Despite the increasingly large collection of fluorescent protein (FP) variants engineered to date, only a few of these were successfully applied in bacteria. Here, we explore the performance of recently engineered variants with the blue (TagBFP), orange , and far-red (mKate2) spectral colors by tagging HU, LacI, MinD, and FtsZ for visualizing the nucleoid and the cell division process. We find that, these FPs outperformed previous versions in terms of brightness and photostability at their respective spectral range, both when expressed as cytosolic label and when fused to native proteins. As this indicates that their folding is sufficiently fast, these proteins thus successfully expand the applicable spectra for multi-color imaging in bacteria. A near-infrared protein (eqFP670) is found to be the most red-shifted protein applicable to bacteria so far, with brightness and photostability that are advantageous for cell-body imaging, such as in microfluidic devices. Despite the multiple advantages, we also report the alarming observation that TagBFP directly interacts with TagRFP-T, causing interference of localization patterns between their fusion proteins. Our application of diverse FPs for endogenous tagging provides guidelines for future engineering of fluorescent fusions in bacteria, specifically: (1) The performance of newly developed FPs should be quantified  Soon after the first cloning of the green fluorescence protein  demands fast-folding of FPs. This is clearly indicated by the fact that mutations improving the folding properties of EGFP generation , enhanced their effective brightness by several folds in bacteria, while this was much less the case when expressed in mammalian cells , 2007. Afixation . The fasfixation , and havfixation . FurtherE. coli by labeling the cytosol, the nucleoid, as well as the division proteins. HU-2, encoded by hupA gene, is a subunit of nucleoid-associated proteins HU, which serves as a marker for the nucleoid that was previously shown to colocalize with DAPI when fused to GFP , orange (TagRFP-T and mKO2), far red (mKate2), and near-infrared (dimeric eqFP670) spectral colors. All FPs used in this study are listed in Table 1. The latter proteins are derivatives of the FPs from the sea anemone Entacmaea quadricolor and Fungia concinna, known for their brightness, photostability, and relatively fast maturation  with four PCR-amplified fragments, which were the backbone of pFB174 with arabinose promoter and chloramphenicol resistance gene, an ftsZ  fragment with an 18 base overhang coding the flexible linker GSGSGS, a GGSGSS flexible linker plus ftsZ  plus aph frt sequence amplified from strain FW1370, two synthesized oligos containing the tetracysteine (TC) peptide coding sequence and the two flanking flexible linkers. The TC coding sequence was then replaced by tagRFP-T, sfGFP, and tagCFP to produce plasmid pFWZ4, pFWZ5, and pFWZ6 through two-fragment Infusion reactions. pFWZ0 was constructed through Gibson assembly of pFB174 backbone and the ftsZ::aph frt sequence amplified from strain FW1370. All plasmids are listed in Table 2.The plasmids were constructed using Gateway cloning kit  and Infusion EcoDry kits , Coding sequences of mCherry in strain RRL189 through λ Red recombination to result in strains FW1965, FW2417, and FW2450. The PCR fragments of pBVS3, pBVS36, and pBVS37 were used to replace the ΔminD minE::cat sacB in strain FW1363 through Red recombination to result in strains FW1480, FW1248, FW1393. Note that in strain FW1363 the minC gene is intact. The above listed strains were cured of kanamycin resistance using pCP20 as described in Table 2. To confirm the functionality of YFP-MinD, Plac::yfp-minDE was transduced from FW1462 into FW1363 to yield strain FW1463. For multi-color imaging, hupA-tagBFP from FW1344 was transduced into strain FW1554 to yield 1561; ΔleuB::eqFP670 was transduced from FW1489 into FW1554 to yield FW1559; ΔleuB::tagRFP-T was transduced from FW1401 into FW1359 to yield FW1406; hupA-mYPet from FW1551 was transduced into a strain with aph cured from FW1965 to yield FW2480; Plac::yfp-minDE was transduced from FW1462 into FW1459 to yield strain FW1503; pFWZ4 was transformed into strain FW1559. All strains used are listed in Table 3.The genomic insertions were constructed using λ Red recombination  and shuﬄE. coli cells were incubated in Lysogeny broth (LB) supplemented, when required, with 100 μg/ml ampicillin (Sigma-Aldrich), 50 μg/ml kanamycin (Sigma-Aldrich), or 34 μg/ml chloramphenicol (Sigma-Aldrich) for plasmid selection, or with 25 μg/ml kanamycin, 20 μg/ml chloramphenicol, or 0.2% sucrose for selection of the genomic insertions of gene cassettes. For imaging strains with fluorescent foci with LacI fusions, we grew cells in liquid M9 minimum medium  supplemented with 2 mM MgSO4, 0.1 mM CaCl2, 0.4% glycerol (Sigma-Aldrich), and 0.01% protein hydrolysate amicase . For imaging other strains, we grew cells either in liquid M9 minimum medium supplemented with 2 mM MgSO4, 0.1 mM CaCl2, 0.4% glucose (Sigma-Aldrich), and 0.25% PHA, or in LB medium. For imaging, overnight cultures were back diluted into the fresh medium described above to an OD  of 0.01 in falcon tubes until an OD of 0.4–0.6 for M9 medium with 0.25% PHA and LB, and OD of 0.1 for M9 medium with 0.01% PHA. The growth conditions for the FtsZ complementation assay are as described in ftsZ.For genetic engineering, ex/λbs/λem wavelengths of the filter cubes are as follows: SBFP2 and EBFP2 (363–391/425/435–485 nm), TagBFP (395–415/420/435–485 nm), mCerulean (426–446/455/460–500 nm), sfGFP (450–490/495/500–550 nm), mYpet and FlAsH (490–510/515/520–550 nm), TagRFP-T and mKO2 (530–560/562/570–620 nm), mCherry and mKate2 (540–580/585/592–668 nm), eqFP670 (589–625/649/655–1200 nm). For imaging with SpectraX, the excitation filters for orange and red proteins are respectively 555/25 nm  and 575/22 nm. The multiband emission filters are respectively 435/26 – 510/40 – 595/40 – 705/72 nm, and 465/25 – 545/30 – 630/60 nm. The fluorescence signal was recorded by an Andor EMCCD camera (iXon Ultra 885), with an EM gain of 100. While our emission filter for the eqFP670 extends to the infrared region, eqFP670 does not fluoresce beyond 850 nm, which is well-within the detection range for most EMCCD cameras, including the one used in this study.Fluorescence imaging was carried out using Nikon Ti-E microscope with 100X CFI Apo TIRF objective with an NA of 1.49. All fluorescent probes were excited using a Nikon Intensilight, except for the photo-bleaching of lacI foci, which was excited by SpectraX LED single-spectrum light sources (Lumencor) with SpectraX filter sets (Lumencor). For imaging with Nikon Intensilight, the λhttp://ceesdekkerlab.tudelft.nl/downloads/.Analysis of fluorescent microscopy images was carried out using Matlab with our customized programs. The background intensity was subtracted for all images individually. For identification of fluorescent LacI foci or nucleoid, the images were Gaussian blurred for subtraction, and a threshold for the expected object size and intensity was applied to eliminate noise. The intensities of the identified objects were then collected for statistics. For the photobleaching data, the mean per-pixel intensity of each nucleoid was calculated independently. To compare the photostability of more than two FPs in the main figure, the total intensities of all identified objects were summed and divided by the total number of pixel for all found objects in the first image, in order to take the completely bleached objects at the later stage of the bleaching period into account. In the latter case, the sum fluorescence intensity of the FPs in individual objects was plotted only in the supplementary figures. The standard deviation values of the intensities were not shown in the main figures for the convenience of display, and are instead plotted in supplementary figures. The matlab scripts used for these measurements can be found at cell = (Icell-Ibg)/SDbg. We use the standard deviation value of the cell-free region as a measure of background noise (SDbg). We use the difference between the mean intensity of a cell (Icell) and the mean background intensity (Ibg) as a measure of signal. The mean and SD values of the SNR calculated from all cells are used for plotting.The signal-to-noise ratio (SNR) of the cytosolic FPs is calculated using SNRTable 1). In principle, if a BFP would be sufficiently bright, it can be imaged with excitation light that is weak enough to avoid photodamage to the cells. It was recently reported that the purified monomeric mTagBFP  has a brightness that is similar to EGFP and 1.8 times that of EBFP2 . The fluorescence signal of the expressed TagBFP is indeed sufficient for full cell labeling of bacteria , indicating that TagBFP may have the brightness and maturation rate suitable for labeling proteins expressed at their endogenous level.The blue variants of the fluorescent proteins (BFPs) have seen few applications in bacteria due to their low brightness and short excitation wavelength . For imaging, we used two customized filter sets for the different excitation peaks of the EBFP2/SBFP2 (379 nm) and TagBFP , and the same emission filter . Importantly, direct FP fusions to the C-termini of genes at their endogenous loci have been confirmed to maintain the endogenous expression level of these genes , i.e., the HU-TagBFP concentration has a standard deviation that is only 13% of the mean value across the cell population during exponential growth in LB.HU-TagBFP outperforms HU-EBFP2 and HU-SBFP2 to a surprising extent in live bacteria, as shown in al phase . Such anFigures 1D,E). Taking advantage of the narrow excitation profile of TagBFP and its excitation peak at 402 nm, we customized a filter set to maximize the excitation efficiency and to collect the majority of the emitted light . This filter set can be combined with regular commercial filter sets for GFP or YFP for two-colors imaging. Here, we illustrate such a combination by transducing the endogenous hupA-tagBFP fusion construct into a strain with a bright endogenous sfGFP-minD fusion , yielding strain FW1561. Shown in Figure 1F, these two fluorescent probes successfully captured both the localizations of the nucleoids and the MinD oscillations, which together define the mid-cell for the localization of the cytokinesis machinery.TagBFP’s excitation/emission spectra are well-separable from green and yellow fluorescent proteins (GFPs and YFPs) for multi-color imaging . In this strain, the expression of the LacI-SBFP2 is constitutive. We found that it can be co-imaged with ter foci label TetR-mCerulean (cyan) and chromosome label HupA-mYpet  . Notably, the LacI-SBFP2 foci are much dimmer than the original LacI-mCherry fusion, requiring 2-s exposure time with Nikon Intensilight, thus less suitable for time-lapse imaging in dynamics studies. Alternatively, Everogen has produced TagCFP (Ex/Em = 457/480 nm) which can be combined with TagBFP, but we did not persue this route due to its potential spectral crosstalk with YFPs, and that our FtsZ-TagCFP fusion did not fluoresce.A potential combination of BFPs with cyan fluorescent proteins (CFPs) can further increase the options for multi-color imaging. Despite the higher brightness of TagBFP compared to SBFP2, it has more spectral overlap with CFP and thus is found less suitable for this application. As shown in  cytosol . However cytosol , resultiFigure 2A). Despite the fact that these two proteins are excited with long-wavelength and hence less phototoxic compared to GFP, frequent time-lapse imaging of strains FW1459 and FW2095, with the same setting for Figure 2A at a frame rate of 5 s for 5 min did lead to cell growth arrest although no significant photobleaching was observed (data not shown), indicating that the photostability is not a limiting factor for imaging live cells using these FPs. Note that this is an overexposure test for whether the photostability is sufficiently high when imaging is carried out at a lower rate where photodamage is avoided. The trade-off between photodamage, fluorescence signal, and temporal resolution requires moderation of the settings to specific cases.By applying the same procedure above to characterize the performance of TagRFP-T and eqFP670  as cytosolic labels, we found that they perform superiorly in brightness and photostability for the orange and near-infrared spectral range, respectively . This is largely owing to the almost full collection of far-red emission lights, while a smaller part of the emitted light is collected for the other proteins to avoid cross-talk. By contrast, a monomeric derivative TagRFP657 (excitation/emission peaks at 611/657 nm), which was produced for flow cytometer applications is not visible in our constructs even when placed under either the same synthetic promoter as above or a T7 promoter in a plasmid with pBR322 origin (pERB004 and pERB005). This agrees with the invisibility of TagRFP657 in a previous attempt in yeast . These customized filter sets allow us to measure the relative brightness of the two proteins in live cells  from yellow to near-infrared spectral region . Both TagRFP-T and eqFP670 are invisible with a YFP filter, while they show an equal brightness when imaged with a regular far-red filter used for mCherry. As expected from the spectral data, TagRFP-T does not show any bleed-through into the near-infrared spectral range. In the orange spectral range, TagRFP-T exhibits an over 10 times higher signal-to-noise ratio than the small bleed-through signal from eqFP670. The bleed-through from the eqFP670 into the orange spectral range is difficult to avoid due to the long tail of its excitation spectrum into the shorter wavelength. From these data, we conclude that the combination of TagRFP-T and eqFP670 is excellent for multi-color imaging at the orange and near-infrared spectral range.TagRFP-T and eqFP670 have the spectral properties that allow multi-color imaging at the red-shifted spectral range. While TagRFP-T has been reported as a red FP, its excitation and emission spectra are more blue shifted compared to conventional RFPs such as mCherry . By comphupA gene to tagRFP-T as well as to another recently reported FP gene, mKO2, which is expected to encode a bright, fast-folding orange/red FP . Indeed, these two fluorescent probes show excellent brightness as an HU-2 tag in live bacteria, clearly improving the performance of orange proteins mKO and mOrange, which were previously shown to be invisible in live cells as fluorescent tags to FtsZ  with lacI-mKate2. Shown in Figure 2F, the average intensity of the fluorescent foci of LacI-mKate2 is 25% enhanced with the same excitation/emission filters as compared to regular far-red proteins. The photostability of the two proteins is similar. In both cases after 300 s of exposure, 95% of all spots were still detectable through our automated software, showing that LacI-mCherry or LacI-mKate2 are both excellent candidates for operator-repressor system in time-lapse imaging. Furthermore, we engineered a LacI-mKO2 fusion based on strain RL189, and its foci are five times brighter in the orange spectral range than in the red spectral range . By contrast, polydimethylsiloxane (PDMS), the most commonly used polymer for microfluidic applications, has a reflective index of around 1.4, which is almost identical to that of a bacterial cell. When imaging bacteria in between PDMS structures and agarose, the cell boundaries display much less contrast . This type of interference between the microfluidic channel boundary and the bacterial boundary is even worsened for PDMS–PDMS interfaces. Furthermore, silicon structures are non-transparent, thus only allowing bright-field or DIC microscopy, in reflection rather than transmission, and bacteria in ∼micron-sized channels are difficult to distinguish . In all these scenarios, the availability of cytosolic labels at the near-infrared (eqFP670) spectral range becomes particularly useful as a third or fourth color .Expanding the spectral range of fluorescence proteins for cytosolic label is also advantageous for studies using microstructures. Microstructures and microfluidics are emerging tools for studying bacterial physiology . While tE. coli with high temporal resolution.As shown above, TagRFP-T can be spectrally well-separable from the near-infrared protein eqFP670 as well as from GFPs/YFPs. This can potentially lead to three-colors imaging at the long-wavelength regime, where phototoxicity can be reduced by avoiding near-UV excitations (such as necessary for CFPs or BFPs). Such an approach is in fact essential when frequent time-lapse imaging is required for tracking dynamic protein localizations over long time. Here, we describe the construction of new FtsZ and MinD fusions for time-lapse imaging of division site selection in Figure 3A; An FtsZ molecule has a globular structure composed of independently folded N-terminal and C-terminal domains both essential for polymerization, followed by an unstructured flexible linker, and a conserved C-terminal tail responsible for interacting with its membrane-bound partners FtsA and ZipA as well as lateral interactions between FtsZ polymers . We hoped that a FP derived from another origin could perform differently during the folding of the sandwich fusion, thus have potential to success in sandwich fusion. In order to confirm the suitability of the custom-designed flexible linkers and insertion site, we first inserted a tetracysteine (TC) sequence (FLNCCPGCCVEP) flanked by the short flexible linkers (GSGSGS-TC-GGSGSS) into the ftsZ gene, yielding pFWZ7. This TC tag can later be labeled by Fluorescein Arsenical Hairpin . Under this growth condition, however, the ΔftsZ strain with pFWZ7 always showed a significant fraction of filamentous cells (30% in biomass). To label the FtsZswTC with the FlAsH dye, we optimized the protocol for live cell staining in E. coli. By incubating an exponentially growing bacterial culture in LB with 2 μM FlAsH dye for 3 h followed by a gentle wash step with EDT2 buffer, we were able to observe the fluorescence of Z-rings localizing at the cell middle in the regular rod-shaped cells . By contrast, the filamentous cells show patches of fluorescence over the whole cell body, indicating that the filamentation is mainly caused by the overproduction of FtsZswTC, which disrupted the ratio between FtsA and FtsZ. It was shown previously that an overexpression of only FtsZ or only FtsA can lead to a filamentous phenotype . The FtsZswsfGFP and FtsZswTagRFP-T were able to form fluorescent Z-rings with or without the existence of native, untagged FtsZ, but they were not able to rescue the lethal effect of FtsZ deletion. The FtsZswTagCFP, on the other hand, did not fluoresce and was not investigated further. Nevertheless, the former two constructs can serve as alternative ectopic FtsZ probes in addition to the existing ones.We further replaced the TC peptide in the pFWZ7 construct with sfGFP, TagRFP-T, and TagCFP, which are derived from three different protein origins . The superior brightness and photostability of both FtsZswTagRFP-T and sfGFP-MinD allowed us to probe the dynamic relations between the localization of FtsZ and MinD with a frame rate of 1–4 s for 2 min. Figure 3D shows that FtsZ filaments can be assembled and disassembled within a time frame of 20–30 s, in a pattern which anti-correlates with sfGFP-MinD localization patterns. Besides the above-mentioned advantages, the use of FtsZswTagRFP-T also results in less phototoxicity due to its long excitation/emission wavelength, in contrast to the previous YFP-MinD/FtsZ-CFP combination . Without the Min system, E. coli often divide asymmetrically, producing anucleated minicells. The Min operon is composed of minC, minD, and minE genes which are positioned sequentially in the genome, with one promoter in front of MinC that drives the transcription of all three genes, and another promoter embedded in the minC gene to only drive MinD and MinE expression . Co-transcription of minD and minE ensures the ratio of their protein products to be rather constant, which is essential for the localization patterns of all Min proteins. Previously, gfp-minDE and yfp-minDE constructs were shown to be able to complement the ΔminDE phenotype when placed under the lac promoter in medium copy plasmids and induced with low concentrations of IPTG . The sfGFP-MinD fusion under the endogenous RBS was expressed at a level almost identical to the wild-type  can be combined with cytosolic TagRFP-T (strain FW1503) for the same applications, yielding the same localization patterns as sfGFP-MinD . This confirms that the effect of symmetry and scale on the Min pattern formation is intrinsic and not dependent on the FPs, and verifies that the previous studies with modest YFP-MinD levels indeed represent well the native Min protein behavior. However, strain FW1503 appeared to be more sensitive to osmotic shock due to unknown reasons and thus less suitable for quantitative studies or for producing cells larger than 4.5 μm × 4.5 μm × 1 μm using the same shaping method.The endogenous fusion of the ructures . By inoc microns . Here, wFigure 4). It appears that the HU-TagBFP recruites FtsZswTagRFP-T to the nucleoid. To confirm such a strong interaction between TagBFP with TagRFP-T, we transduced a tagRFP-T cassette into strain FW1359, and found that HU-TagBFP is also able to recruit the freely diffusing cytosolic TagRFP-T molecules to the nucleoid . Moreover, this recruitment induced elongated cell morphology and chromosome-segregation defects .From the characteristics of all FPs above, we expected that TagBFP and TagRFP-T would be excellent, mutually exclusive partners for multi-color imaging, in combination with the broadly available bright GFPs/YFPs variants. Thus, we transformed pFWZ4 (FtsZswTagRFP-T) into strain FW1359 for co-imaging of FtsZ and chromosome. To our surprise, we observed patchy blobs in the orange channel, which colocalize with the fluorescence signals from the TagBFP channel , orange (TagRFP-T and mKO2), red (mKate2), and near-infrared FPs (eqFP670) into localization studies in bacteria. These proteins generally showed the superior brightness and photostability in bacteria, consistent with previous reports of their For single-color imaging with high temporal resolution, TagRFP-T appears to be an excellent probe for bacterial studies. Besides its extremely strong photostability, it is excited with a much longer wavelength compared to the common GFPs. Using TagRFP-T reduces the phototoxicity compared to GFPs, allowing imaging at higher frequency and intensity. Adding to a previous study which successfully used its ancestor TagRPF as N-terminal tag , we showTable 4), whereas SBFP2 can supplement the latter. However, eqFP670 is a dimer, and thus often unsuitable for protein tagging, while SBFP2 shows a very low brightness, thus requiring either high expression levels, or strong excitation at around 380 nm.For two-colors imaging, we have shown that a combination of TagRFP-T and sfGFP can allow imaging at high frequency without significant photobleaching. For three-colors protein tagging, TagBFP/GFP/mKO2 can be a good alternative to the commonly used set of CFP/YFP/mCherry. In particular, cyan FPs are mostly dimmer and less photostable than GFPs, whereas TagBFP is a fast-folding, bright and photostable variant. However, we note that the excitation spectrum of TagBFP is closer to the UV range compare to that of CFPs, which imposes a trade off between brightness and phototoxicity in time-lapse imaging. When a fourth color is required, one can add eqFP670 to the former combination (Table 5). Note that the above-mentioned limitations regarding the brightness of SBFP2 and the dimer property of eqFP670 are evident. The brightness in the blue/cyan spectrum can be improved by either engineering a bright FP that is more blue-shifted than TagBFP, or by engineering a cyan FP with a narrower excitation/emission spectrum that reduces spectral cross-talk with TagBFP. Regarding monomeric near-infrared or infrared FPs, there have been reports of newly evolved versions with an improved brightness (For five-colors imaging, it is possible to combine SBFP2/mCerulean/mYPet/TagRFP-T/eqFP670 (see ightness . They weThe observed direct interaction between TagBFP and TagRFP-T prohibits the combination of these two proteins in the same cell. While the dimerization or aggregation of monomeric FPs at high concentration has been recognized , the posΔftsZ but not in the case of sfGFP and TagRFP-T agrees with the finding that the unstructured region of the FtsZ has a limited tolerance to the size of a insertion (Our efforts to produce HU-2, FtsZ, and MinD fusion proteins for fully replacing the endogenous ones exemplify the multi-faceted challenge regarding fluorescent tagging, which naturally depend on the structural properties of the native proteins and the transcriptional/translational read-through at their genomic loci. The fact that a short TC peptide insertion into the unstructured region of FtsZ successfully rescued the lethal effect of nsertion . For visnsertion . Regardinsertion , they wensertion . Here weThe expansion and detailed study of the FP reported in this paper will help imaging cell division in bacteria. The understanding of the processes involved in chromosome organization and cell division in bacteria has seen great development owing to the use of FPs. Current challenges in microscopy imaging of these processes lie in increasing the spatiotemporal resolution, uncovering the native behavior, and simultaneously inspecting the multiple interacting sub-components. We expect that our quantitative evaluation of novel FPs and the fusion strategies will facilitate tackling these challenges.FW, JK, and CD conceived the experiments, FW, EVR, and BVS did the experiments, FW analyzed the data, FW and CD wrote the paper.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.


Major efforts for malaria prevention programs have gone into scaling up ownership and use of insecticidal mosquito nets, particularly in sub-Saharan Africa where the malaria burden is high. Socioeconomic inequities in access to long lasting insecticidal nets (LLINs) are reduced with free distributions of nets. However, the relationship between social factors and retention of nets after a free distribution has been less studied, particularly using a longitudinal approach. Our research aimed to estimate the ownership and use of LLINs, and examine the determinants of LLIN retention, within an Indigenous Batwa population after a free LLIN distribution. Two LLINs were given free of charge to each Batwa household in Kanungu District, Uganda in November 2012. Surveyors collected data on LLIN ownership and use through six cross-sectional surveys pre- and post-distribution. Household retention, within household access, and individual use of LLINs were assessed over an 18-month period. Socioeconomic determinants of household retention of LLINs post-distribution were modelled longitudinally using logistic regression with random effects. Direct house-to-house distribution of free LLINs did not result in sustainable increases in the ownership and use of LLINs. Three months post-distribution, only 73% of households owned at least one LLIN and this period also saw the greatest reduction in ownership compared to other study periods. Eighteen-months post distribution, only a third of households still owned a LLIN. Self-reported age-specific use of LLINs was generally higher for children under five, declined for children aged 6–12, and was highest for older adults aged over 35. In the model, household wealth was a significant predictor of LLIN retention, controlling for time and other variables. This research highlights on-going socioeconomic inequities in access to malaria prevention measures among the Batwa in southwestern Uganda, even after free distribution of LLINs, and provides critical information to inform local malaria programs on possible intervention entry-points to increase access and use among this marginalized population. Insecticide-treated nets (ITNs) and long-lasting insecticidal nets (LLINs) are well recognized as cost-efficient and highly effective interventions for reducing the burden of malaria in endemic regions in sub-Saharan Africa . TreatinA core focus of free mosquito net distribution campaigns has been equitable coverage–within and between populations–with the goal of reducing disparities in access to preventive measures for economically or socially marginalized sub-populations. Previous studies show that large-scale, free net distribution campaigns can reduce inequities in household net ownership across socio-economic gradients, relative to other forms of distribution –10. Ineqet al . Further, 19% of households reported that their net(s) had been treated with insecticide. For households reporting net ownership, the median number of nets owned was 1 [range: 1–5]. There was on average 5 people per net [CI 4.0–5.8] in Batwa households approximately a year before LLIN distribution.LLIN ownership decreased notably over the year and a half following LLIN distribution. In January 2013, three months after the LLIN distribution, only 73% [95% CI 64–80] of Batwa households reported owning a LLIN. In the following surveys, 61% [CI 51–69] of households in July 2013, 45% [CI 35–55] of households in November 2013, 34% [CI 26–44] of households in January 2014, and 32% [CI 24–42] of households in April 2014 reported owning a LLIN , Fig 2. Three months after the LLIN distribution (January 2013), 53% [95% CI 46–60] of the population had access to a LLIN within their own household. This proportion decreased steadily over the observation period with only 19% [95%CI 14–25] of the population having access to a LLIN 18-months post distribution. A similar trend was observed with net use: 47% [95% CI 43–51] of the population at 3-months post-distribution, and 21% [95% CI 18–25] of the population at 18-months post-distribution, reported sleeping under a LLIN the night before the survey. The ratio of LLIN use to access was 0.89 at 3-months, and 1.10 at 18-months, post distribution . Among nThere was notable variation in household ownership and individual use of LLINs amongst the 10 Batwa communities (p<0.05) at the end of the study period (April 2014). Community-specific rates of household LLIN ownership varied, with two communities achieving >50%, while others had lower LLIN ownership rates of 20–50% (6 communities) and <20% (2 communities). Individual use of nets also varied considerably amongst communities. In half of the communities (n = 5), 20–35% of community members reported sleeping under a LLIN the previous night, while usage rates were <20% in the other five communities.Three- and 18-months following distribution, results indicate a rapid decline in LLIN use among the Batwa . As mentThree months post-distribution, 50% of children under five years old reported sleeping under a LLIN the previous night. Under-five LLIN usage decreased to 20% at 18-months post-distribution, with 62% living in households that did not own a LLIN. The usage gap was most marked for children 6–12 years old across both time periods; this age group consistently comprised the largest proportion of individuals who lived in a household with a LLIN hanging, but who did not sleep under a LLIN the previous night . Three months post-distribution, half of adults 13–34 years were sleeping under a LLIN, but a year and a half later, 62% were living in households not owning a LLIN. Adults 35 years and older consistently comprised the greatest proportion of individuals who slept under a LLIN, relative to other age groups. No significant difference in LLIN usage by males and females was observed in any LLIN usage category. Details are provided in an Additional file .Inequities in household retention of LLINs along socioeconomic tertiles were pervasive throughout the post-distribution study period. Consistent throughout each survey post-distribution, the least poor and poor Batwa households comprised a greater proportion of those owning a LLIN compared to the poorest Batwa households . The equp<0.01). Highest adult female education in the household (p = 0.69), number of rooms in the household (p = 0.07), number of people (p = 0.38), and household roofing material (p = 0.16) were not significant predictors but improved the fit of the model and were retained as covariables. As expected, there was a significant global time effect (p<0.01) on the retention of LLINs in the model, with decreased odds of owning a LLIN with every subsequent survey from baseline. Model comparison using BIC values as the criterion indicated that controlling for household effects and including community as a higher level of clustering improved the model, even though the variance estimate between communities was lower than the variance within communities. The post estimation diagnostics indicated that the model was a good fit for the data.LLIN retention in the model was further defined as self-reported ownership of at least one IHACC distributed LLIN. In the best-fit random effects multivariable logistic regression model, controlling for household and community-level effects, socioeconomic factors were significant predictors of households that retained at least one LLIN, keeping other included variables in the model constant . The oddFollowing a targeted, house-to-house distribution of LLINs in ten Indigenous Batwa communities in southwestern Uganda, Batwa households reported a decline in LLIN ownership over an 18-month period. The greatest reduction in net ownership occurred within the first three months following distribution, similar to Koenker et al 2014) in a multi-country study . In our  in a mulWhile Uganda has made notable gains in increasing overall ownership, access, and use of ITNs and LLINs over the past decade, our data suggest that the poorest and most marginalized populations, notably Indigenous populations, may still be disadvantaged from ownership and use of LLINs compared to the general Ugandan population . An accuJust three months after the targeted Batwa LLIN distribution, which provided two LLINs to every household, less than half of the population reported sleeping under a LLIN the previous night; 18-months post LLIN distribution this proportion dropped as low as 21%. While overall use of LLINs decreased over the study period due to non-ownership, it is possible that the remaining nets were better used over time, consistent with our observation that the proportion of people living in a household hanging a net but themselves not sleeping under one decreased. However, this observed usage trend could also be explained by the seasonality of malaria transmission in the area, as the 3 and 18-months post distribution surveys were conducted during the dry and rainy seasons, respectively. For example, the 18-months post-distribution season was conducted in a period of heavy rains, likely resulting in higher mosquito densities, which has been shown to coincide with higher net hanging and usage rates . ConsistIt is now well established that both global disease burden and access to preventive measures are patterned by socioeconomic gradients ,54. The While this study did not explore the reasons why nets were not kept, local informants working closely with the Batwa and past researchers in the field indicated that nets were often either sold/given away, used for other purposes, or discarded due to infrastructural limitations of thatched, semi-permanent housing ,17,18. EThis study has several limitations: first, our low sample size limited the power for statistical inference, even though our attempted-census surveys of the Batwa in Kanungu successfully covered a significant proportion of the total population. Conducting this research among a unique Indigenous population is both a strength and weakness of our study: it limits the generalizability of our results but offers critical insights into the situation of malaria prevention for a highly vulnerable population. Our study relies on self-reported measures, which could suffer from recall bias or be influenced by participant’s specific motivations or interpretation and understanding of a question . It is pDirect house-to-house distribution of free LLINs did not result in sustainable increases in the ownership and use of LLINs among a population of Indigenous Batwa in Kanungu, Uganda. A year and a half after LLIN distribution, only one-third of Batwa households in Kanungu reported owning at least one LLIN, with levels of ownership reverting to pre-distribution estimates. Even after a targeted distribution, which aimed to reduce social, monetary and distance barriers in access to LLINs for these marginalized Indigenous communities, the use and ownership of LLINs among the Batwa remained below national and sub-national estimates, largely as a result of low LLIN retention. Within the Batwa communities, socioeconomic gradients were reinforced: the least poor Batwa households were significantly more likely to retain a LLIN compared to the poorest Batwa households. This research indicates that retention of freely distributed LLINs, particularly for impoverished populations, may remain subject to patterning by socioeconomic gradients over time. More effective longitudinal monitoring and evaluation programs are needed to assess the long-term impact of free LLIN distributions, and to identify contextually appropriate interventions to maximize the retention of nets, particularly for marginalized and vulnerable populations.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.


A few studies showed that long-term methotrexate (MTX) use exacerbates liver fibrosis and even leads to liver cirrhosis in rheumatoid arthritis (RA) patients. We therefore conducted a population-based cohort study to investigate the impact of long-term MTX use on the risk of chronic hepatitis B (CHB)-related cirrhosis among RA patients. We analyzed data from the National Health Insurance Research Database in Taiwan and identified 631 incident cases of RA among CHB patients (358 MTX users and 273 MTX non-users) from January 1, 1998 to December 31, 2007. After a median follow-up of more than 6 years since the diagnosis of CHB, a total of 41 (6.5%) patients developed liver cirrhosis. We did not find an increased risk of liver cirrhosis among CHB patients with long-term MTX use for RA. Furthermore, there was no occurrence of liver cirrhosis among 56 MTX users with a cumulative dose ≧3 grams after 97 months’ treatment. In conclusion, our data showed that long-term MTX use is not associated with an increased risk for liver cirrhosis among RA patients with CHB. However, interpretation of the results should be cautious due to potential bias in the cohort. Methotrexate (MTX) is a commonly used immunosuppressant in the treatment of many autoimmune diseases such as rheumatoid arthritis (RA), psoriasis and psoriatic arthritis346810111213Alcohol consumption, diabetes, obesity, chronic viral hepatitis and hepatotoxic medications have been reported to be etiological factors of liver cirrhosisThis retrospective cohort study analyzed data obtained from the National Health Insurance Research Database (NHIRD)20The study flowchart is illustrated in Risk factors for liver cirrhosis, such as non-alcoholic fatty liver disease , diabetes mellitus (ICD-9-CM code 250) and dyslipidemia (ICD-9-CM code 272), as well as comorbidities such as hypertension (ICD-9-CM codes 401–405) were documented for each patient. Diagnosis of decompensated liver cirrhosis in all cases was confirmed by inclusion in the CIPD. One of the following criteria is needed for patients with liver cirrhosis to be registered in the CIPD: (1) intractable ascites, (2) variceal bleeding, or (3) hepatic coma or liver decompensation.Statistical analysis was conducted using SAS software version 9.3 . All quantitative data are presented as means plus the standard deviation unless otherwise specified. For numerical variables, comparisons between subgroups of patients were performed using Student’s t test. For categorical variables, comparisons between subgroups of patients were performed using Chi-squared test. Hazard ratios of developing liver cirrhosis since the diagnosis of CHB between subgroups of RA patients with CHB were calculated using the Cox proportional hazards model after adjustment for age, gender and comorbidities such as NAFLD, diabetes mellitus, dyslipidemia and hypertension. Kaplan-Meier survival analysis and the log rank test were used to compare the probability of liver cirrhosis-free survival since the diagnosis of CHB between MTX non-users and MTX users with different cumulative doses. A two-tailed p value less than 0.05 was considered statistically significant.This population-based cohort study investigated the impact of long-term MTX use on the risk of liver cirrhosis among RA patients with CHB. Our results demonstrate no significant increase of liver cirrhosis in RA patients with CHB who received long-term MTX treatment. However, no firm conclusion can be drawn due to potential bias in the cohort.Among RA patients with CHB in Taiwan, 358 (57%) of 631 patients (excluding those patients who had received biologics) had received MTX to treat RA. The proportion of MTX used in our study was lower than that reported in a multi-national cross-sectional study of RA patients24After a median follow-up of more than 6 years since the diagnosis of CHB, 41 (6.5%) of 703 RA patients with CHB developed liver cirrhosis. The incidence of liver cirrhosis in our study was higher than that in the general population with CHB (3.0% after a mean follow-up of more than 9 years)Previous studies indicated that the risk for liver cirrhosis after long-term MTX therapy is low among RA patientsHowever, it is difficult to draw a firm conclusion from our data since such cohort is easily biased despite efforts to adjust for confounding variables. Nevertheless, analysis using a real-world database can still provide insight regarding the issue. We gave a possible explanation for our observations29Our study has a number of advantages. First, it is difficult to conduct a randomized control trial to evaluate the impact of long-term MTX use on the risk of CHB-related liver cirrhosis. Therefore our data, based on a population-based cohort from a database containing the healthcare records of 24 million people, can still provide important insight. Second, unlike previous studies that mainly focused on liver fibrosis8910111213Our study has some limitations. First, a longer follow-up period with a higher cumulative MTX dose may be required to observe the contribution of MTX to the risk of HBV-related liver cirrhosis. Therefore, a study with a longer follow-up is needed to confirm our results. Second, previous studies demonstrated that the adherence rate to MTX ranged from 59% (underuse) to 107% (overuse)In conclusion, our data showed that long-term MTX use is not associated with an increased risk for liver cirrhosis among RA patients with CHB. However, interpretation of the results should be cautious due to potential bias in the cohort.How to cite this article: Tang, K.-T. et al. Methotrexate is not associated with increased liver cirrhosis in a population-based cohort of rheumatoid arthritis patients with chronic hepatitis B. Sci. Rep.6, 22387; doi: 10.1038/srep22387 (2016).


Recent negative life events (RLE), childhood adversity (CHA), lifetime depression, Brief Symptoms Inventory (BSI) depression and anxiety scores were determined in each subject. Besides traditional statistical analysis we calculated Bayesian effect strength and relevance of 5-HTTLPR genotypes in specified models.In the present study we investigated how age, type and intensity of life-stressors modulate the effect of 5-HTTLPR. Regarding current (BSI) and lifetime depression 5-HTTLPR-by-RLE interactions were confirmed. Main effect, with other words direct association, was supported with BSI anxiety. With more frequent RLE the prevalence or symptoms of depression increased in ss carriers. Although CHA failed to show an interaction with 5-HTTLPR, in young subjects CHA sensitized towards the depression promoting effect of even mild RLE. Furthermore, the direct association of anxiety with the s allele was driven by young (≤30) individuals.The short (s) low expressing allele showed association with increased risk of depression related phenotypes, but all nominally significant effects would turn to non-significant after correction for multiple testing in the traditional analysis. Bayesian effect strength and relevance analysis, however, confirmed the role of Our study is cross-sectional and applies self-report questionnaires.5-HTTLPR has only weak/moderate effects, the s allele is directly associated with anxiety and modulates development of depression in homogeneous subgroups.Albeit  One o5-HTTLPR is a repeat length polymorphism that has been shown to affect the efficiency of serotonin uptake and thus synaptic and extracellular serotonin concentrations in the brain. The short (s) allele of 5-HTTLPR which shows a reduced transcriptional efficiency compared to the long (l) variant predisposes to cognitive vulnerability to stress-sensitivity including anxiety-related personality traits such as neuroticism [5-HTTLPR s allele. Subsequently, primate studies demonstrated that the effect of the 5-HTTLPR s allele on serotonin function in the central nervous system and on behavior is modulated by early rearing conditions providing the first evidence for GxE [roticism , or amygroticism  which arroticism . Collierroticism  suggeste for GxE ,9.5-HTTLPR s allele modulates the effects of stressful life events in the development of depression. Although numerous genetic epidemiological studies replicated the initial findings, there are also non-replications and part replications, and even meta-analyses draw differing conclusions  statistical GxE analysis methods which have limited power to detect biological interactions [5-HTTLPR genotypes in specified models and the ratio of risk of these phenotypes conveyed by the ss genotype after low, medium and high exposure to life stressors.In the case of human depression, two main environmental psychosocial factors were identified, childhood adversity (CHA) and recent negative life events (RLE), which usually precede the development of episodes . Caspi eee, e.g. ,12). Theee, e.g.  further ee, e.g. ,15. To eractions , we calcThe reported studies were part of the EU funded NewMood study  approved by the local Ethics Committees  and carried out in accordance with the Declaration of Helsinki. All participants provided written informed consent before participating in the study. All the relevant data are included in the paper and Supporting Information file .Subjects aged 18–60 years and of European white origin were recruited through general practices, advertisements and a web-site from Greater Manchester, UK (n = 1355) and through general practices and advertisements from Budapest, Hungary (n = 1003). All willing subjects were included who filled out the NewMood questionnaire pack, English or Hungarian version as appropriate, and provided DNA by using a genetic saliva sampling kit. Details of responses have been published previously ,18.The NewMood questionnaire included items covering background information , personal and family psychiatric history and brief standard and validated questionnaires covering current mood and anxiety, and life events. A description of the questionnaires has been published previously ,19.In the present study, we analyzed reported lifetime depression (DEP) that was derived from the background questionnaire and was validated in a subpopulation using the Structured Clinical Interview for DSM-IV (SCID) . ValidatWe used the List of Life Threatening Experiences questionnaire  to ident5-HTTLPR genotype was performed as previously described [For genotyping we used buccal mucosa cells collected using a cytology brush  and 2.0 ml of collection buffer in 15-mL plastic tubes. Genomic DNA was extracted according to a published protocol . Determiescribed . All labhttp://pngu.mgh.harvard.edu/purcell/plink/) to calculate Hardy-Weinberg equilibrium, and build logistic regression models for binary outcome variable (DEP) and linear regression models for continuous outcome variables . Additive genetic model was applied, sex and age were covariates in all analyses. The two population data were analyzed together to increase the power, especially for GxE interaction tests. For power calculation, see Table A in 5-HTTLPRxlife events (RLE or CHA) interaction results were very similar in the first analysis  and for those who were above 30 (>30). Other statistical analyses were performed with IBM SPSS 21.0 for Windows. We also used R Project [Statistical analyses were performed with PLINK v1.07  we applied Bayesian relevance analysis ,27 basedADD = 0.154) which allowed us to carry out mega-analyses in the combined sample. Description of the population can be seen in The total population and all sub-populations  were in Hardy-Weinberg equilibrium (p>0.05). The genotype frequency was not significantly different in the Budapest and Manchester samples  or high  RLE (Bayesian≈1) in subjects with low RLE. The relatively flat posterior distribution curves in case of high RLE indicate several possible Bayesian Odds Ratio values with moderate or low certainty. Bimodal distribution for medium RLE subjects suggests that one group of models supports one, and another group of models supports another odds ratio.Bayesian effect strength estimation confirmed that the effect of 2.4) RLE  but it h5-HTTLPR genotypes on lifetime depression or on BSI depression scores  RLE groups also support the LE group . The RLEBayesian = 1.31–1.39) effects, in the total population (5-HTTLPR is modest (ORBayesian = 1.13–1.19), in the younger subpopulation, however, the effect is strong with a firm credible interval ORBayesian = 1.55–1.82  of e groups  and chile groups , in thos5-HTTLPR weakly modulated the effect of RLE on lifetime depression and current BSI-depression, while in BSI-anxiety it showed a weak main effect, namely a direct association with the risk s allele even in individuals having experienced low life stress, and stress exposure caused only a minor additional increase in anxiety. These weak genetic associations are in line with the hypothesis that depression and anxiety are highly polygenic and multifactorial disorders [5-HTTLPR on any of the phenotypes, it had an important influence on the 5-HTTLPRxRLE interaction. Namely, in young subjects it sensitized towards the effect of RLE even if RLE was mild, when a combined multivariate outcome was used. In older subjects (>30) 5-HTTLPR was only relevant when more RLE were reported irrespective of childhood adversity. The results suggest that the modulatory effects of serotonin transporter gene variation on the risk of depression may involve different mechanisms in different age groups.According to our results, exposure either to RLE or CHA caused a marked, dose-dependent increase in all three depression-related phenotypes .Interestingly, and in contrast to previous results, we could only demonstrate an effect of recent life events in our interaction models, but not of childhood maltreatment ,11,14,38urrences . This isurrences . In our 5-HTTLPR genotype and environmental adversities on the development of depression appears to be a function of several other variable conditions, and also seems to be an age dependent effect as it is reported as a significant finding in studies in young adults but not in adolescents or the elderly [5-HTTLPR is strongly relevant in older adults (>30) only in the presence of high number of RLE suggesting that tonic effect of this polymorphism might weaken with age but robust stress could still elicit phasic effects. As the incidence of depression is also age-dependent, it is not unlikely that in different ages and stages of development different factors play a role in the development of depression, and thus this risk is also influenced by different genetic factors.The GxE interaction between  elderly . Based oBesides advantages, there are several limitations of our study to be noted. In our cross-sectional population sample almost half of the willing participants reported lifetime depression that might introduce sampling bias. Current (BSI) or lifetime depression and current BSI-anxiety were assessed via self-report, with no psychiatric screening that might result inaccurate phenotype calling. However, our data were validated ,19 in a 5-HTTLPR might be better captured in specific subgroups of the population than in large but heterogeneous population samples. Our Bayesian Odds Ratio calculations demonstrated that in these subgroups the genetic ORs are increased. Such stratified analyses should result in greater power in future GxE interaction studies Table A. Required sample sizes for 90% powerTable B. Similar effects of continuous and grouped life event scores in the PLINK analysis5-HTTLPR on RLE or CHATable C. Effect of 5-HTTLPRxRLE interactionTable D. Effect of age and childhood adversity on Fig. A. Likelihood ratio of lifetime depression according to the life event groupsEffects of life events (description)5-HTTLPR and RLE on different phenotypes, in two age subgroupsFig. B. Interaction between Bayesian analysis of relevance (description)Bayesian Odds Ratio (description)5-HTTLPR and RLE (description)The analysis of the joint effect of 5-HTTLPR (ss vs. ll) with respect to phenotypes BSI-ANX, BSI-DEP and DEPTable E. Comparison of logistic regression models of (PDF)Click here for additional data file.S2 FileData(PDF)Click here for additional data file.


The majority of patients were male . Most patients (92% and 77%) were still receiving ruxolitinib at the medical record abstraction date . The proportion of patients with moderate or severe palpable splenomegaly (≥10 cm) decreased from diagnosis (56%) to best response (12%). Fatigue was reported in 47% of patients and was the most common constitutional symptom. For most symptoms in both risk groups, shifts in the distribution of severity from more to less severe from diagnosis to best response were observed. Both patients with low-risk and intermediate-1-risk MF experienced a substantial decrease in spleen size with ruxolitinib treatment in real-world settings. For most symptoms examined, there were distinct improvements in the distribution of severity during ruxolitinib treatment. These findings suggest that patients with lower-risk MF may benefit clinically from ruxolitinib treatment.Few trial-based assessments of ruxolitinib in patients with lower-risk myelofibrosis (MF) have been conducted, and no studies have made such assessments in a real-world population. We assessed changes in spleen size and constitutional symptoms during ruxolitinib treatment using a retrospective, observational review of anonymized US medical record data of patients diagnosed with IPSS low-risk ( Myelofibrosis (MF) is a myeloproliferative neoplasm (MPN) characterized by cytopenias, splenomegaly, constitutional symptoms , and progressive bone marrow fibrosis . It is aUntil recently, medical and surgical options for patients with MF have been limited . Most phThe recent identification of mutations associated with the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway and a new appreciation of the role of cytokines signaled through JAK1 and JAK2 in the pathogenesis of MPNs –16 has rTo date, few trial-based assessments of ruxolitinib in patients with lower-risk MF have been conducted, and no studies have made such assessments in a real-world setting. In this study, we sought to understand whether symptomatic patients with lower-risk MF would also benefit from ruxolitinib treatment, as was seen in patients with intermediate-2- and high-risk disease studied in the registration trials, by retrospectively assessing changes in spleen size and disease-related symptoms in routine clinical practice.This was a retrospective, observational review of anonymized medical record data collected in January 2014 by 49 hematologists and oncologists in the United States. Participating physicians were recruited from an existing research panel maintained by All Global, Ltd. Data were collected with secure, online case report forms (CRFs) administered to the selected physicians who had treated ≥2 patients with MF with ruxolitinib since November 2011 (US launch date for the drug). Patient inclusion criteria were as follows: (1) being diagnosed with lower-risk MF (IPSS score of 0 [low risk] or 1 [intermediate-1 risk]); (2) being first treated with ruxolitinib ≥3 months before the medical record abstraction date; (3) being ≥18 years of age at ruxolitinib initiation; (4) having a medical history from MF diagnosis until the medical record abstraction date; and (5) never being enrolled in an MF-related interventional trial. Minimum quotas of 25 and 50 were set for patients with low- and intermediate-1-risk disease, respectively, with a predetermined maximum of 110 patients in the combined total. To increase generalizability of the sample, each physician was limited to 3 patient entries . Furthermore, when >3 patient records were available for a physician, patients with birth months nearest to a birth month randomly generated by the electronic CRF were selected.Spleen size and constitutional symptoms were the key measures, retrospectively extracted at MF diagnosis, at ruxolitinib initiation, and at best response while receiving ruxolitinib treatment. Symptoms of interest included those described in the validated Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF) , which w9/L) or grade ≥3 anemia (hemoglobin <8 g/dL) as measured at any point after ruxolitinib initiation through last ruxolitinib dose.Although this study was not designed as a safety evaluation of ruxolitinib, we additionally report the frequency of thrombocytopenia and anemia, which are the 2 most common adverse events associated with ruxolitinib (as expected based on the drug's mechanism of action), as well as the proportion of patients who required a change in ruxolitinib treatment  as a result of an adverse reaction. Following previous safety reporting from the ruxolitinib COMFORT-I trial , we specAll statistical analyses were carried out using SAS  statistical software. Because this study was exploratory in nature with no proposed hypotheses to test, only descriptive analyses were implemented. These analyses entailed the tabular display of means, SDs, medians, and value ranges for continuous variables and the frequency distribution of categorical variables. Conduct of this study was approved by an authorized institutional review board  and carried out in accordance with the 1996 Helsinki Declaration regarding the ethical conduct of human subject research. Because anonymized retrospective patient data were collected, an informed consent waiver was granted. JAK2 V617F mutation (72%) compared with patients with low-risk disease (56%). The prevalence of comorbidities in the selected patients appeared to be consistent with that in the general population, with diabetes and hypertension the most common conditions recorded at ruxolitinib initiation. Finally, most patients in both risk groups (92% for low risk and 77% for intermediate-1 risk) were still receiving ruxolitinib treatment at the time of data abstraction or last available follow-up.A total of 49 physicians were recruited for the data abstraction, of whom 82% specialized in hematology/oncology. Mean (SD) duration of practice experience was 12.7 (5.9) years, and the majority (73%) of the physicians practiced in community-based group clinics. A total of 108 patients were included (25 with low-risk and 83 with intermediate-1-risk disease) in this study  from MF diagnosis to the time of best response during ruxolitinib treatment . Among pGrade ≥ 3 thrombocytopenia was observed in 7% of all patients at some point during ruxolitinib treatment (12% of low-risk patients and 6% of intermediate-1-risk patients); grade ≥ 3 anemia was observed in 22% of patients (20% of low-risk patients and 23% of intermediate-1-risk patients) . A reducIn light of robust trial data showing that ruxolitinib improved both splenomegaly-related and non-splenomegaly-related constitutional symptoms in patients with intermediate-2-risk and high-risk MF , 17, theNCT01558739) in the United Kingdom showed that half of the patients with intermediate-1-risk MF treated with ruxolitinib achieved a reduction in spleen size of ≥50% at week 48 (versus baseline) after initiation of ruxolitinib. Mead et al. [To our knowledge, only 1 previous study  sought td et al.  also repn = 6) without splenomegaly at MF diagnosis. To the extent that absence of splenomegaly is a proxy for lower-risk MF, a comparison of findings on symptom improvement between that study and ours may be useful. Benjamini et al. [N = 28) found substantial improvements in constitutional symptoms and in spleen size [Although the study by Mead et al.  represeni et al.  found a een size . Howevern = 74 in Benites et al. [n = 1000 in Tefferi et al. [In our study sample, we also found that more than half (53%) of patients with intermediate-1-risk MF had moderate to severe splenomegaly  at MF diagnosis. Two previous medical record reviews , 23 alsos et al.  and n = i et al.  and 21% i et al.  and TeffOur findings on the 2 most common adverse events associated with ruxolitinib (thrombocytopenia and anemia) were consistent with safety data reported in the ruxolitinib COMFORT-I trial , in whicThis study is subject to several limitations. As in many retrospective medical record abstraction studies, patients selected for inclusion represent a convenience sample. Our study findings therefore may not be generalizable to the overall low- or intermediate-1-risk MF populations in the United States, and although participating physicians were recruited from all geographic regions, it was not possible to construct sampling weights that allowed for generalization to the national population. Only physicians who agreed to participate in the study contributed data; these physicians therefore may not be representative of all physicians treating low-risk or intermediate-1 MF in the United States. Finally, although no time limit was imposed on physicians for the completion of individual CRFs, the CRF was designed to limit physicians' time burden to help ensure full and accurate responses. Therefore, the scope of information that could be collected in this study was limited, and it is possible that additional information could have contributed further context to the study findings.Despite the noted limitations, findings from this study indicated that patients with lower-risk MF in routine clinical practice may benefit from ruxolitinib treatment, specifically for spleen size reduction and improved splenomegaly-related and constitutional symptoms. Furthermore, ruxolitinib has been shown to prolong overall survival in patients with intermediate-2 or high-risk MF and to reduce the risk of death among high-risk patients receiving ruxolitinib to that of intermediate-2-risk patients receiving placebo or best available therapy . These r


PREDICT is an online prognostication tool for early-stage breast cancer, which incorporates human epidermal growth factor 2 (HER2) status and stratifies absolute treatment benefits for hormone therapy, chemotherapy and trastuzumab. The present study compared historical multidisciplinary team (MDT) decisions regarding adjuvant treatment with PREDICT estimates, to determine whether certain patients are being over- or undertreated, particularly when stratified by age and oestrogen-receptor (ER) status. HER2-positive early-stage breast cancer cases over a five-year period at the Cambridge Breast Unit  were retrospectively reviewed. Patients receiving neo-adjuvant therapy were excluded. Adjuvant chemotherapy/trastuzumab recommendations based on PREDICT  were compared with actual MDT decisions. In total, 109 eligible patients were identified. The average age at diagnosis was 59.6 years, with 21 patients older than 70 years (19%). Four patients were predicted to gain an absolute benefit of >5% from chemotherapy/ trastuzumab, but were not offered treatment . Amongst the 19 patients aged >70 years predicted to benefit >3%, six were not offered treatment (32%). In the patients aged <69 years, there was evidence of overtreatment with adjuvant chemotherapy/trastuzumab in 8 out of 12 cases with <3% benefit using PREDICT. For all 20 patients with ER-negative tumours, the MDT and PREDICT decisions correlated, whilst for ER-positive cases, more than half (8 out of 14) were offered treatment despite a <3% predicted benefit. PREDICT can aid decision-making in HER2-positive early-stage breast cancer by identifying older patients at risk of undertreatment with chemotherapy/trastuzumab, and by reducing the overtreatment of patients with little predicted benefit, particularly in ER-positive disease. In early-stage breast cancer, accurate prognostication and the analysis of treatment benefit is important in selecting the appropriate adjuvant therapy following surgery. Treatment decisions traditionally made by multidisciplinary teams (MDTs) can now be aided by predictive models based on large patient cohorts. A variety of validated tools are available that provide estimates of survival and treatment benefit.Initial prognostication tools have provided survival estimates within defined prognostic groups. The Nottingham Prognostic Index (NPI), originally developed in 1982 and updated in 2007, was derived from data on symptomatic patients in the UK, pre-dating the screening era (www.adjuvantonline.com/index.jsp) was introduced in 2001 and is a free web-based tool for use by health care professionals, based on data from the USA Surveillance Epidemiology and End Results Program  is an alternative freely available web-based breast cancer prognostication and treatment benefit tool for use by health professionals and the public, which was introduced in 2010  is an oncogene amplified in up to 30% of breast cancers and associated with a poorer prognosis and higher likelihood of recurrence following initial treatment . TrastuzPREDICT was updated in October 2011 to incorporate the prognostic effect of HER2 status, using data from >10,000 patients in the Breast Cancer Association Consortium . The updBreast cancer is increasingly recognised as a spectrum of disease with tumour heterogeneity requiring individualised treatment regimes. Moreover, patients are increasingly involved in the clinical decision-making process and require clearly presented personalised data to aid them. Tools which estimate prognosis do not necessarily predict which tumours will respond well to adjuvant chemotherapy, leading to over- and undertreatment of early-stage breast cancers. Several commercial tests have been developed that provide more accurate predictions of response to chemotherapy, including gene expression profiling  and expanded immunohistochemical analysis (NPI Plus) . HoweverAt present, the majority of patients with HER2-positive tumours receive chemotherapy and trastuzumab, regardless of their ER status. However, adjuvant chemotherapy is associated with a greater benefit in reduction of disease recurrence for ER-negative tumours . TherefoThe aim of the present study was to compare historical MDT decision-making for adjuvant therapy with novel estimates derived from PREDICT to determine whether clinical judgement alone is effective, and whether certain patient groups with early-stage HER2-positive disease are being over- or undertreated, particularly when stratified by age and ER status. Elucidation of the most accurate prognostication and treatment benefit techniques in this patient group is clearly important, as up to one-quarter of early-stage breast cancer diagnoses are HER2-positive .Prior to October 2011, the combination of adjuvant chemotherapy and trastuzumab was routinely recommended for all patients presenting to the Cambridge Breast Unit  with HER2-positive tumours, unless they had significant co-morbidities. Since October 2011, the PREDICT absolute 10-year survival benefit from chemotherapy has been routinely used in the MDT decision-making process as follows: <3%, chemotherapy not recommended; 3–5%, chemotherapy discussed as a treatment option; and >5%, chemotherapy recommended. These values were selected to maximise the benefit to risk ratio of chemotherapy treatment, and to ensure that individual patient variables were taken into account.This study includes all females presenting to the Cambridge Breast Unit with HER2-positive early-stage breast cancer diagnosed between January 2007 (when routine testing of HER2 status commenced) and October 2011 (when the updated PREDICT model became available). Exclusion criteria included evidence of metastatic disease at presentation, patients treated in the private sector (problems with access to records) and patients receiving neo-adjuvant therapy.Calculated adjuvant chemotherapy/trastuzumab recommendations based on PREDICT treatment benefits were compared with MDT decisions recorded contemporaneously.Statistical analysis was performed using Fisher’s exact testing and Microsoft Excel . P<0.05 was used to indicate a statistically significant difference.Over the study period of 57 months, 186 females with HER2-positive early-stage breast cancer were treated in the public sector. In total, 77 patients received neo-adjuvant chemotherapy and trastuzumab, and were therefore excluded from this study. Therefore, 109 patients were eligible for inclusion.A total of 69 cases presented symptomatically (63%), with the remainder detected by routine breast screening. Patient and tumour characteristics are summarised in The average patient age was 60 years, with a median age of 60 years and 21 patients aged ≥70 years (19%). Tumours were generally of high grade (III) and ER-positive.All patients underwent primary surgical treatment with successful breast conserving surgery  or mastectomy . Axillary surgery was undertaken for all cases, except three who refused any surgical staging procedure. Almost two-thirds of patients underwent sentinel lymph node biopsy as the only axillary procedure , with the remaining 42 patients requiring axillary node clearance due to nodal involvement detected pre-operatively with core needle biopsy, or as a completion procedure due to at least one sentinel node containing macro- (>2 mm) or micro-metastases (0.2–2 mm).A comparison of the MDT decision regarding adjuvant chemotherapy and trastuzumab, and the PREDICT estimate of treatment benefit is shown in The historical MDT decision and the PREDICT estimate was concordant in 91 cases (83%). Six patients were not offered treatment, as the benefit was believed to be minimal, and this was confirmed in all six cases by the PREDICT analysis, which gave a benefit estimate of <3% over 10 years. Chemotherapy and trastuzumab were offered to 85 patients following MDT discussion, and a benefit of >3% over 10 years was confirmed by PREDICT.Of the 18 discordant cases . A further six patients were not offered adjuvant therapy following MDT recommendation, but had a moderate indication of benefit (3–5%) using PREDICT. Overall, 8 patients were offered adjuvant chemotherapy and trastuzumab, although PREDICT estimated the benefit to be <3% at 10 years. All of these patients were aged <70 years , and all had ER-positive disease.In a subgroup analysis of those patients (n=21) aged ≥70 years, the majority were predicted to benefit >3% from adjuvant therapy , despite 6 not being offered adjuvant chemotherapy and trastuzumab (32%). Furthermore, when comparing the likelihood of undertreating patients, an age of ≥70 years is a significant risk factor (P=0.0033). In the remaining 88 patients aged <70 years, 12 cases were predicted to benefit <3% from adjuvant treatment (14%), amongst whom, eight were offered chemotherapy and trastuzumab (67%). Nonetheless, age was not a statistically significant factor for overtreatment of the disease (P=0.3495).All 19 patients with ER-negative tumours were offered adjuvant chemotherapy and trastuzumab following MDT discussion. This was fully concordant with PREDICT estimates of benefit, which is most likely associated with a paucity of other treatment options. By contrast, of the 90 patients with ER-positive disease, 14 were predicted to gain <3% benefit from adjuvant therapy and eight of these were offered treatment following MDT discussion (57%). However, ER status alone was not an independent risk factor for overtreatment of the disease (P=0.3461).The results of this retrospective study indicate that MDT decisions regarding adjuvant therapy for HER2-positive early-stage breast cancer without the use of predictive tools are appropriate in the majority of cases. This is conditional upon the MDT comprising an appropriate group of experienced clinicians from relevant disciplines. However, the present study also identified selected groups of patients for whom application of a predictive tool, which includes HER2 status, may improve adjuvant decision-making. These include older patients (≥70 years) who are more likely to be offered suboptimal treatment, and those patients with ER-positive disease whose absolute benefit is less from chemotherapy and trastuzumab due to endocrine therapy sensitivity.However, there are limitations to the present study. Firstly, the number of patients in certain subgroups was comparatively small (≥70 years and ER-negative disease), which may limit detection of any statistically significant differences between groups. Secondly, the choice of adjuvant therapy was based on factors other than those included in the PREDICT model of calculation, for example, patient co-morbidities. Of the four patients predicted to gain >5% benefit from adjuvant therapy, but who were not offered treatment following MDT discussion, one had significant cardiac morbidity precluding the use of chemotherapy, with a survival time of only 8 months following surgery. However, the remaining three patients, all ≥70 years, did not have any specific contraindication to adjuvant chemotherapy and trastuzumab and their median overall survival time following surgery was 57.7 months. The inclusion of the American Society of Anaesthesiologists grade in PREDICT (as in Adjuvant!) may therefore affect decision-making in only a minority of cases.In total, 78 patients received adjuvant chemotherapy and trastuzumab, with one-quarter of patients (n=20) experiencing treatment-related morbidity, ranging from nausea to neutropenic sepsis and cardiac dysfunction. The combined use of adjuvant chemotherapy and trastuzumab carries a significant risk of side-effects, which should be borne in mind in patients with ER-positive/HER2-positive early-stage breast cancer that may derive minimal benefit from more aggressive therapy. Decisions may be aided by additional tumour markers that are now available, including the proliferative factor, Ki67, which predicts poorer survival and response to chemotherapy. This latter factor has recently been incorporated into the PREDICT tool and awaits validation .A significant proportion of patients (41%) presenting to the Cambridge Breast Unit with HER2-positive early-stage breast cancer received neo-adjuvant endocrine therapy or chemotherapy on MDT recommendation, and were therefore excluded from the present study. Amongst these 77 patients, 72 received a combination of chemotherapy and trastuzumab, and five received endocrine therapy alone. PREDICT is unable to calculate estimates of survival and treatment benefit for these patients as post-operative pathological tumour size and lymph node involvement are frequently downstaged. Of these 77 neo-adjuvant patients, 21 (27%) were found to have a pathological complete response (pCR) on final histopathological analysis, with no viable tumour remaining. However, it remains a challenge to select subgroups of patients who are most likely to benefit from neo-adjuvant therapy. A recent study of HER2-positive breast cancer treated with neo-adjuvant chemotherapy and trastuzumab suggested that patients with ER-negative disease are more likely to achieve a pCR than those with ER-positive disease ,20. PatiThe present study has highlighted the potential impact of age on the type of adjuvant therapy offered, with patients aged ≥70 years significantly less likely to be offered adjuvant chemotherapy and trastuzumab. The contemporary female life expectancy in the UK is 82 years , with approximately one-third of females presenting with newly diagnosed breast cancer when aged >70 years . In the Notably, although PREDICT was originally derived from data on females aged between 23 and 95 years, it has been shown in validation studies to underestimate 10 year mortality in the >75-years age group (In conclusion, PREDICT is a validated prognostication and treatment benefit tool that can effectively aid decision-making for adjuvant chemotherapy and trastuzumab in HER2-positive early-stage breast cancer, and in turn potentially reduce the over- and undertreatment of the disease. The PREDICT model continues to evolve and most recently includes the tumour maker Ki67. In the future, as more data becomes available, PREDICT will be a particularly useful tool for estimating the benefit from adjuvant treatments in the older patient age group.


There is increased interest in using microRNAs (miRNAs) as biomarkers in different diseases. Present in body fluids, it is controversial whether or not they are mainly enclosed in exosomes, thus we studied if urinary miRNAs are concentrated inside exosomes and if the presence of systemic lupus erythematosus with or without lupus nephritis modifies their distribution pattern. We quantified specific miRNAs in urine of patients with systemic lupus erythematosus (n = 38) and healthy controls (n = 12) by quantitative reverse-transcription PCR in cell-free urine, exosome-depleted supernatant and exosome pellet obtained by ultracentrifugation. In control group, miR-335* and miR-302d were consistently higher in exosomes than in exosome-depleted supernatant, and miR-200c and miR-146a were higher in cell-free fraction. In lupus patients, all urinary miRNAs tested were mainly in exosomes with lower levels outside them . This pattern is especially relevant in patients with active lupus nephritis compared to the control group or to the SLE patients in absence of lupus nephritis, with miR-146a being the most augmented . Among the exosomal miRNAs tested, only the miR-146a discriminates the presence of active lupus nephritis. In conclusion, urinary miRNAs are contained primarily in exosomes in systemic lupus erythematosus, and the main increment was found in the presence of active lupus nephritis. These findings underscore the attractiveness of exosomal miRNAs in urine, a non-invasive method, as potential renal disease markers. Over the last few years, there has been increasing interest in detecting body fluid micro RNAs (miRNAs) as biomarkers of activity in several diseases. MiRNAs, a family of small non-coding RNAs, play an important role in a variety of biological processes –3, mainlExtracellular miRNAs released into urine can circulate bound to RNA-binding proteins or packaged into microvesicles such as exosomes –11. ExosIn the kidney, miRNAs are indispensable to the regulatory mechanisms for renal development, maintenance of renal function and homeostasis processes. Likewise, their role in the progression of kidney disease has recently been emphasized ,18. UrinThe study protocol was approved by the Ethics committee of the Hospital Clinico Universitario of Valencia in accordance with the Declaration of Helsinki of 1975 as revised in 2008. All subjects have signed a written informed consent.The study included 28 consecutive patients with systemic lupus erythematosus (SLE) . SLE and Lupus nephritis were diagnosed following the KDIGO Clinical Practice Guideline for Glomerulonephritis diagnostic criteria , the preav for 45 min at 4°C to eliminate large microvesicles . The supernatant was spun in an ultracentrifuge at 160,000 gav for 70 min at 4°C, obtaining exosome-depleted supernatant. Exosome pellets were washed with 2 mL of sterile RNase-free PBS and ultracentrifuged again at 160,000 gav for 70 min . A 50 mL-pellet was resuspended in 100 μL of sterile PBS for protein quantification and electron microscopy and the other 50 mL-pellet was resuspended in 100 μL of Exosome Resuspension Buffer  for RNA isolation. The three urine fractions: cell-free urine (CFU), pellet containing exosomes (Exo) and the exosome-depleted supernatant (Sn) were then immediately processed to extract RNA.Urinary cells and debris were removed by centrifugation at 2250 g for 30 min at 4°C, obtaining the first fraction, cell-free urine. Then, 100 mL of the collected supernatant were transferred to clean tubes with 4.2 mL of protease inhibitor cocktail  and was centrifuged at 20,000 gRNA was extracted from cell-free urine (CFU) and exosome-depleted supernatant (Sn) fractions using the mirVana PARIS kit  using the manufacturer’s recommended protocol for total RNA. Briefly, 500 μL of sample were mixed with an equal volume of 2x Denaturing Solution and 1 mL of Acid phenol-chlorophorm. After centrifugation at 10,000 x g for 5 min, the aqueous phase was transferred to a new tube and, using silica-membrane RNeasy spin columns, purified RNA was extracted in 100 μL of elution buffer and stored at -80°C. From exosome pellets, RNA was extracted using the Total exosome RNA and protein isolation kit  from 100 μL of exosome suspension and stored at -80°C. To normalize the technical variability (sample-to-sample variation), we spiked-in a synthetic and non-homologous to human C. elegans miRNA, cel-miR-39 , after the addition of the denaturing solution to the samples. This miRNA, went through the entire RNA isolation and was measured by RT-qPCR, providing an internal control. To confirm that the RNA was confined to the exosomes, urinary exosomes were treated with 0.1 μg μl-1RNase A (Biosharp). Quantification of total RNA was performed by NanoDrop ND-1000 , whereas quality and size distribution were examined by capillary electrophoresis  with the RNA 6000 Pico chip.Because a consensus internal reference is lacking for the RT-qPCR analysis of serum or urine miRNAs, concentrations were normalized to the sample volume as described . Five μL(-ΔΔCt) after normalization to the cel-miR-39.The target microRNA data were normalized across samples using a median normalization procedure . For eacThe CFU, Exo and Sn of urine were lysed in RIPA lysis buffer on ice for 20 min, sonicated 5 min and then centrifuged at 16,100 g for 10 min. The supernatant was collected and transferred to a new tube. Total protein quantity were calculated by the Lowry method. An equal amount of total soluble protein (20 μg) were electrophoresed on 4–12% Bis-Tris NUPAGE SDS-PAGE  and transferred to a PVDF membrane for each sample analysed. The blotting membrane was incubated with exosomal primary antibodies CD9 mouse monoclonal dilution 1:300  and TSG101 mouse monoclonal dilution 1:500 , and with non-exosomal primary antibodies: calnexin rabbit polyclonal dilution 1/600 , GM-130 rabbit monoclonal dilution 1/1200  and nucleoporin 62 rabbit polyclonal 1/500 , followed by incubation with anti-mouse IgG Alkaline Phosphatase peroxidase secondary antibody . The immunoreactive bands were visualized using an acid phosphatase-conjugated secondary antibody and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate  substrate system and quantified using the TotalLab TL-100 (v.2008) program.We placed a drop, approximately 30 μL of exosomes resuspended in PBS on the paraphilm. Then, a formvar carbon-coated nickel grid was positioned on top of each drop for 15 minutes. Finally, a drop of 2% uranyl acetate was placed on the paraphilm and incubated the grid on top of the drop for 5 minutes in the dark; thus, the grids were negative stained. After air drying, the grids were examined with a CM-10 Phillips transmission electron microscope. The size distribution of captured exosomes in a total of 10 micrographs from two samples was analysed using an image processing program .U-test was used to compare differences in relative fold changes of miRNAs between urinary fractions and disease groups. For exosomal miRNAs, we constructed ROC curves and calculated the area under the ROC curve (AUC) to identify their associations with SLE and lupus nephritis. Frequency tables and ROC curves were then used to evaluate the diagnostic effects and to find the appropriate cut-off point. A P value <0.05 was considered statistically significant.The statistical analyses were performed with Statistical Package for the Social Sciences . Student’s t-test and Fisher’s exact test were used to determine the difference of clinical characteristics between two groups where appropriate. The miRNA data was presented as the mean (SEM) or as the fold change relative to control group (SEM) and other variables were expressed as the mean (SD). The nonparametric Mann–Whitney The total SLE patients was slightly older than controls , and two groups had similar gender distribution . In order to validate the exosome purification protocol, urinary exosome morphology, shape and size were analysed by transmission electron microscopy. The isolated microvesicles had a spherical shape with a size ranging from 21–140 nm . QuantitThe relative amount of the microRNAs was determined by calculating the difference of Ct values between the Exo and Sn or CFU. In total SLE patient group, we have shown that Exo fraction was significantly increased for all miRNAs compared to Sn fraction, especially miR-146a  . MoreoveSLE patient group showed a significant increase for all miRNAs in exosome fraction compared to that in controls, especially for miR-146a . Furthermore, only CFU and Sn fractions of miR-146a were also significantly increased  in SLE group . The selROC curve analyses were conducted to determine the diagnostic usefulness of the exosomal selected miRNAs for SLE and lupus nephritis. For all the SLE patients  and control groups, the AUCs ranged from 0.705 to 0.839. Furthermore, for LN samples (active and inactive), the AUCs ranged from 0.788 to 0.948 , with thIn the present study, two questions were addressed, if urinary miRNAs are mainly in exosomes and if lupus nephritis (LN) changes the miRNA distribution pattern in urine. As a tool to measure mature miRNAs, individual TaqMan miRNA assays for four common miRNAs, two ubiquitously expressed in human urine (miR302d and miR-335*) , and othSeveral studies analysed miRNAs in urine to identify potential biomarkers for renal diseases. Most authors use the urine cellular sediment for miRNA analysis ,37. NeveNevertheless, we have found that the highest increase of exosomal miRNAs was in patient group with active LN compared to inactive LN, SLE in absence of LN and controls. Additionally, CFU showed an increase in miRNA levels, but lower than exosome pellet. At variance with non-exosomal urinary RNAs, exosomal miRNAs are in a remarkably stable form. The association between miRNAs and renal disease has been emphasized ,25,40. TFurthermore, the high increase of exosomal miRNAs could be interesting in the diagnosis of SLE because of the important differences found between lupus patients and the control group, and between the different pathological conditions (active or inactive) of lupus patients. ROC curve analyses also revealed a strong relationship between these miRNAs and SLE, mainly in active lupus nephritis, with miRNA-146a being the most discriminatory. In addition, such information will not only increase the number of novel biomarkers for molecular diagnostics and assessment in SLE, but also provide mechanistic insight regarding the pathogenesis and progression of this disease. The miR-146a is involved in renal inflammation, fibrosis, and negatively regulate the interferon-γ pathway ,43. TissSeveral limitations of the present study need to be acknowledged. First, studies involving a high number of miRNAs analysed, a larger patient cohort and patients with more active SLE are needed for further confirmation of urinary exosomal miRNAs as SLE diagnostic tool. Secondly, the inclusion of a different disease control group than SLE, with similar degree of renal impairment, would be interesting to analyse the specificity of these miRNAs for lupus nephritis. Thirdly, functional experimental studies are required to verify and establish the causal association between increased exosomal miRNAs and SLE.The current investigation has shown that mature miRNAs are enriched in urinary exosome fractions than in the other components of urine. In the presence of active LN, the quantity of miRNAs increased in cell-free urine and more highly in exosome pellet than that for controls or SLE patients without renal disease. In addition, these results indicated that levels of urinary exosomal miRNAs might be used as new markers of SLE disease. Therefore, these findings, together with the accurate detection of miRNAs by RT-qPCR and the use of urine, a non-invasive method, underscores the attractiveness of exosomal miRNAs in urine as potential biomarkers for renal diseases and could be providing a new therapeutic approach.