the patient’s desire for autonomy (on the part of nurses and relatives) in light of opportunities for involvement in their own physical assessment, and (3) doing the physical examination while simultaneously making it visible to the nurse . Nurses remained responsible for making a clinical judgment of the adequacy of the examination and the trustworthiness of the data. In sum, despite significant challenges, selected participants in heart failure consultations managed to successfully complete video examinations.Video examinations are possible in the context of heart failure services. However, they are limited, time consuming, and challenging for all involved. Guidance and training are needed to support rollout of this new service model, along with research to understand if the challenges identified are relevant to different patients and conditions and how they can be successfully negotiated. Video consultations using technology such as FaceTime (Apple Inc) offer potential benefits to patients  -4 and hevideo examination is possible . Studies on, for example, chronic obstructive pulmonary disease [In video consultations, patients and clinicians have no shared physical environment . This ma disease  and asth disease , show thnormality of what they saw and felt [Little is currently known about when it is (and is not) possible to conduct a video examination. Clinicians and patients appear cautious , with viand felt . Anotherand felt . Finallyand felt . Cautionand felt .Current research suggests that video examinations may be possible. However, questions remain about how they might be accomplished in practice and with which patients and conditions. Participants have to accomplish the same tasks they would in a face-to-face consultation, maintaining at least the same quality of care, but they cannot rely on the practices and procedures they would conventionally use. They are thus faced with the challenge of developing methods for completing a physical examination over video in real time. In this paper, we explored the interactional and technological challenges of conducting video examinations and how they are overcome.good video consultation . Our fo details ,23 with  details  to exami details  allowed  details , how and details ,28, and  details .Video consultations involved all 5 members of a community heart failure specialist nurse team who were piloting the use of tablet devices for video consultations. All 7 patients had heart failure with reduced ejection fraction , were known to the nurses (having regularly attended follow-up appointments in community clinics), were considered clinically stable, and had sufficient health/digital literacy to participate in a video consultation. As this was a new and potentially risky service model, a doctor-researcher visited each patient at home at the time of the video consultation to troubleshoot the technology, repeat the examination, and check if the patient had any concerns.We recorded both ends (clinic and patient’s home) of each video consultation, using either small digital camcorders  or a handheld iPad (Apple Inc), capturing as much as possible of each individual and their screens as well as contextual details .interactional repair [Initial exploration of data raised questions about how the technology was being used in video examinations , problems experienced when using the technology , and changes in participant roles  . Throughl repair ,32) or tl repair ). Other collections of all instances of each challenge [We transcribed video examinations following CA conventions  Multime, allowinhallenge  . All participants consented to anonymized data being used for research, teaching, and reporting.Video examinations were new to all participants. All seven video consultations were successfully completed but involved clinicians and patients working collaboratively to perform examinations and provide results, sometimes with the help of a relative. In three cases, a doctor-researcher provided assistance (once when a blood pressure monitor battery ran out and twice to position the patient’s tablet or laptop to aid examination of edema). The average duration of a video examination was 6.8 min (range 4.7-11.3 min), in consultations of 21 to 48 min.Below, we focus on three challenges of completing an examination and discuss communication strategies that participants used to negotiate these successfully. Our analysis hones in on successful negotiation of challenges, but that is not to say these video examinations were straightforward. In recipient design [oximeter and instead used the descriptive formulation "little oxygen thing" while simultaneously moving her index finger and thumb together and apart repeatedly, depicting how the oximeter is a hinged, crocodile clip–like device opening at one end to enable fingertip insertion . In theNurses consistently provided upward of 20 instructions in a single consultation. In all seven consultations, patients accepted instructions and explanations and successfully completed the examination. The challenge for nurses was to make correct assumptions about what patients knew to instruct them. These assumptions were not always correct: in eight instructions, nurses assumed that the patient knew more or less than they did. This did not appear to cause issues for patients or relatives who simply sought clarification ,32.Our dataset contained one example of when a patient overestimated their own expertise. The extract in okay to show that she wants to move on with the next step of the consultation [In line 3 , the nurultation -43. She ultation . But theThe cause of the low readings was that the patient held his left hand on his right arm , and there was an apparent tension between supporting the patient’s autonomy over their own body and illness and the role of the relative in enabling a video examination.We identified three cases of patients actively resisting challenges to their autonomy and competence. In the example in before the patient had a chance to perform the examination. In doing so, she revealed doubts about the patient’s capacity to manage the blood pressure meter and attempted to mobilize the relative to help. The patient interrupted, resisting the challenge to her autonomy and competence. Moreover, the patient confirmed that she is “gonna have a go.” By saying, “give me a moment,” she treated the nurse’s request for the relative to help as coming too soon. As she subsequently explained, she was fine.The nurse asked the relative to help out participation framework of a consultation  [bendopnea). At this point of the consultation, the patient’s daughter had barely been involved—she was not visible to the clinician, and the interaction had largely been between nurse and patient. However, at the start of the extract, the nurse calls the daughter by name. And when she appears on screen, the nurse informs her of what she wants her to do, essentially bypassing the patient.Part of the challenge around autonomy relates to the bserver) . Consultbserver) . Consideparticipation framework is noteworthy. By addressing the daughter and stating that she wanted to check her mum, the patient’s role was changed from active coparticipant to clinical object [The exclusion of the patient from the l object ,48. Althl object ). WhetheOf the 18 cases we identified where the nurse or relative spoke on behalf of a patient or assumed responsibility for an examination , 15 were not explicitly challenged by the patient. We did find that patients have alternative ways of resisting their exclusion from the interaction, as with the patient in The third challenge we identified was related to how nurses observed and evaluated video examinations. One way was for the patient or relative to tell the clinician what they saw or felt. Verbal communication of some aspect of a physical examination  was largely unproblematic. Examinations involving physical observation and/or manipulation of a patient’s body were more problematic, with patients and relatives at times struggling to make bodies visible and nurses struggling to observe and assess.professional vision [Patients and relatives do not have l vision  ; for exOur findings demonstrate that physical examinations in video consultations are sometimes possible but are not straightforward replacements of in-clinic examinations. The combination of multimodal recordings of video consultations with microlevel analysis of video examinations, using an established, systematic approach, has allowed us to do the following: First, we have shown that accomplishing video examinations involves a collaborative process with patients, clinicians, and (sometimes) relatives. In the context of heart failure services, this involves rethinking the interactions that typically take place in a face-to-face clinical consultation. Second, we have shown how video examinations are inherently shaped by technology in use. Patients and those supporting them need to understand and manipulate the technology to enable observation and evaluation on the part of the clinician. This involves a complex process of giving and receiving instructions, manipulating technology (the affordances of which can subtly impact the examinations), and ensuring visual presence. This combination can be physically challenging (particularly for older people with limited mobility), practically tricky, and time consuming. For heart failure specialist nurses, it also involves an awareness (built up over time) of patients’ knowledge and experience of their condition, the technology, and the requirements of physical examination. Third, we have highlighted the potential of video examinations to extend patient autonomy and self-management. The lack of physical copresence and the use of video medium requires patients (and sometimes relatives) to take an active role in assessment. Some patients appear to value video examinations as an opportunity to learn how to do self-assessment and manage their own condition. However, caution is needed as some patients may overestimate their expertise, potentially leading to incorrect assessment (and inaccurate results).In sum, our data suggest that participants in heart failure consultations develop new communication practices that enable them to successfully negotiate the interactional and technological challenges of video examinations. This confirms that, at least in some cases, video examinations are feasible.Research on video consultations typically focuses on the feasibility and acceptability of video technology and allied services, with limited appreciation of video examinations. To date, only one study has been published focusing on video examinations, but patients were collocated with a nurse who assisted the specialist with video examination . EvidencOur study, therefore, offers a small but important contribution. To our knowledge, it is the first study to focus specifically on video examinations. As such, we confirm previous work  that thPrevious studies have shown that lack of appreciation of patients’ desired autonomy on the part of the clinician, combined with a focus on relatives over patients, can be detrimental to patient engagement, self-management, and quality of care . We haveThis was an exploratory study, drawing on a small sample of video-recorded consultations in a single heart failure service. Data have allowed us to examine whether video examinations are possible, and our microanalytic approach has enabled us to identify key challenges experienced by clinicians, patients, and relatives as well as strategies for potentially overcoming them. Our methodology is transferable to the study of physical examinations in other clinical conditions and settings.However, there are clear limits to the transferability of our findings. Our focus on patients with heart failure meant that we examined the use of video examinations with a group that are typically older, dependent, and have limited mobility (often because of breathlessness associated with the condition) and multimorbidity. Many struggled with the physical, practical, and technological challenges of video examinations and needed help from a relative to successfully complete the examination. It is likely that a sample of patients with a different condition  would not have the same struggles. Younger patients might have a particular aptitude. Further research is needed to appreciate whether video examinations might be less challenging with those experiencing other conditions.Recordings were made early in the piloting of a remote consulting service. This meant that clinicians, patients, and relatives received no training or preparation for conducting a video examination. Given the complex collaborative process involved in performing video examinations and the need for clear instructions (that likely differ from those in face-to-face consultations), those replicating or extending this work are advised to build in adequate training and support, particularly for those new to the video medium.It is sometimes possible to conduct a physical examination in a video consultation. Video examinations appear feasible for some patients with heart failure, some of the time, but there are significant interactional and technological challenges for all involved. Clinicians and patients require sound appreciation of the technology involved and need to work together to perform video examinations. Further research is needed to understand if other patients with other conditions would find video examinations less challenging. Developers in this space need to work with providers to consider how their devices/software can facilitate video examination. Decision makers would do well to appreciate the challenges of video examinations and the time involved (in setting up as well as doing). Given patient and clinician caution around video examinations and the challenges participants encounter in developing new ways of working, guidance and training are urgently needed to support patients and clinicians in gaining the appropriate experience, knowledge, and interactional skills necessary to successfully manage video examinations. Without this, widespread uptake of video consultations is unlikely.


CRC patients with high lincRNA-p21 expression had shorter DFS (p = 0.0372) and shorter OS (p = 0.0465). Of note, the major prognostic impact was observed in the subset of rectal cancer patients where patients with high lincRNA-p21 levels had worse DFS (p = 0.0226) and OS (p = 0.0457). Interestingly, rectal cancer patients with high lincRNA-p21 benefited from post-operative chemoradiotherapy, as indicated by a longer OS in the group of high lincRNA-p21 patients receiving post-operative chemoradiotherapy (p = 0.04). Finally, patients with high lincRNA-p21 levels in mesenteric vein (MV) had shorter OS (p = 0.0329). LincRNA-p21 is a marker of advanced disease and worse outcome in CRC. Moreover, rectal cancer patients with high lincRNA-p21 levels could benefit from post-operative chemoradiotherapy, and plasmatic-lincRNA-p21 is a promising liquid biopsy biomarker.LincRNA-p21 is a long non-coding RNA involved in the p53 pathway and angiogenesis regulation that acts as prognostic marker in several tumors. In the present study, we aimed to analyze the clinical value of lincRNA-p21 in 177 resected stage I–III colorectal cancer (CRC) patients. Tumor and normal paired tissue and plasma samples from tumor-draining mesenteric veins and paired peripheral veins were analyzed. LincRNA-p21 expression was determined by RTqPCR and correlated with disease-free (DFS) and overall survival (OS). LincRNA-p21 was downregulated in tumor versus normal tissue ( Colorectal cancer (CRC) remains the third most common cancer worldwide, with 1.8 million new cases per year 10.2%), and it is the second leading cause of cancer-related death, with almost 900,000 deaths annually (9.2%) %, and it. In contNon-coding RNAs comprise 97% of the transcriptome, while protein-coding mRNAs account for only 3% —have beeTP53-activated regulator of p53-mediated apoptosis [The long intergenic non-coding RNA p21 (lincRNA-p21) was first described in 2010 as a poptosis . In recepoptosis ,18 and rpoptosis ,20. Howepoptosis . Howeverpoptosis .In the present study, we have explored the potential use of lincRNA-p21 expression levels in tumor tissue from resected CRC patients as a prognostic biomarker. We have assessed the expression levels of lincRNA-p21 in tumor and adjacent normal tissue exploring its association with overall survival (OS) and disease-free survival (DFS) in 177 CRC. We additionally performed an exploratory study to evaluate the role as a liquid biopsy biomarker of lincRNA-p21 using plasma samples obtained from the tumor-draining mesenteric vein (MV) or paired peripheral vein (PV).g and stored at −20 °C until use. The study was approved by the hospital Ethics Committee, and all participants provided their signed informed consent in accordance with the Declaration of Helsinki.Tumor and paired adjacent normal tissue samples were obtained from 177 adult CRC patients at the time of surgical resection in the Hospital Municipal de Badalona, and all samples were stored at −80 °C until use. In addition, on the day of surgery, 5 mL of blood was drawn from the PV and stored in heparinized tubes. During surgery, with vascular ligation before tumor resection, an additional 5 mL of blood was drawn from the inferior tumor-draining mesenteric vein (MV) in 20 rectal cancer patients. Blood samples were collected in BD Vacutainer EDTA tubes (k3) and plasma were separated by centrifugation for 10 min at 2000× RNA was isolated from tumor and normal tissue using TriZol™ Reagent  and from plasma with TriZol™ LS Reagent (Life Technologies) supplemented with MS2 RNA using the Total RNA purification kit  according to the manufacturer’s instructions. cDNA was obtained from 500 ng of RNA using the High-Capacity cDNA Reverse Transcription Kit . Relative lincRNA-p21 expression was determined by real-time polymerase chain reaction (RTqPCR) using the StepOne™ Real-time PCR System Thermal Cycling Block (Applied Biosystems). Quantification of lincRNA-p21 expression levels was performed with Custom qPCR Probes  using the primers and probes described by Hall et al. . Taqman p < 0.1 in the univariate analysis were included in the Cox multivariate regression analyses of DFS and OS to determine hazard ratios (HR) with their 95% confidence intervals (CI). Paired (when necessary) or non-paired t-tests were used for comparisons between groups. Optimal cutoffs of lincRNA-p21 expression data for DFS and OS were obtained using X-Tile software [DFS was defined as the time between surgical resection and recurrence, death without recurrence, or last follow-up. OS was calculated from resection to the date of death or last follow-up. Kaplan–Meier curves for DFS and OS were plotted and compared with log-rank test. All factors with software . All staFrom 2003 to 2017, 177 surgically resected CRC patients not having received any treatment prior to surgery were included in the study. Neoadjuvant treatment was the unique exclusion criteria used in the patient selection, since it could affect lincRNA-p21 basal levels. Ninety-six patients had CC and eighty-one had RC. All patients had tumor and adjacent normal tissue samples and 20 RC patients had blood samples from the MV obtained during surgery and paired PV. Ninety-two (52%) received adjuvant treatment . Among the patients with CC, 51 received adjuvant CT , while among those with RC, eight received adjuvant CT  and 33 received CRT . Median follow-up time for CRC patients was 97.6 months . During follow-up, 33 CRC patients relapsed, 22 of whom died. n = 62). LincRNA-p21 was downregulated in tumor compared to normal tissue samples (Paired t-test p = 0.0012) (p = 0.016). Moreover, lincRNA-p21 levels were significantly different among N0, N1, and N2 patients (p = 0.0482) (p = 0.016) (p = 0.002)  A. The co 0.0482) B. These = 0.016) C. Finall= 0.002) D.p = 0.0372; p = 0.0465; Using the optimal cutoff values identified by X-Tile, we classified the patients as having high or low lincRNA-p21 expression A. Of thep = 0.0226; p = 0.0457; When we performed a sub analysis according to tumor location (CC versus RC), we observed that of the 96 CC patients, 47 had low and 49 high expression; and of the 81 RC patients, 50 had low and 31 had high expression. In the subgroup of 96 CC patients, despite the CC patients with high lincRNA-p21 levels having worse outcome, there was no significant association between lncRNA-p21 levels and either DFS or OS c,d. Intep = 0.025) and in the subgroup of rectal cancer patients . The multivariate analysis of OS identified lincRNA-p21 levels as an independent prognostic marker in all patients . The multivariate analysis of DFS identified lincRNA-p21 as an independent prognostic marker in the overall cohort  or OS (p = 0.4463) in samples from the PV , those with high plasmatic–lincRNA-p21 expression in plasma obtained from MV a showed m the PV e,f.In the present study, we have studied the clinical utility of lincRNA-p21 in CRC. The analysis of 177 CRC patients showed that despite lincRNA-p21 being downregulated in tumor tissue compared to normal tissue, its higher levels in tumor tissue positively correlated with advanced disease characteristics, including lymph node involvement and an advanced stage, and related to worse outcome. Moreover, we observed that lincRNA-p21 expression levels were associated with tumor location where RC patients had the highest lincRNA-p21 levels. In relation with this, we observed that the major prognostic impact was achieved in RC patients, whereas it was not significantly achieved in CC patients.Several studies have explored the function and clinical role of lincRNA-p21 in CRC. An early study  includinAlthough no correlation with survival was observed in previous CRC studies, which was probably due to the small size of the cohorts analyzed, the prognosis impact of lincRNA-p21 has been confirmed in other tumors. In line with our results, high lincRNA-p21 levels were correlated with worse outcome in non-small-cell lung cancer  and prosSince previous in vitro findings have linked lincRNA-p21 expression to radiation response . Taken tFinally, we performed an exploratory analysis to evaluate the potential utility of lincRNA-p21 as liquid biopsy biomarker. For this, we analyzed lincRNA-p21 levels in plasma obtained from two different sources: tumor-draining vein and PV. Our group have demonstrated previously the benefit of analyzing tumor-draining vein samples in front of PV in CCR ,30 and aTo date, the role of lincRNA-p21 in CRC has been studied in small patient cohorts or in vitro, and the present study is the first to explore its impact in a relatively large number of patients pointing out their relevance as prognostic biomarker, especially in RC patients. In summary, our findings indicate that lincRNA-p21 may be a useful prognostic biomarker in CRC, especially in RC where it also could play a role as an indicator of survival benefit from radiotherapy. Of note, these findings will benefit from validation in an independent cohort and from further studies to elucidate their mechanism of action, since validating the role of lincRNA-p21 as a prognostic marker could lead to the development of novel therapeutic strategies in CRC.LincRNA-p21 is a marker of advanced disease and worse outcome in CRC with a major impact in RC patients, where it is clearly overexpressed. Moreover, lincRNA-p21 acts as a marker of radiotherapy response, since RC patients with high lincRNA-p21 levels benefit from post-operative CRT. Finally, the analysis of lincRNA-p21 levels in plasma from tumor-draining vein is a prognostic biomarker for resected RC patients.


It is now clear that thymus function is largely determined by the availability of stromal microenvironments. These specialized areas emerge during thymus organogenesis and are maintained throughout life. They are formed from both epithelial and mesenchymal components, and collectively they support a stepwise program of thymocyte development. Of these stromal cells, cortical, and medullary thymic epithelial cells represent functional components of thymic microenvironments in both the cortex and medulla. Importantly, a key feature of thymus function is that levels of T cell production are not constant throughout life. Here, multiple physiological factors including aging, stress and pregnancy can have either short- or long-term detrimental impact on rates of thymus function. Here, we summarize our current understanding of the development and function of thymic epithelial cells, and relate this to strategies to protect and/or restore thymic epithelial cell function for therapeutic benefit.The thymus is unique in its ability to support the maturation of phenotypically and functionally distinct T cell sub-lineages. Through its combined production of MHC-restricted conventional CD4 BaseWhile TEC populations are known to be key regulators of these distinct T cell development programs, recent studies have uncovered significant new TEC heterogeneity that must be considered in relation to our understanding of microenvironmental control of T cell development –11. Sign−CD8− precursors to the CD4+CD8+ stage, and the positive selection of CD4+CD8+ cells expressing αβTCRs capable of MHC recognition. Interestingly, interactions between CXCR4 and its ligand CXCL12, a chemokine expressed by cTEC, have been reported to play a role in the regulation of both these events  are large epithelial cell complexes in which single cTECs encase viable thymocytes. This is a unique feature of cTEC, and ~10–15% of cTEC form such complexes; each containing between four and eight DP thymocytes . TNC areAtg5−/− embryonic thymus lobes, grafted into nude mice, and showed that host mice generated symptoms of systemic autoimmunity  compartment represent directly progenitors of later mTEC stages, including mTEChi. Indeed, although immature mTEC progenitors are known to reside within the bulk mTEClo compartment, the expression of CCL21 by some of these cells suggests that they are already functionally mature , namely Tspan8 and GP2 protein. To determine the likely developmental progression , cluster medulla . Howeverpression . Interespression  suggeste are not . More ree mTEClo . While ty mature , and so  of RANK , 39 . The transcription regulator Aire is required for efficient promiscuous gene expression of TRAs by mTEC, which is vital for the deletion of self-reactive thymocytes. This contributes to the multi-organ autoimmunity in Aire deficient mice and Aire deficient  patients. The function of Aire exceeds TRA expression. For example, Aire regulates the expression of XCL1 in mTEC; a chemokine important for the medullary localization of thymic DC and Treg generation. Mice deficient in XCL1 exhibit symptoms of autoimmunity, suggesting that Aire promotes central tolerance via multiple mechanisms  TEC  mouse. These grafts contained both Ly51+ cTEC, and Keratin-5+ mTEC, each anatomically segregated into distinct compartments, thus demonstrating that one cell can produce both TEC lineages  TEC , 63. Howlineages . Similarlineages . Althouglineages  confirmehs later .loα6+Sca-1+ cells at the corticomedullary junction (CMJ) could generate both cTEC and mTEC lineages in reaggregate grafting experiments  in adult TECs revealed MHCIIeriments . Howevereviously . In the eviously  and IL7 eviously . In addieviously  fate mapeviously  showed <eviously , 26. SucCre+ cells, that thymosphere forming cells are neural crest derived, and such structures can incorporate bystander TECs, thereby producing the results seen in the initial study , which progressed to become mTEC . In addial study . As suchBoth chronic and acute damage to the thymus have detrimental effects on its ability to support T cell development. In particular, changes that take place within thymic epithelial microenvironments result in a reduction in T cell production, and such events can take place in several ways. For example, both age-related thymic involution and therapeutic cytoablative treatments erode TEC microenvironments which then impair rates of thymocyte development. Importantly however, regeneration processes can occur within the thymus, and several efforts have been made to understand this process and to harness it for therapeutic benefit.de novo T cell production during the first 5–6 months of life , cause apoptosis of radiosensitive cells including thymocytes and TEC . As a reLtbr−/− and TekCreLTβRfloxed mice  of IL-22 deficient mice resulted in defective thymus regeneration compared to WT mice. Furthermore, when irradiation was targeted to the thymus, an increase in IL-22 was also observed suggesting a direct intrathymic recovery mechanism. Interestingly, IL-22 levels were recorded at the highest level when the thymus had the smallest cellularity, suggesting an inverse relationship between levels of IL-22 and thymic size. Production of IL-22 following damage was attributed to radioresistant thymic LTi/ILC3 which were present in increased numbers following thymic insult. Interestingly, irradiated RORγt deficient mice that lack LTi/ILC3 did not increase their levels of intrathymic IL-22 after damage, suggesting a need for RORγt-dependent cells, including LTi/ILC3, during thymus recovery following damage. Importantly, IL-22R is expressed by TEC, and IL-22 increased thymus cellularity due to increased proliferation of TEC and increased frequencies of all developing thymocyte subsets. These positive effects of IL-22 on thymus regeneration is limited to damage, as steady state mice treated with IL-22 showed no increase in total thymus cellularity . It is iby RANKL .Dll4, Kitl, and Cxcl12, thus implicating Bmp4 by endothelial cells to initiate thymus recovery. Moreover, tamoxifen induced deletion of Bmp4 in endothelial cells prevented thymus recovery following TBI. Similar to the radioresistance of ILC3/LTi post-TBI, endothelial cells are also proposed to be radioresistant, as their frequencies remained unchanged in the thymus post-TBI.In addition to the potential of IL22, a recent study highlighted the involvement of bone morphogenic protein 4 (Bmp4) in thymus recovery following damage . Mice th+ T cells, infiltrating the thymus was shown to be reduced following KGF treatment. Despite this, absolute numbers of cells were not reduced suggesting that abrogation of thymic GVHD by KGF treatment was not due to decreased infiltration of donor transplanted T cells. Analysis of T cell development showed a loss of DP thymocytes in mice with GVHD, which was restored with KGF treatment. Although KGF treatment was able to protect the thymus from GVHD, it did not prevent donor T cell infiltration into the spleen which resulted in acute GVHD. The receptor for KGF (FGFR2IIIb) is expressed by TECs, thus the thymic microenvironment was analyzed post-GVHD induction. Thymic cortex/medulla organization was found to be severely disrupted, however organization was maintained following KGF treatment suggesting KGF acts on TEC to protect thymic microenvironments and subsequently promote thymopoiesis  treatment prior to transplant , 101–103Fgf7−/−) mice were sub-lethally irradiated to dissect the role of endogenous KGF on thymus regeneration, they displayed significant reductions in all thymocyte subsets. In addition, Fgf7−/− hosts that received allogeneic or syngeneic BM showed impaired regeneration of thymus as well as reduced peripheral donor and host T cells compared to WT hosts, suggesting host KGF is necessary to mediate thymus regeneration post-BMT (+ were reduced compared to the placebo group. In addition, frequencies of RTE were reduced following treatment with palifermin up to 6 months later. CD4+ effector memory cells were increased post-palifermin suggesting decrease in TCR repertoire (Other studies have also assessed the impact of KGF treatment in a mouse models that are aimed to mimic clinical settings. Here, mice were pre-conditioned with both irradiation and cyclophosphamide then reconstituted with MHC-mismatched bone marrow. Strikingly, KGF treatment allowed for sustained increased numbers of thymocytes for at least 3 months. In addition, KGF treatment increased frequencies of peripheral donor derived naive T cells, suggesting increased thymic output rather than peripheral T cell expansion . Furtherpost-BMT . Similarpost-BMT  studied post-BMT . In relapertoire . Despitelo population, that constitutes the majority of mTEC in the adult thymus, is now known to contain multiple populations that include progenitors of mTEChi, CCL21+ mTEC, and stages that represent post-mTEChi cells. Thus, and in contrast to initial thoughts, mTEClo do not simply represent ‘immature mTEC'. Approaches that enable the isolation and study of individual mTEClo subsets will be needed to understand the functional importance of this diversity. Relevant to this, gaining a better understanding of the functional properties of recently described thymus tuft cells, that reside within mTEClo and represent mTEC developmental stages that occur beyond the mTEChi stage, may be important in revealing how the thymus medulla is able to support the development of multiple T cell lineages that include conventional αβT-cells, Foxp3+ T-Reg and CD1d-restricted iNKT cells.The importance of thymic epithelial microenvironments for T cell development is well-established. Despite this, we still lack a clear understanding of how TEC populations are established during development, and how they change during the life-course. Importantly, we still do not have a clear picture of the changes that take place in TEC populations in response to thymus damage, and how they are restored either naturally or following therapeutic intervention. As such, understanding the cellular makeup of TEC subsets is a key initial step to gain a clearer view of TEC biology, and also inform and improve approaches to manipulate TEC microenvironments with the longer-term goal of boosting immune system recovery. Relevant to this, several studies have now reported previously unrecognized heterogeneity within TEC compartments, and single cell RNA sequencing approaches represent a powerful approach to initially describe new TEC populations. A key goal of future studies will be to examine the potential functional importance of these newly described subsets, and to place them in a developmental sequence that will provide a detailed roadmap of stages in TEC development. For example, the mTECFinally, and as previously noted, while it is clear that TEC recovery occurs in damage settings that include bone marrow transplantation, it is not known whether such recovery involves a complete restoration of all TEC subsets that are now known to exist. Indeed, it is not known whether therapeutic treatments such as IL22, RANKL and KGF impart their effects via TEC progenitors, or via individual or multiple stages in the TEC developmental program. Again, understanding the relationships between newly described TEC populations, and identification of the factors that control their development, survival and expansion will be an important step in optimizing approaches to target TEC populations for therapeutic benefit.AA and BL reviewed the literature. AA, BL, and GA wrote and edited the manuscript.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.


Preterm prelabour rupture of membranes beyond the 34th week of gestation (late PPROM) is frequently associated with the risk of the microbial invasion of the amniotic fluid (MIAC) and histological chorioamnionitis (HCA). Hence, we employed a Tandem Mass Tag-based approach to uncover amniotic fluid proteome response to the presence of MIAC and HCA in late PPROM. Protein dysregulation was associated with only five cases in the group of 15 women with confirmed MIAC and HCA. Altogether, 138 amniotic fluid proteins were changed in these five cases exclusively. These proteins were particularly associated with excessive neutrophil responses to infection, such as neutrophil degranulation and extracellular trap formation. We believe that the quantification of these proteins in amniotic fluid may assist in revealing women with the highest risk of excessive inflammatory response in late PPROM. Late PPROM, occurring between 34 and 37 weeks of gestation, represents more than 50% of cases2. Despite being considered as “near-term,” late PPROM is associated with significant short-term neonatal morbidity, mainly respiratory distress syndrome and a higher risk of early-onset neonatal sepsis3. Concerns about the long-term consequences have also been raised because children born late preterm are at a higher risk of adverse developmental outcomes up to 7 years of age5.Preterm prelabour rupture of membranes (PPROM) is defined as leakage of amniotic fluid prior to 37 weeks of gestation and the leakage of amniotic fluid happening at least two hours prior to the onset of regular uterine contractions6. A recent meta-analysis did not reveal evidence of a higher incidence of early-onset neonatal sepsis in the group with expectant management7. In addition, neonates in the active-management group were more prone to develop the respiratory distress syndrome7. The authors therefore suggest that expectant management is an acceptable alternative to immediate delivery even in the near-term pregnancy.Women with late PPROM can be managed actively with an early planned delivery or expectantly through an attempt to prolong the pregnancy. The aim of the active approach is to avoid prolonged fetal exposure to a potential intra-amniotic infection. On the other hand, expectant management may reduce the incidence of neonatal morbidity  as the neonates are more matured9. The main rationale for the guidelines is the possible presence of subclinical complications such as microbial invasion of the amniotic cavity (MIAC) and intra-amniotic inflammation such as acute histological chorioamnionitis (HCA). These complications are related to the highest incidence of fetal inflammatory response syndrome and short-term neonatal morbidity12. Moreover, MIAC and HCA are frequently asymptomatic and therefore difficult to being detected. The current dilemma with the delivery timing suggests that a universal approach may not offer an apparent benefit in all cases. Early identification of cases with intra-amniotic and intrauterine complications and personalized clinical management might improve the outcomes of late PPROM pregnancies.Nonetheless, expectant management for all women is still controversial and most national guidelines recommend planned delivery for PPROM shortly after 34 weeks of gestation13. However, little is known about the intra-amniotic environment in terms of other proteins, when MIAC and HCA are present in late PPROM. Moreover, the concentration of amniotic fluid IL-6 in the presence of MIAC and HCA may vary from hundreds to hundreds of thousands pg/mL14. Thus, the heterogeneity of individual cases may hamper proper assessment of how severe the intra-amniotic inflammation is.Our group has previously shown that the presence of both MIAC and HCA in women with late PPROM was associated with an increase of amniotic fluid interleukin-6 (IL-6) in comparison with women with either HCA alone, MIAC alone or with absence of both conditions15. One of the main and widely used approaches of protein quantification is multiplexed isobaric tagging allowing simultaneous protein quantification in multiple samples. In this approach, peptides processed from previous protein digestion are labeled with a chemical tag (reporter) in each sample. Differently-labeled samples are then combined before LC–MS/MS analysis. The fragmentation of peptide precursors separates the reporter. Subsequently, the difference in peptide level among samples is assessed according to the relative abundance of reporters in the fragmentation spectra16. Finally, protein quantification is based on the relative quantification of tagged peptides, unique to identified proteins  enables identification of thousands of proteins in different kinds of biological samplesThe aim of this study was to take advantage of the modern methodology to assess how the amniotic fluid proteome responds to the presence of MIAC and/or HCA in women with late PPROM. We believed that these findings might improve our understanding of the molecular processes taking place due to MIAC and HCA in late PPROM pregnancies, and hence facilitate personalized clinical management.Unless otherwise indicated, chemicals and reagents were purchased from Sigma-Aldrich  in the highest available grade.This study was approved by the Institutional Review Board—Ethics Committee, University Hospital Hradec Kralove . All research was performed in accordance with relevant guidelines and regulations. Informed consent was obtained from all participants.17. A total of 60 samples of amniotic fluid were collected by transabdominal amniocentesis. Women older than 18 years with a singleton pregnancy, with gestational age between 34 and 37th week, and admitted with a diagnosis of PPROM were included. The diagnosis of PPROM was conditioned by pooling of amniotic fluid in the vagina and detection of insulin-like growth factor-binding protein 1  in the vaginal fluid. Pregnancy complications such as intrauterine growth restriction, structural malformation or chromosomal abnormality of the fetus, maternal hypertension, diabetes mellitus, pre-eclampsia, thyroid disease, vaginal bleeding, and fetal hypoxia were exclusion criteria. All clinical characteristics are listed in Table 2 test and are presented as numbers (%).The late PPROM cohort was completed considering the following criteria as described previouslyUreaplasma spp., Mycoplasma hominis, Chlamydia trachomatis, and positivity for bacterial DNA identified by PCR targeting 16S rRNA and analyzed by sequencing, and aerobic/anaerobic culture of amniotic fluid. Detection of DNA of Ureaplasma spp., M. hominis, C. trachomatis and other bacteria and aerobic and anaerobic cultures of amniotic fluid was carried out as in previously published work18. HCA was diagnosed on the basis of the degree of neutrophil infiltration that was inspected separately in the amnion, chorion-decidua, chorionic plate, and umbilical cord. The degree of infiltration was assessed according to the criteria described by Salafia19. A diagnosis of HCA was conditioned by the grades of infiltration 3–4 for the chorion-decidua, 3–4 for the chorionic plate, 1–4 for the umbilical cord, and/or 1–4 for the amnion. Samples were divided according to the MIAC and HCA status and were distributed into four subgroups: subgroup with the presence of both MIAC and HCA, subgroup with MIAC alone, subgroup with HCA alone, and subgroup with absence of both MIAC and HCA , or tocolytics. Up to five mL of amniotic fluid was supplemented with a protease inhibitor cocktail . The treated samples were then centrifuged at 300 × g for 15 min and passed through a 0.22 μm syringe filter  to remove cells and debris. The total protein concentration was determined by Bicinchoninic acid assay and ballast proteins were removed as described in the Amniotic fluid samples were prepared according to the workflow successfully applied in our previous reportsEnzymatic digestion of proteins was processed in 200 mM triethylammonium bicarbonate buffer, pH 8.5  with 1% of sodium deoxycholate. Disulfide bonds were reduced with 5 mM tris(2-carboxyethyl) phosphine hydrochloride at 60 °C for 1 hour (h), and the free thiol groups were blocked using 10 mM methyl methanethiosulfonate at room temperature for 30 min. Amniotic fluid proteins were digested with lysyl endopeptidase  at 37 °C for 4 h, followed by digestion with trypsin  at 37 °C overnight. Both enzymes were added at a 1:50 enzyme to protein ratio (w/w).Tandem Mass Tag (TMT) 10plex isobaric label reagents  were dissolved according to the manufacturer’s instructions. All the amniotic fluid samples were evaporated to dryness and re-suspended in 8.57 µL of 100 mM TEAB, and 1.57 µL was taken from each sample and combined to prepare a global internal standard (GIS). The total volume of the GIS was split into 10 aliquots. Individually digested samples and the GIS aliquots, both at concentration 1 µg/µL, were incubated with 10 μL of the TMT reagents at room temperature for 1 h. The reaction was terminated by adding 5% hydroxylamine. Nine amniotic fluid samples and one GIS aliquot were mixed in 1:1 ratio to create seven multiplexes  at 1.8 kV spray voltage. Positive ion full scan MS spectra were acquired in the range of m/z 350–1600, with 3 × 106 AGC target in the Orbitrap at 70,000 resolution with a maximum ion time of 50 ms. A lock mass of m/z 445.12003 ([C2H6SiO]6) was used for internal calibration of the mass spectra. The fragmentation (MS/MS) spectra were acquired for the 15 most intense precursors. Other parameter settings were assumed from the literature22. Isolation window of 1.2 m/z and normalized collision energy of 33 was used. Each fragmentation spectrum was acquired at a resolution of 35,000, with a 2 × 105 AGC target and a maximum injection time of 100 ms. The first mass was fixed to 120 m/z.The multiplexes were redissolved in 0.1% trifluoroacetic acid (TFA), 2% acetonitrile (AcN) to a concentration of 1 µg/µL and injected twice on an UltiMate 3000 RSLCnano System  in three technical replicates. The analytical system consisted of a PepMap100 C18, 3 µm, 100 Å, 75 µm × 20 mm trap column and a PepMap RSLC C18, 2 µm, 100 Å, 75 µm × 500 mm analytical column  and was previously used in our proteomic analyses23 and R24. Potential contaminants and proteins identified by site and by reverse sequence were removed. The reporter intensity of proteins in each sample was divided by reporter intensity of proteins in the GIS, both adjusted to correction factors. Resulting ratios were log2 transformed and normalized to the median. One-way analysis of variance (ANOVA) with Benjamin–Hochberg false discovery rate (FDR) correction was applied to compare normalized protein ratios in the subgroups of late PPROM. Perseus Tukey’s significant honest difference post-hoc test on the significance level of 0.05 was used on the ANOVA significant hits. Gene ontology (GO) analysis was carried out using StringApp plugin25 in the Cytoscape platform26 . Violin plots, principal component analysis (PCA) plot and bubble plots were designed in the R software. Bar graphs of GO terms were prepared in GraphPad Prism 6.Recorded spectra were processed and searched in MaxQuant 1.5.5.1 against a reviewed SwissProt human protein database including contaminants downloaded in 2016. Trypsin digestion specificity with up to two missed cleavages allowed was used. Cysteine thiomethylation was set as a fixed modification and oxidation of methionine was selected as a variable modification. The mass tolerance in MS and MS/MS mode was left at the default value for the initial search and 6 ppm was set as mass tolerance in MS mode for the main search. The false discovery rate for protein identification was left at the default value. Output files from MaxQuant were processed in PerseusELISA kit for human IL-6  and human histone H1.5  were used to determine IL-6 and H1.5 levels in amniotic fluid. Samples with IL-6 concentration above the top calibration point were diluted to fit the calibration range. Resulting absorbance was read at 450 nm with correction to 540 nm. Data was evaluated in GraphPad Prism 6.27. Hence, the late PPROM cohort was carefully designed to include women in similar gestational age at admission and delivery. The median time of latency between late PPROM and amniocentesis was 4 h. We found a significant difference only between the subgroup with the presence of both MIAC and HCA  compared to the subgroup with the absence of both MIAC and HCA . The median time of latency between amniocentesis and delivery was 10 h and the median time of latency between late PPROM and delivery was 16 h. There was not any significant difference in the duration between amniocentesis/late PPROM and delivery . Based on these results, we infer that the changes of protein levels were not due to differences in the gestational age.A late PPROM cohort of 60 amniotic fluid samples with the presence or absence of MIAC and/or HCA was designed Fig. A and imp28. The immunoaffinity depletion of abundant ballast proteins allowed us to detect a total of 1132 amniotic fluid proteins. The protein quantity was expressed as the ratio of TMT reporter ion intensity of each identified protein to the TMT reporter ion intensity of the global internal standard (GIS) in the respective multiplex  is the most abundant protein in amniotic fluid and may smother information on important but far less abundant proteinsThe initial comparison was based on determination of the protein difference in the subgroups divided according to MIAC and/or HCA . Firstly, the multiple comparisons of protein levels were adjusted by Benjamin-Hochberg correction with the FDR threshold 0.05. However, no significant protein changes related to MIAC and/or HCA were found. The histone H1.5 (H1.5) was found as the first protein above the FDR threshold . Thus, violin plots based on the level of H1.5 in amniotic fluid were constructed to explain the uniformity of the late PPROM subgroups despite distinct MIAC and HCA status  and protein S100A12. The size of bubbles are set according to the concentration of IL-6 and the color of bubbles represents the presence or absence of HCA and funisitis, the most severe form of HCA characterized by the presence of neutrophils in the umbilical cord  or the presence of Haemophilus influenzae and Sneathia sanquinegens was confirmed  The levels of amniotic fluid proteins in the subgroup with both MIAC and HCA were surprisingly uniform compared to the other late PPROM subgroups where both conditions weren’t confirmed. We found only five cases of late PPROM women with a substantial protein dysregulation related to both MIAC and HCA. We speculate that particularly these women can profit from an active management beyond the 34th week of gestation. (2) Proteome change was associated with a high concentration of IL-6 in amniotic fluid. (3) The positive correlation of proteomic findings with IL-6 concentration identified five women with strong intra-amniotic inflammation in late PPROM.32. However, to the best of our knowledge, a global proteomic study focused on PPROM beyond the 34th week has not been carried out yet. In TMT 10plex-based quantification, peptides of each sample are tagged with one different isotopic variant from a set of ten mass tags33. However, since the number of patients in our late PPROM cohort exceeded the number of TMT channels, we designed seven multiplexes and analyzed them in different LC–MS/MS runs  and proteins integrated in neutrophil extracellular traps  were shown to be considerably upregulated in the presence of both MIAC and HCA. Besides the CysTRAQ article, recent papers have discussed the elevation of IL-6 in the presence of both MIAC and HCA in cases of both PPROM and late PPROM36. The authors also concluded that the presence of both MIAC and HCA and a higher level of IL-6 were related to early-onset sepsis and neonatal morbidity.This study was inspired by published results which indicated that a change of amniotic fluid proteome is associated with the presence of both MIAC and HCA in PPROM37. However, protein inflammatory response to MIAC and HCA is not a homogeneous phenomenon and a pooling strategy may disguise the variance of inflammatory protein concentration in individual samples. Keeping in mind the heterogeneity of MIAC and HCA, we required information about levels of proteins in each individual sample of the patient cohort. Moreover, we included also the subgroups with MIAC alone and HCA alone. Histological chorioamnionitis itself may evoke the pro-inflammatory protein response, but only the presence of both MIAC and HCA is apparently related to a higher number of cases of early-onset sepsis and fetal inflammatory response syndrome38. Hence, it is desirable to distinguish women with both MIAC and HCA from those with HCA alone.In the CysTRAQ study, a pooled sample strategy was used. The pooling of samples is a time-efficient strategy compensating for limited amounts of the samples or high biological variationUreaplasma spp. and/or M. hominis and/or the presence of Haemophilus influenzae and Sneathia sanquinegens , DEFA3, cathelicidin are released from neutrophil granules41. The lumen of neutrophil granules was the most over-represented cellular component in our GO analysis 43. The ligand-RAGE interplay is a key mechanism of reactive oxygen species (ROS) generation and oxidative burst44. Other target molecule of S100 proteins is glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme of glycolysis45. Glycolysis and the pentose phosphate pathway (PPP) are the key metabolic pathways in neutrophils and we found both of them as significant pathways in GO enrichment (Fig. Besides the granular proteins mentioned above, we found other 34 proteins present in neutrophil granules and most of them were highly upregulated in the five discussed samples (Fig. 48. The presence of NETs in intra-amniotic infection and inflammation has been already established49. Unambiguous confirmation of NETs in amniotic fluid for instance using immunofluorescence microscopy50 was beyond the scope of this study. Nevertheless, we found alterations in the redox homeostasis affecting the neutrophil activity towards to NETs formation as well as the substantial upregulation of proteins being a part of NETs defense mechanisms such as MPO, DEFA3, MMP9 and histone isoforms representing strong indicia of the NETs presence (Fig. 51.Besides energy generation, glycolysis and PPP directly affect also the formation of ROS by providing NADPH. A total of 19 proteins related to the redox process were upregulated in the five samples. We found upregulation of enzymes important for formation and elimination of ROS, including MPO, mitochondrial superoxide dismutase, catalase, glutaredoxin-1, peroxiredoxin-1, etc. , Fig. 3C49. In the five outlying samples from the subgroup with both MIAC and HCA, histones H1.5, H2B, H4 and H3.2 were highly upregulated. The level of H1.5 in amniotic fluid determined by ELISA also showed a significant increase in the five outlying samples with the presence of both MIAC and HCA compared to the other 55 samples (Fig. 52. NETs have been even signed as a link between inflammation and thrombosis53. We found twenty up- or downregulated proteins related to platelet activation, signaling and aggregation (Fig. 55. On the other hand, another platelet protein thrombospondin-1 was downregulated in the five outlying samples with the presence of both MIAC and HCA compared to the other 55 samples. Thrombospondin-1 inhibits the activity of neutrophil elastase and cathepsin G and mitigates the inflammatory response56. We suggest that downregulation of thrombospondin-1 may promote the activity of neutrophil proteases as a consequence of excessive intra-amniotic inflammation in the specific subgroup with the presence of both MIAC and HCA in late PPROM.Histones are frequently discussed as key part of NETs and potential biomarkers of MIAC and HCA in PPROMles Fig. . Beyond ion Fig. . From th57, and the conclusive cut-off value has not been established. Future research should thus unambiguously define the concentration of IL-6 representing the threshold for the confirmation of intra-amniotic inflammation.In this study, we analyzed a unique collection of late PPROM amniotic fluid samples without a significant difference in gestational age at PPROM, amniocentesis, and delivery. We revealed the dysregulation of 138 proteins along with the high level of amniotic fluid IL-6, the presence of funisitis and MIAC. This study was initially aimed at discovery of protein changes associated with MIAC and HCA. We had presumed that the presence of neutrophils in the placenta, umbilical cord, and fetal membranes would be reflected by the change in the amniotic fluid proteome. However, we only detected five samples in the subgroup with the presence of both MIAC and HCA with substantial change in proteome composition. Consequently, the original stratification of the late PPROM cohort based on the status of HCA turned out to be a limitation of this study because the number of MIAC/HCA women with a strong proteome response was very low. However, these women can particularly profit from active management compared to other women with late PPROM. With respect to the achieved results, we recommend diagnosing the intra-amniotic inflammation rather based on the level of amniotic fluid IL-6 than as the diagnosed HCA. Nevertheless, the threshold values of intra-amniotic inflammation in PPROM vary from 2.6 to 11.3 ng/mL in previous publicationsSupplementary information.Supplementary table.