Caenorhabditis elegans homolog of the SETDB1 histone methyltransferase, has a noncatalytic function that contributes to gene repression. Subnuclear foci of MET-2 coincide with H3K9me deposition, yet these foci also form when MET-2 is catalytically deficient and H3K9me is compromised. Whereas met-2 deletion triggers a loss of silencing and increased histone acetylation, foci of catalytically deficient MET-2 maintain silencing of a subset of genes, blocking acetylation on H3K9 and H3K27. In normal development, this noncatalytic MET-2 activity helps to maintain fertility. Under heat stress MET-2 foci disperse, coinciding with increased acetylation and transcriptional derepression. Our study suggests that the noncatalytic, focus-forming function of this SETDB1-like protein and its intrinsically disordered cofactor LIN-65 is physiologically relevant.Transcriptionally silenced heterochromatin bearing methylation of histone H3 on lysine 9 (H3K9me) is critical for maintaining organismal viability and tissue integrity. Here we show that in addition to ensuring H3K9me, MET-2, the  Caenorhabditiselegans reveals that MET-2 promotes transcriptional silencing and fertility through both H3K9 methylation and focus formation, which blocks histone acetylation.Genetic and genome-wide analysis of a catalytically deficient SETDB1-like enzyme, MET-2, in  Constitutive heterochromatin is associated with H3K9me, which is necessary for the repression of satellite repeats and transposable elements3. Tissue-specific genes can also be repressed by H3K9me (refs. 6). Finally, H3K9me mediates chromatin anchoring at the nuclear periphery7 and helps prevent interaction between euchromatic and heterochromatic compartments9. In mammals, the loss of H3K9me is associated with aging, cancer and the impaired maintenance of tissue integrity13.The proper segregation of active and inactive regions of the genome during differentiation is critical for the establishment and maintenance of tissue and genome integrity in eukaryotes. Heterochromatic regions of the genome are condensed and sequestered at the nuclear periphery or around nucleoli and are typically not transcribed19. In budding yeast, a function has been attributed to these stable perinuclear foci containing the Silent Information Regulator complex: the clustering promotes histone deacetylation and contributes to the stability and spread of repressed chromatin19. In mammals, the H3K9 histone methyltransferases (HMTs) SUV39H and SETDB1 remain bound to clusters of heterochromatin throughout the cell cycle21. In the case of SUV39H, this localized accumulation of the HMT requires an N-terminal chromodomain, which recognizes H3K9me2/me3 and thereby sequesters the enzyme at its chromatin targets21. To date, however, no function was assigned to the localized retention of H3K9 HMTs.The enzymes that establish and maintain heterochromatin form subnuclear clusters or foci in many species23. In Caenorhabditis elegans, however, foci of MET-2, the SETDB1 homolog responsible for H3K9me deposition, and its disordered domain cofactor LIN-65, remain stable despite downregulation of the worm HP1 homologs14. Moreover, MET-2, unlike SUV39H, has no chromodomain. We therefore set out to examine what drives the focal clustering of heterochromatin proteins such as MET-2, and the relationship of such foci to histone H3K9me.Recently, heterochromatic focus formation has been proposed to depend on disordered protein domains and multivalent interactions that promote local phase separation, as exemplified by the H3K9me reader HP1 proteinC. elegans proteins have been implicated in methylation of H3K9 (refs. 27), only the double deletion of genes encoding two SET domain-containing proteins, MET-2 and SET-25, eliminates all detectable H3K9me, both in embryos28 and at later stages of somatic development29. The loss of H3K9me compromises the transcriptional repression of tissue-specific genes and repetitive elements30, although no other common histone methylation marks were altered upon met-2 and set-25 ablation7. The SET domain of MET-2 has 50% identity to that of SETDB1 (refs. 32), which has been shown to methylate histone H3K9 both in vivo33 and in vitro35. Moreover, MET-2 harbors a domain organization very similar to SETDB1, including a methyl CpG binding domain, Pre-SET domain and bifurcated SET domain at the C terminus , and the poorly characterized but conserved ARLE-14, form foci that coincide with H3K9 methylation18. Not only the MET-2 partners, but also their regulatory interactions, are conserved in flies (Windei and CG14464 bind dSETDB1)37 and mammals (ATF7IP and ARLE14EP bind SETDB1)40. Ablation of lin-65 (or atf7ip) leads to a loss of MET-2 (or SETDB1) in nuclei, respectively41, and in worms, lin-65 deletion compromises the formation of MET-2 foci and derepresses a large subset of MET-2-sensitive targets18. Consistent with the notion that the unstructured domains present in LIN-65 contribute to MET-2 focus formation, these foci are sensitive to interventions that disrupt phase-separated condensates, such as treatment with 1,6-hexanediol and heat14.In cells, MET-2 and its two cofactors, the intrinsically disordered protein LIN-65 . LIN-65, similar to its homolog ATF7IP, is composed of two very large regions of disorder surrounding a coiled-coil domain . ARL14EP, the mammalian homolog of ARLE-14, requires its conserved cysteine-rich C-terminal domain to bind SETDB1 (ref. 43). We examined the interdependence of MET-2 complex formation using mutants and mass spectrometry. In wild-type  embryos expressing MET-2::FLAG::mCherry, only ARLE-14 and LIN-65 were highly enriched in the MET-2 pulldown ) nor of arle-14 (arle-14Δ(tm6845)) affected binding of the other cofactor to MET-2. We conclude that ARLE-14 and LIN-65 interact independently with MET-2.To understand how MET-2 is regulated, we examined in greater detail its interaction with ARLE-14 and LIN-65  to the endogenously tagged met-2::mCherry locus by CRISPR (NLS::met-2). The NLS::MET-2::mCherry nucleoplasmic signal was stronger than the WT MET-2::mCherry that traffics between the cytosol and nucleus18, but in both cases foci were clearly distinguishable , the loss of arle-14 did not reduce MET-2::mCherry levels . Remarkably, only 14% of the genes derepressed by loss of met-2 (met-2Δ genes) were upregulated in arle-14Δ  restricts gene derepression.Because the loss of H3K9me is associated with a failure to silence heterochromatin14Δ Fig. , while 5arle-14 and lin-65 mutants had different effects on transcription and focus formation, we performed epistasis analysis for the two genes, monitoring H3K9 methylation. We found that the lin-65Δ;arle-14Δ double mutant displayed a greater loss of H3K9me2 than either single mutant alone, achieving the same drop in methylation as in met-2Δ embryos . This loss of germline arises largely from the formation of R-loops at repeat elements, which are promiscuously transcribed upon loss of met-2 (refs. 30). To test whether ARLE-14 and LIN-65 differentially affect worm fertility, we quantified the number of progeny in arle-14Δ and lin-65Δ mutants. We observed an enhanced loss of viable progeny in both met-2Δ and lin-65Δ animals at 25 °C, while the arle-14Δ population was only modestly decreased in comparison with WT worms, despite having a near equal drop in H3K9me2  (hereafter met-2-CD) alters a highly conserved motif that is essential for the catalytic activity of SET domain proteins generally47 . Consistently, we find that H3K9me2 and H3K9me3 levels dropped to the same level in met-2-CD as in met-2Δ  into the met-2::mCherry locus to track catalytically deficient MET-2 in live embryos. We found that MET-2-CD forms foci, and the number of foci did not differ from WT MET-2 . However, there were 271 met-2Δ targets that were upregulated in met-2Δ but not in met-2-CD (‘met-2Δ-specific’ genes) and 179 genes that exhibited a mitigated response  in early embryos. We concentrated on H3K27ac due to its well-described role in transcriptional activation and enhancer function50. Relative to WT embryos, we defined 565 differentially enriched H3K27ac peaks in met-2Δ, while only 38 changes were detected in met-2-CD . Compared with WT, these loci were enriched for H3ac in met-2Δ but not in met-2-CD embryos  had equally low levels of H3K9me1 at these loci , but arle-14 did lead to the loss of MET-2-CD at these same sites 51. Intriguingly, although the HSR is clearly associated with well-described signaling pathways that dampen transcription while specifically upregulating protein chaperone genes53, earlier studies in other species also linked acute heat stress to the transcriptional derepression of silent chromatin58. Thus, HS conditions provided a physiological system in which we could monitor a transcriptional response that coincides with the induced dispersion of MET-2 foci, without genetic alteration.Having shown by genetics that MET-2 has a noncatalytic function that correlates with its concentration into foci, we wondered whether foci dynamics might be physiologically relevant in animals containing normal levels of H3K9me. We previously showed that MET-2 foci disperse upon transient exposure to 37 °C heat shock (HS) Fig. 14, coincmet-2Δ-upregulated genes, had very low expression levels in embryos cultured at 20 °C  between the transcriptional changes induced by 37 °C versus those induced by the loss of met-2 at 20 °C versus WT (20 °C) embryos  or HS (1 h at 37 °C). We observed a broad upregulation of genes at 37 °C relative to embryos kept at 20 °C  > 4, false discovery rate (FDR) < 0.01, Fig. yos Fig. . In factnes Fig. . The majnes Fig. . These dnes Fig. . On the met-2Δ mutants present enhanced phenotypes at 25 °C (refs. 30), we wondered whether the shift from 20 °C to 25 °C also induces a similar stress response as exposure to 37 °C. However, no HSF-1 foci formed in either WT or met-2Δ embryos at 25 °C, nor did the transcriptome of animals grown at 25 °C correlate with the pattern of genes induced by HS , we scored a ~24% reduction in MET-2 protein level  by ChIP-seq, and compared these peaks with data from our met-2Δ and met-2-CD animals. HS (30 min at 37 °C) led to an enrichment of H3K27ac on 1646 peaks , consistent with the idea that HS mobilizes a broad transcriptional response, as well as destabilizing MET-2 foci. Other regions of the genome of HS-treated embryos actually lost H3K27ac, reflecting the fact that a certain degree of transcriptional downregulation occurs during the canonical HSR59. The sites that gained acetylation in both HS and met-2Δ embryos were mainly promoters and repeats, while met-2-CD embryos showed almost no gain in acetylation over WT  showed particularly pronounced derepression during HS-induced loss of focus formation , we have been able to distinguish a repressive role for MET-2 focus formation distinct from the catalytic function of MET-2’s SET domain. H3K9me is clearly the key regulatory mark for the stable silencing of lineage-specific genes and repetitive elements across animal species, and the majority of loci repressed by heterochromatin in worms depend on the activity of MET-2’s SET domain. Yet the careful analysis of the met-2-CD allele has revealed a second, noncatalytic role for MET-2 that requires its binding partners LIN-65 (H.s. ATF7IP) and ARLE-14 (H.s. ARL14EP), and which correlates with the accumulation of MET-2 and its ligands in subnuclear foci   Clr3 and Sir2 are essential to restrict RNA Pol II access to heterochromatin63, and the treatment of murine cells with HDAC inhibitors resulted in a widespread loss of heterochromatic repression64 and impaired the establishment of lineage-specific expression patterns in differentiating mouse embryos65. Finally, histone acetylation itself is known to promote chromatin decompaction, weaken the interaction between histones and DNA, and antagonize phase separation of chromatin arrays in vitro69. Therefore, MET-2-CD’s ability to prevent hyperacetylation could explain how it maintains the semirepressed state that we observe by RNA-seq. MET-2-CD may block acetyltransferase access directly or recruit the factors, such as HDACs, that ensure that heterochromatin remains silent.The mechanism of focus-mediated repression appears to be largely structural: we found that MET-2-CD foci were able to block the increase in histone tail acetylation found in 14 , the DREAM complex72 and SMARC-1 chromatin remodelers73. While we do not exclude it, the hypothesis that these factors serve as effectors of MET-2’s noncatalytic function is made less likely by the fact that each of the others was shown to act either redundantly with MET-2 or through H3K9me. Alternatively, recent investigations into the function of subnuclear condensates suggest that these foci may physically impair the access of transcription activating complexes, such as RNA Pol II, Mediator, basal transcription factors or histone acetyltransferases74. Because foci of MET-2 and its cofactors share properties of condensates, they may also block recruitment of the transcription machinery and thereby inhibit the activation and acetylation of silenced genes, despite reduced levels of H3K9me.We cannot yet determine whether MET-2/LIN-65/ARLE-14 preserve hypoacetylation directly or indirectly. Although no HDACs were enriched in our MET-2 pulldown14 Fig. , it was 76. This precluded in vitro tests with recombinant protein for direct binding and/or HMT activity, which would complement the extensive genetic and biochemical evidence implicating MET-2 as the primary H3K9me1/me2 HMT in C. elegans embryos82. Nevertheless, we were able to demonstrate H3K9me activity of MET-2 recovered in a pull-down assay. We note that MET-2-CD has the same protein stability, localization, dynamics and protein interactions as WT MET-2, and yet H3K9me2 levels drop strikingly. This supports a direct role of MET-2 catalytic activity in H3K9me deposition, and a direct role for MET-2-CD in a secondary repressive function associated with LIN-65 dependent focus formation.We note that we were unsuccessful in expressing recombinant MET-2 or LIN-65, potentially due to their large size and highly disordered domains43. We therefore propose that ARLE-14 helps recruit MET-2 to chromatin, acting independently of the H3K9me mark. It is possible that ARLE-14 has other roles in gene activation that are independent of MET-2, or else that ARLE-14 also prevents MET-2 mistargeting, given that some genes actually acquire, rather than lose, repression upon loss of arle-14 . HS resulted in hyperacetylation and activation of silenced genes, coincident with MET-2 focus dispersion, even though the loss of genome-wide H3K9me was less severe than that triggered by arle-14Δ or met-2Δ strains. The dissolution of MET-2 foci by HS illustrates a rapid transcriptional response that promotes chromatin opening while retaining H3K9me, potentially allowing a quick post-HS recovery of the prestress transcriptome.Our study is not the first to suggest noncatalytic functions for a diverse group of HMTs. In mammalian cells, the H3K9 HMT G9a is able to promote repression by recruiting DNA methylation enzymes, independently of its catalytic activity14), they do depend on LIN-65, a protein rich in low-complexity sequence and disordered domains18. They are also stabilized by ARLE-14 in a manner redundant with H3K9 methylation, and they attenuate histone tail acetylation. This indicates that the SETDB1-like complex containing MET-2, ARLE-14 and LIN-65 contributes to gene repression beyond the targeted methylation of histone H3K9, with physiological consequences for organismal growth and fertility.In summary, we have uncoupled the effect of MET-2 concentration into subnuclear foci from its lysine methyltransferase activity. While H3K9me deposition is the primary driver of gene silencing, both H3K9me and MET-2 focus formation contribute to the repression of transcription and may mutually reinforce the heterochromatic state. Whereas MET-2 foci do not require the HP1-like factors HPL-1 and HPL-2  on nematode growth media supplemented with 1 mM IPTG and 100 µg ml−1 carbenicillin. Embryos isolated from these animals were analyzed as indicated. Synchronized animals fed either vector or arle-14 RNAi were grown to L4 stage at 20 °C before being shifted to 25 °C overnight, and their progeny were imaged , arle-14(gw1584) and met-2(gw1660). CRISPR RNA (crRNA) and homology directed repair (HDR) repair templates are listed in Supplementary Table arle-14(gw1584) was later confirmed not to be a null allele by mass spectrometry.We used CRISPR as previously described14. Briefly, all images were captured using Visiview software on a confocal spinning-disk microscope, the AxioImager M1 (Zeiss) with a Yokogawa CSU-X1 scanhead (Yokogawa), a Rolera Thunder camera (Photometrics) and an α plan-NEOFLUAR ×100/1.45 oil objective (Zeiss). Live embryos were mounted on 2% agarose pads in M9 buffer. For immunofluorescence, embryos were fixed for 5 min in 2% formaldehyde, then transferred to poly-l-lysine-coated slides and snap-frozen. Embryos were freeze-cracked and immediately treated with 100% ethanol precooled to −20 °C for 2 min then allowed to dry. After three 5-min washes using PBS with 0.25% Triton X-100 (PBS-X), slides were blocked in PBS-X + 2% milk for 1 h at room temperature and subsequently exposed to antibodies overnight at 4 °C in a humid chamber. After washing as above, slides were treated at room temperature with secondary antibodies diluted in PBS-X + 2% milk. Antibody dilutions are as follows: 1:500 mouse anti-H3K9me2 (MBL), 1:5,000 mouse anti-H3K27ac (gift from H. Kimura), 1:500 rabbit anti-H4 (Abcam), 1:1,000 goat anti-mouse Alexa Fluor 488  and 1:1,000 donkey anti-rabbit Alexa Fluor 555 . After DAPI  staining in PBS-X for 10 min, and three washes, slides were mounted with ProLong Gold Antifade (Thermo Fisher Scientific). Images were deconvolved using the Huygens remote manager (http://www.huygens-rm.org/wp/). Nuclear mean intensity and number of foci were quantified using KNIME87. Nuclei were first identified using MET-2::mCherry fluorescence, then foci were detected with a Laplacian-of-Gaussian detector from TrackMate 88.Imaging was performed as previously describedg for 10 min. Then, 10 µg of total protein was separated on Bis-Tris gels (Bio-Rad or Invitrogen) using MES buffer and transferred to 0.2-µm PVDF (Bio-Rad). Membranes were blocked with Protein-Free Blocking Buffer (Pierce) or PBS plus 0.5% Tween-20 (PBS-T) with 5% powdered milk (Sigma). Antibody dilutions: 1:2,000 mouse anti-H3K9me2 (MBL), 1:1,000 rabbit anti-RFP antibody, preadsorbed (Rockland), 1:100,000 mouse anti-H3K9me3 (MBL), 1:60,000 rabbit anti-H3K9me1 , 1:40,000 mouse anti-H3K9acetyl (a gift from H. Kimura), 1:40,000 rabbit anti-acetyl-Histone H3 (Millipore), 1:2,000 rabbit anti-acetyl-Histone H4 (Millipore), 1:20,000 rabbit anti-H2B (Abcam) and 1:40,000 rabbit anti-MRG-1 . After overnight rotation with antibodies diluted in blocking buffer at 4 °C, blots were washed three times in PBS-T, re-blocked and exposed to HRP-conjugated secondary antibodies for 1 h , goat rabbit IgG HRP ). After three further washes in PBS-T, ECL (Millipore) signal was detected with an Imager 600 (GE).All lysates were prepared at 4 °C and first processed using a Fast Prep-24 5G Benchtop Homogenizer  using a 4:1 ratio of lysate to 0.5-mm zirconia/silicon beads (BioSpec). For histone methylation analysis, embryos were lysed in RIPA buffer. Lysates were treated with 5 µl of benzonase (Sigma) for 1 h at 4 °C with rotation. For acetylation analysis, embryos were lysed in TAP buffer , 1× Deacetylase Inhibitor (Active Motif) and 1 mM DTT) and sonicated using a Bioruptor Plus (Diagenode) for 14 cycles, 15 s on, 30 s off. TAP lysates were cleared at 21,00014. Intensity-based absolute quantification values were used as a measure of protein abundance in immunoprecipitation89. For H3K9 post-translational modification profiling, we employed the Histone Purification Kit Mini (Active Motif). Embryos isolated by bleaching were homogenized as above in histone extraction buffer. After overnight extraction at 4 °C with rotation, we proceeded with histone purification according to the manufacturer’s instructions. Purified histones were propionylated twice before and once after overnight trypsin digestion to prevent cleavage at lysine residues and facilitate separation of peptides. Samples were dried in a speedvac and resuspended in 0.1% trifluoroacetic acid (TFA), 2% acetonitrile. LC–MS was performed as follows: samples were loaded onto a PepMap 100 C18 2-cm trap (Thermo Fisher) using an EASY nLC-1000 system (Thermo Fisher). On-line peptide separation was then performed on a 15-cm EASY-Spray C18 column  by applying a linear gradient of increasing acetonitrile concentration at a flow rate of 150 nl min−1. An Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) was operated by alternating between MS1 survey scan mode, recording spectra at 120,000 resolution in the Orbitrap, and data-independent parallel reaction monitoring (PRM) mode, by targeting previously identified doubly charged histone H3 peptide isoforms of the sequence ‘KSTGGKAPR’ (9–17) containing lysines K9 and K14 , and two endogenously unmodified reference peptide precursors (YRPGTVALR (41–49) at m/z = 544.8109 and YQKSTELLIR_K56me0  at m/z = 681.8817) for higher-energy collisional dissociation (HCD) fragmentation at a normalized collision energy of 33%, recorded at 15,000 resolution in the Orbitrap analyzer. Acquired data from strains indicated in Supplementary Table 90, diagnostic fragment (z = 1) ions y6, y7 and y8 for each histone H3K9 peptide isoform (mark) of interest were selected and retention time windows were manually chosen to avoid quantifying background noise from coeluting isoforms or other unrelated analytes. Integrated fragment ion abundances were aggregated by peptide and histone mark for all samples and replicates and then divided by the total sum of all histone H3 peptide abundances per sample.For immunoprecipitation experiments, embryos isolated by bleaching gravid adults were lysed at 4 °C in TAP buffer. Proteins from homogenized, sonicated and cleared TAP lysates (above) were immunoprecipitated with anti-FLAG-M2 beads (Sigma). Proteins were digested, bound to beads and subjected to LC–MS/MS exactly as described previouslyFor brood size, L4 animals grown from egg at the indicated temperature were singled out onto individual plates. They were transferred to fresh plates every 24 h until egg laying ceased, after which their total progeny was counted. For developmental rate, synchronized L1s were plated and the numbers of L4 and pre-L4 (delayed) animals were counted 42 h post feeding.6. Embryos were freeze-cracked five times, then RNA was extracted with choloroform followed by isopropanol precipitation. Further purification was performed with the RNA Clean and Concentrator kit (Zymo). Libraries were produced using the Smart-Seq2 mRNA sequencing kit (Illumina). Ribosomal RNA was depleted using Ribo-Zero Gold kit (Epicentre), and libraries were produced using the Total RNA Sequencing Scriptseq kit (Illumina). Equimolar amounts of indexed libraries were pooled and sequenced on a HiSeq 2500 (Illumina).RNA was extracted from early embryos using Trizol as previously described6. Adapters were trimmed using Trimmomatic v.0.39. Reads were aligned to the C. elegans genome (ce10) with the R package QuasR v.1.22.0 . The command ‘proj < -qAlign’ instructs hisat2 (ref. 92) to align using default parameters, considering unique reads for genes and genome-wide distribution. Count tables of reads mapping within annotated exons in WormBase (WS220) were constructed using the qCount function of the QuasR package to quantify the number of reads in each window ) and normalized by division by the total number of reads in each library and multiplied by the average library size. Transformation into log2 space was performed after the addition of a pseudocount of 8 to minimize large changes in abundance FC caused by low count numbers. The EdgeR package v.3.24 was applied to select genes with differential transcript abundances between indicated genotypes (contrasts) based on FDRs for genes. Replica correlations are shown in Extended Data Fig. 93.Reads were analyzed as described previously6. In brief: early embryos were harvested from synchronized animals in three replicates. Then, 40 μg of chromatin was incubated overnight with 2 μg of anti-H3K9me2 (MBL), anti-H3K27ac , anti-H3K9me1  or anti-acetyl-Histone H3 (Millipore) antibody coupled to Dynabeads Sheep Anti-Rabbit IgG (Invitrogen), in FA buffer ) containing 1% SDS. For MET-2::mCherry ChIP, 100 μg of chromatin was incubated with RFP-Trap magnetic agarose (Chromotek). Antibody-bound chromatin was washed 3 × 5 min with FA buffer; 5 min with FA buffer with 1 M NaCl; 10 min with FA buffer with 500 mM NaCl; 5 min with TEL buffer  and 2 × 5 min with Tris-EDTA-buffer. Complexes were eluted in 1% SDS in Tris-EDTA buffer with 250 mM NaCl at 65 °C for 15 min. Samples and inputs were treated with 20 μg of RNAse A for 30 min at 37 °C and 20 μg of proteinase K for 1 h at 55 °C. Crosslinks were reversed by overnight incubation at 65 °C. DNA was subsequently purified using Zymo DNA purification columns (Zymo Research). Primers used in quantitative PCR are listed in Supplementary Table ChIP experiments were performed as previously described6 using the NEBNext Ultra DNA Library Prep kit for Illumina (NEB no. 7370) and the NEBNext Multiplex Oligos for Illumina (NEB no. E7335), according to the manufacturer’s recommendations, without size selection. Libraries were indexed and amplified using 12 PCR cycles, following the manufacturer’s recommendations. Libraries were further purified with Agencourt AmPure XP beads (Beckman no. A63881). Library size range and concentration were determined using a BioAnalyzer 2100 (Agilent Technologies) and Qubit (Invitrogen) instrument, respectively. Equimolar amounts of indexed libraries were pooled and sequenced on a HiSeq 2500 (Illumina) in rapid mode (Paired-End 50). Reads were aligned to the C. elegans genome (ce10) with the R package QuasR v.1.22.0 (www.bioconductor.org/packages/2.12/bioc/html/QuasR.html). The command ‘proj < -qAlign’ instructs bowtie to align using the parameters ‘-m 1–best–strata–phred33-quals’, considering unique reads for genes and genome-wide distribution.Libraries were prepared as previously describedhttps://bioconductor.org/packages/release/data/annotation/html/BSgenome.Celegans.UCSC.ce10.html). Differences in read depths between samples were normalized by dividing each sample by total reads and multiplying by average library size. The various count tables used throughout this study were normalized according to the total genome count. log2 expression levels were determined after addition of a pseudocount of 2 (y = log2(x + 2)) to minimize large changes in FC caused by low count numbers. Results are displayed as the mean enrichment of immunoprecipitation − input (log2). H3K27ac peaks were called using MACS2 (ref. 94) with the following parameters: -g 93260000 --broad. Differential peaks among the genotypes were called using the Diffbind package v.3.0.11 (http://bioconductor.org/packages/release/bioc/html/DiffBind.html)95, normalizing on the total read counts and using the implemented DESeq2 analysis with standard parameters. Read density was calculated by tiling the genome into 500-bp nonoverlapping windows, or specifically at promoters (defined as 1 kilobase upstream and 100 bp downstream of transcription start site), and using the qCount function of the QuasR package to quantify the number of reads in each window ). Differences in read depths between samples were normalized by dividing each sample by total reads and multiplying by average library size. The various count tables used throughout this study were normalized according to the total genome count.Read density was calculated by tiling the genome into 500-base-pair (bp) nonoverlapping windows and using the qCount function of the QuasR package to quantify the number of reads in each window ). H3K9me-positive domains were determined as regions with a consecutive enrichment of H3K9me2 over input. For gene quantification, gene annotation from WormBase was used (WS220). Quantitation for each gene was performed by counting the reads overlapping the exons. Genome annotation was based on BSgenome.Celegans.UCSC.ce10 package (Further information on research design is available in the Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41594-021-00712-4.Supplementary InformationSupplementary Tables 1 and 2.Reporting Summary.


IgG4-related disease (IgG4-RD) is increasingly recognised in rheumatology practice. We describe a case of orbital swelling initially diagnosed as IgG4-RD. It was exquisitely steroid responsive and she was treated with 6 years of immunosuppression but her symptoms later deteriorated. She developed worsening left eye swelling that required debulking surgery; histopathology demonstrated IgG4-positive lymphoma. She had an excellent response to localised radiotherapy and remains in remission. Although her initial presentation was typical of IgG4-RD, her subsequent lack of response to steroids prompted reconsideration of the initial diagnosis and re-examination of her previous biopsy specimen, confirming IgG4-positive Iymphoma at both timepoints.A 25-year-old lady presented in 2008 with a swollen left eye. She had a history of childhood eczema, asthma and sinusitis. Diagnostic excision was undertaken; histology was considered to show chronic inflammation, scattered eosinophils, dense fibrosis and no granulomas. She was diagnosed with a ‘granulomatosis with polyangiitis-like’ orbital pseudotumour. It was exquisitely steroid responsive; mycophenolate mofetil was added from an early stage of treatment.The patient was referred to rheumatology 3 years after diagnosis as she was unable to reduce her prednisolone below 5mg daily. Her immunology screen was negative. Although her plasma IgG4 levels were normal at 0.14g/L, the original histology was reviewed and immunostained for IgG4. This demonstrated more than 80% of plasma cells to be IgG4 positive, with the previously noted dense fibrosis and chronic inflammatory infiltrate. Her diagnosis was revised to orbital IgG4-RD. She commenced azathioprine as she was planning a pregnancy.Following an uncomplicated pregnancy in 2014, her symptoms significantly deteriorated, with worsening pain, discharge and swelling in her left eye. A repeat MRI scan showed a new cuff of abnormal tissue within the lateral aspect of the left orbit. She was referred to ophthalmology and underwent left anterior orbitotomy with debulking. Histopathology at this time was consistent with extranodal marginal zone lymphoma (EMZL) of MALT (mucosa-associated lymphoid tissue) type, IgG4 positive. An extraordinary feature was an abundance of crystal-storing histiocytes, presumed to contain ingested IgG4.To investigate whether she had an IgG4-secreting lymphoma from the beginning or whether there had been malignant transformation of her orbital IgG4-RD, the histology from 2009 was reviewed. This was considered to indicate a MALT-type EMZL.The patient was referred to haematology and she was treated with localised radiotherapy with an excellent response. She remains in remission following this and she has subsequently been discharged from haematology follow-up.IgG4-RD is a condition involving fibroinflammatory lesions that can affect any organ in the body. As a newly recognised condition, the role of IgG4 in IgG4-RD is still not fully understood. Questions that remain unanswered include whether or not IgG4 is directly pathogenic and whether there is an increased risk of malignancy in patients with IgG4-RD.Our patient’s case demonstrates how IgG4-positive lymphoma can mimic IgG4-RD. It is important to keep a broad differential diagnosis in complex cases, and to support the eventual diagnosis with blood results, imaging studies and histology. It is essential to involve colleagues in radiology and histopathology so that a multidisciplinary decision can be made with regards to management. IgG4-RD is a steroid-responsive condition. When there is poor response to conventional treatment, this should raise doubts regarding the diagnosis, and it would be prudent to consider a repeat biopsy as well as obtaining previous pathology specimens so that they can be re-reviewed.Ocular adnexal lymphomas form 1—2% of non-Hodgkin lymphomas and 8% of extranodal lymphomas. Over recent decades the incidence has increased, partly reflecting better diagnostic techniques. The most common subtype of ocular adnexal lymphomas are EMZL of MALT type, as in our patient.There are several case reports of lymphoma arising in patients with established IgG4-RD, raising the question of potential lymphomagenesis in IgG4-RD. The earliest reports come from Asia and most have been reported in patients with ocular IgG4-RD. However, the link between malignancy and IgG4-RD is still being investigated. It is important to consider a potential malignancy when managing patients with IgG4-RD, especially if there is a lack of response to steroids.There is no single diagnostic test for IgG4-RD. Diagnosis should be based on the overall clinical picture, supported by blood results, imaging and histopathology.IgG4-RD is a steroid-responsive disease. If there is a poor response to treatment, the diagnosis should be re-considered.Re-examination of previous biopsy specimens can be invaluable, especially in patients refractory to standard treatment.The link between IgG4-RD and malignancy has not been established and it is important to remain vigilant. As ocular adnexal lymphomas are not uncommon, it is important to keep this differential diagnosis in mind when reviewing patients with eye swelling thought to be secondary to IgG4-related ocular disease.


When patented, brand-name antibiotics lose market exclusivity, generics typically enter the market at lower prices, which may increase consumption of the drug. To examine the effect of generic market entry on antibiotic consumption in the United States, we conducted an interrupted time series analysis of the change in the number of prescriptions per month for antibiotics for which at least one generic entered the US market between 2000 and 2012. Data were acquired from the IQVIA Xponent database. Thirteen antibiotics were analyzed. Here, we show that one year after generic entry, the number of prescriptions increased for five antibiotics (5 to 406%)—aztreonam, cefpodoxime, ciprofloxacin, levofloxacin, ofloxacin—and decreased for one drug: cefdinir. These changes were sustained two years after. Cefprozil, cefuroxime axetil and clarithromycin had significant increases in trend, but no significant level changes. No consistent pattern for antibiotic use following generic entry in the United States was observed. Generics contribute to increased availability of antibiotics, benefiting healthcare systems but potentially leading to increased consumption with implications for antibiotic stewardship and resistance. Here, the authors found no consistent changes in prescribing patterns of the 13 antibiotics that entered the US market as generics from 2000–2012. Antibiotic use is a primary driver of AMR3 and remains at a high level in the United States, despite efforts to improve prescribing practices and discourage inappropriate use5. As much as 30% of oral antibiotic use in the United States may be unnecessary6. Apart from factors such as patients’ and physicians’ expectations, physicians’ training, and patient characteristics8, the price and introduction of generics are potential drivers for overconsumption14. Generic drugs enter the market shortly after exclusivity rights  for the original patented drug end14. This was made possible by the Hatch-Waxman act, allowing companies to obtain approval for generic drugs without conducting additional clinical trials14. As a result, availability and access increase, while individual and national health expenditures fall, benefiting healthcare systems16. In the case of antibiotics, generics could potentially increase inappropriate antibiotic consumption and hasten the development of antibiotic resistance17.Antimicrobial resistance (AMR) is a global challenge that raises mortality from infectious diseases and increases healthcare costs14. In Denmark, study findings showed a correlation between generic entry of ciprofloxacin and increased cases of ciprofloxacin-resistant Escherichia coli from urine isolates9. In Germany, generic entry of cephalosporins and fluoroquinolones increased the use of these antibiotic classes13. A more recent study examining the effect of generic entry of levofloxacin on fluoroquinolone use and meropenem on carbapenem use in five European Union countries and in the United States18 showed significant increases in some cases and significant decreases in others, with no discernible patterns between different antibiotics or different countries. These previous studies have focused only on a few antibiotics, not addressed alternative explanations to their findings (co-interventions)18 or not accounted for secular trends14.After generic entry, amoxicillin-clavulanate use in the United States and ciprofloxacin use in Denmark increasedIn this study, we analyze the effect of loss of exclusivity on the use of antibiotics for systemic use that had at least one generic enter the US market between 2000 and 2012, using an interrupted time series (ITS) design. We hypothesized that a level increase in antibiotic prescriptions would be visible by 6–12 months after the entry of the first generic and by 24 months at the latest. This was the case for five antibiotics , whereas one (cefdinir) showed a decrease, one year after generic entry. These changes were sustained two years after. Our second hypothesis was an increase in trend, which was the case for three antibiotics . These changes represented use leveling out. Together, these findings indicate no consistent pattern for antibiotic use following generic entry in the United States.We identified 13 antibiotics that met the inclusion criteria Figs. . CefuroxPrescriptions for five antibiotics—aztreonam, cefpodoxime, ciprofloxacin, levofloxacin, and ofloxacin—had a statistically significant increase 6–12 months after generic entry, which was sustained two years after generic entry. Cefdinir was the only antibiotic showing a significant decrease in prescriptions against the historical trend, sustained over time , ofloxacin prescriptions had increased significantly by 0.17 per one million population , whereas cefdinir prescriptions had decreased by −0.27 per one million population . Twelve months after generic entry, aztreonam prescriptions had increased significantly by 0.04 per one million population , cefpodoxime prescriptions increased by 0.22 per one million population , and levofloxacin prescriptions increased by 672.41 per one million population  , ofloxacin prescriptions by 0.38 per one million population , whereas cefdinir prescriptions had decreased significantly by −0.85 per one million population . Aztreonam prescriptions had significantly increased by 0.05 per one million population , cefpodoxime prescriptions by 0.45 per one million population , and levofloxacin prescriptions by 1989.14 per one million population   after 12 months and 803.76 per one million population  by 24 months . This could have promoted the IV formulation of aztreonam as a treatment option for patients with cystic fibrosis and increased its use, as the IV formulation of aztreonam has clinical benefits for cystic fibrosis patients as well25, and as IV antibiotics are sometimes used for preparing inhalations. Another plausible explanation for the observed changes in prescriptions for aztreonam, cefpodoxime, and ofloxacin is that consumption leveled out when approaching zero, as “negative consumption” is not possible. In the case of ciprofloxacin, the anthrax attacks and resulting stockpiling seem to have affected prescriptions. We did not find alternative explanations for the significant changes seen in the other antibiotics. FDA reported safety issues for fluoroquinolones in October and December 2008 due to an increased risk for tendinitis and tendon rupture, and azithromyzin in May 2012 and December 2013 due to an increased risk for fatal irregular heart rythms26. None of these events coincided with time of generic entry for these drugs. Unrelated to generic entry, it is possible that the decrease in levofloxacin, beginning in 2008, was partly due to safety issues, as that was the year FDA issued a safety warning. One of the effects of vaccines is a decrease in the use of antibiotics27. Five vaccines were added to the vaccination program in the United States between 2000 and 2012; pneumococcal vaccines in 2000  and 2010 , influenza vaccine in 2004, quadrivalent meningococcal vaccine in 2005, and rotavirus vaccine in 200628. The 7-valent pneumococcal vaccine had a major impact on infections caused by the bacteria strains covered by the vaccine, with the main effect seen between 2000 and 200229. This coincided with the generic introduction of cefuroxime axetil and could have masked a significant increase in use, which, according to our results, had an insignificant increase. Based on the same logic, the increase in the use of levofloxacin could have been reduced by the introduction of the 13-valent pneumococcal vaccine causing a decrease in upper respiratory tract infections. However, decreases should then be visible in other respiratory tract antibiotics as well at this point in time, which was not the case. In contrast to the other vaccines, which target mainly the younger population, the influenza vaccine targets all age groups. Although this makes it relevant to this study, the impact of the influenza vaccine is more difficult to assess given that both effectiveness and coverage differs each season. Therefore, we cannot rule out that the effect of generic entry of an antibiotic used for respiratory tract infections was masked by the influenza vaccine. In 1995, the Centers for Disease Control and Prevention (CDC) launched the National Campaign for Appropriate Antibiotic Use in the Community, which was renamed Get Smart: Know when antibiotics work campaign in 200331. Get Smart promotes responsible use of antibiotics and supports states and communities to develop and implement stewardship programs32. We did not find any programs that were implemented simultaneously in all states, and which could be linked in time with the dates for generic entry of the antibiotics included in this study. Although statewide and community-based programs have contributed to improvement in antibiotic use, it is unlikely that they would be able to cause a significant change in national use of one specific antibiotic at a specific point in time. Between 2000 and 2012, CDC reported increases of macrolide-resistant bacteria, including Streptococcus pneumoniae, Carbapenem-resistant Enterobacteriaceae, extended spectrum Betalactamase, and fluoroquinolone-resistant bacteria33. Although resistance develops gradually, an immediate impact on use could happen if the resistance levels led to nationwide guideline changes. We were not able to detect a guideline change that coincided with our dates for generic entry. On the contrary, all three fluoroquinolones included in the study were among the antibiotics with significant increases in prescriptions and in 2012 azithromycin was the most prescribed antibiotic in outpatient care in the United States, despite growing levels of resistance towards these antibiotics34.Other factors coincident with generic entry, so-called co-interventions, could alter the trajectory of prescriptionsIt is possible that the antibiotics included in this reseach impacted each other by competing for the same market shares. This would be relevant primarily for antibiotics with similar indications, formulation, and generic entry at close proximity in time. The three fluoroquinolones included in the study all showed an increase in use, so regardless of whether or not they had an impact on each other it should not have affected the outcome. However, a number of the oral antibiotics, where generic entry had a mixed effect, have respiratory tract infections as indications—azithromycin, cefdinir, cefpodoxime, cefprozil, cefuroxime axetil, clarithromycin, as well as ciprofloxacin, levofloxacin, and ofloxacin  and resistance is multifactorial and complex, and models should not assume that generic entry will automatically increase antibiotic prescriptions in settings like the United States. Nevertheless, some antibiotic classes, namely fluoroquinolones, could be more sensitive. Interestingly, a significant increase in fluoroquinolone use was also observed by Stephens18, by Jensen et al.9, and by Kaier13. However, it is difficult to know whether this is coincidental, as the three studies did not include more than one other antibiotic or class and did not discuss alternative explanations for their findings. Also, the increase in fluoroquinolones was notable, given the well-known problems related to both resistance and side effects38. Arguably, this increase could be considered inappropriate (even if the anthrax attacks were a likely contributing factor), but without looking at overall antibiotic use and use by antibiotic class, this is difficult to judge.Our findings suggest that, consistent with evidence from other countries17, with a small effect on price after the first generic entry39; the biggest price reduction occurs after the second generic entry40. We did not have access to dates of subsequent generic entry. However, for eight of the 13 antibiotics in our study, three or more generic products were approved one year after the first generic entry , it could have delayed a potential price reduction by limiting competition. To account for a delay in the effect of generic entry, we included analysis of the effect 24 months after the event. Information regarding the number of manufacturers includes only approvals to market and depends on companies actively notifying the FDA if products are taken off market. This means that the number of manufacturers could have been overestimated. We did not examine class-based substitution effects, which could explain some of the trends observed—e.g., if increases in the use of a particular antibiotic were due to declines in sales in a substitutable product. Finally, we addressed the issue of co-interventions by searching for events that could have affected use. However, given the multitude of factors that influence antibiotic use, there could be co-interventions that we have overlooked. Additional research is needed to explore the effect of generic entry on antibiotic prices and to consider differences among antibiotic classes. Research is also needed to determine the effects, including access and antibiotic resistance, when generics enter the market in low- and middle-income countries.Our study included all antibiotics for which generics were introduced between 2000 and 2012, which allows for analyzing differences among classes, formulations, and target indications. Nevertheless, several limitations should be considered when interpreting our findings. Research shows that price reductions depend on the number of generics introduced to market24, to assess the effect of loss of market exclusivity on antibiotic use. The intervention being studied was generic entry, defined as the month when prescriptions of the first generic were first recorded. We chose generic entry to mark the end of exclusivity because alternatives, such as patent expiration date or approval date of the first generic product, do not guarantee that a generic has entered the market. Moreover, companies manufacturing brand-name antibiotics may contract with generic manufacturers to distribute a generic version of the product prior to the end of exclusivity. We therefore compared the date of the first recorded generic prescription with FDA approval dates to ensure that approval preceded distribution of generics. This was the case for all but one antibiotic (ciprofloxacin), for which the intervention date was set to the month when both generic prescription and approval had occurred . A change in trend represent an increase or decrease in the slope of a time series segment after the intervention compared with the time series segment preceding the intervention23 /Abbreviated New Drug Application (ANDA) status, obtained from the FDA website49. The statistical model used for this model ist, t and is coded 0 pre-intervention and 1 post-intervention. “Time after intervention” is a continuous variable, time t is coded 0 pre-intervention and is time − time point when the intervention occurred. t, “Level effectt” is the change in level at time t, and “Predicted valuet” is the counterfactual at time t50. Analysis was done using Stata1551. To identify potential co-interventions (events that coincide with the intervention and affect the outcome measure thereby impacting the result), we conducted a systematic search using open-access internet resources and published articles24. Factors considered possible co-interventions included safety issues, infectious disease outbreaks, changes in treatment guidelines, vaccination programs, stockpiling, drug shortages, price negotiations, stewardship programs, and antibiotic resistance. Safety issues for each of the 13 antibiotics were identified using the FDA drug safety communications26. Infectious disease outbreaks and development of antibiotic resistance were identified searching reports by the CDC33. We searched for changes in treatment guidelines developed by the Infectious Diseases Society of America and in treatment guidelines posted on the CDC webpage53. Changes in the US vaccination program were identified using the CDC “Immunization schedules”28. In addition, we searched PubMed Google and Google Scholar using the following combinations of search terms: [pathogen] AND vaccine; outbreak bacteria USA; antibiotic prescriptions USA; antibiotic stewardship program AND [antibiotic]; antibiotic shortage; antibacterial drug shortages; antibiotic stockpiling; antibacterial stockpiling; price negotiations AND [antibiotic].Segmented regression analysis, a statistical method commonly used in ITS design, was conducted and uses time-series data to measure change in level and change in trend, allowing for underlying trends before and after the event of interest (intervention)entclass1pt{minimaFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Conventional µ-CGGs fabricated via photolithography-based micromachining processes, however, are fundamentally limited to two-dimensional fluidic routing, such that only two distinct antimicrobial drugs can be tested at once. This work addresses this limitation by employing Multijet-3D-printed microchannel networks capable of fluidic routing in three dimensions to generate symmetric multidrug concentration gradients. The three-fluid gradient generation characteristics of the fabricated 3D µ-CGG prototype were quantified through both theoretical simulations and experimental validations. Furthermore, the antimicrobial effects of three highly clinically relevant antibiotic drugs, tetracycline, ciprofloxacin, and amikacin, were evaluated via experimental single-antibiotic minimum inhibitory concentration (MIC) and pairwise and three-way antibiotic combination drug screening (CDS) studies against model antibiotic-resistant Escherichia coli bacteria. As such, this 3D µ-CGG platform has great potential to enable expedited combination AST screening for various biomedical and diagnostic applications.Microfluidic concentration gradient generators ( μ-CGGs) have been used to identify optimal drug compositions through antimicrobial susceptibility testing (AST) to treat antimicrobial-resistant infections, and a new method has been developed to overcome previous 2-D limitations. Conventional μ-CGGs fabricated through photolithography-based micromachining processes are limited to 2-D fluidic routing, which allows only two distinct antimicrobial drugs to be tested simultaneously. However, a team headed by Eric Sweet at the University of California, Berkeley, United States employed 3-D-printed microchannel networks that are capable of 3-D fluidic routing to generate symmetrical multi-drug concentration gradients. The authors were able to confirm their 3-D μ-CGG prototype by means of both theoretical simulations and experimental validations. The team believes that its 3-D μ-CGG platform offers considerable potential for conducting multi-drug AST evaluations for a variety of biomedical and diagnostic applications.Microfluidic concentration gradient generators ( In the context of antibiotic resistance, for instance, more than 18 distinct bacteria, including pathogenic Escherichia coli (E. coli), Listeria monocytogenes (L. monocytogenes), and Staphylococcus aureus (S. aureus), have developed biological resistance to one or more of the world’s essential first-line-of-defense antibiotic agents5. The susceptibility of AMR organisms to antimicrobial compounds is assessed in clinical and biomedical research settings through antimicrobial susceptibility testing (AST) methods, predominantly minimum inhibitory testing (MIC) and combination drug screening (CDS), which are particularly useful in the fight against antibiotic-resistant infections, such as urinary tract infections (UTIs)6. Conventional MIC testing7  infections places a significant economic burden on the worldwide economy, upwards of $35 billion per year in the United States alone, and is projected to be the cause of over 10 million deaths per year by year 205013. Furthermore, the only effective treatment against certain AMR infections is the use of combinations of multiple antimicrobial agents17. Conventional antibiotic CDS , the most widely adopted class of microfluidic AST technologies46 for MIC and CDS studies, employ branching microchannel networks comprised of nodal units to produce diluted concentrations representing a gradient between input species. Discrete concentration gradients, for example, one antibiotic and one buffer solution48, are represented by the independent outputs from the µ-CGG network itself49, often referred to as discrete µ-drug cocktails15. Several discrete µ-CGG devices have been demonstrated for both single-antibiotic MIC testing and CDS studies50 of clinically relevant antibiotics against laboratory-standard bacteria51, bioengineered strains52, and cells isolated from biological fluid samples53. The µ-drug cocktail solutions are either used to perform on-chip bacteria culture54 or are collected as discrete approximately microliter-volume antibiotic solutions for use in off-chip bacteriological experiments39. As a result, a typical duration for rapid AST analysis is on the order of ~6–8 h55.Various microfluidic-based AST platformsµ-CGG enabled devices toward AST involving more than two antimicrobial compounds, however, is fundamentally limited. Traditional MEMS-based microfluidic fabrication approaches are monolithic in nature; as a result, conventional microchannels have inherently two-dimensional geometric complexity and are therefore capable of fluidic routing in essentially only two dimensions56. Symmetric fluidic gradients, those capturing all possible combinations of the inputs, are limited to only two distinct fluidic species at a given time  3D-printing technology and additive manufacturing have garnered significant interest recently toward various microfluidic applicationsµ-CGG prototype employing a unique 3D microchannel network that is only possible to fabricate using an additive manufacturing-based approach. The only way to accomplish a symmetric gradient of three or more fluids is to perform fluidic routing in truly three dimensions  structures to best elucidate the analytically predicted behavior; then, the performance of the fabricated prototype was experimentally validated through fluorescence imaging. Finally, antibiotic gradients collected from the device were used to demonstrate its proof-of-concept utility as an AST tool for MIC testing and pairwise and three-antibiotic CDS bacteriological experiments for three clinically relevant antibiotics against antibiotic-resistant E. coli bacteria.This paper reports the design and development of a truly 3D ons Fig. . A tetraµ-CGG microchannel network  integrated into the sidewall, into a hollow spherical bulb  and concentration of each input fluidic species (Ci) at each nodal unit  and the lower bulbs (where the fluids split off from) was investigated by studying three different 3D µ-CGG designs incorporating (i) smooth-walled vertical channels, serving as a reference, and imbedded (ii) 3D bulbous and (iii) 3D rifled microstructures, both based on a previously demonstrated 3D-printed µ-mixer66 to induce chaotic advective fluid motions for enhanced mixing efficiency67. The analytically calculated normalized concentrations (Nc) of a single-input fluidic species (C1) at each outlet of a conceptual device  output concentrations representing practically useful proportions of each input fluidic species, i.e., ~1, ~7/10, 5/10, ~3/10, and ~0, in addition to an output capturing a nearly equivalent proportion of all three inputs fluidic species, i.e., ~1/3, and was therefore chosen as the most appropriate design for prototype fabrication, experimental characterization, and bacteriological demonstrations.The reference ign Fig.  exhibiteign Fig.  exhibiteign Fig. , on the µ-CGG prototype were assessed by using the device to generate a gradient between one rhodamine fluorescent dye solution and two DI water fluidic inputs. Three experiments were performed with the rhodamine solution input into one of the device inlets, i.e., inlet 1, then the process was repeated with the rhodamine solution input into each of the other two inlets, i.e., inlets 2 and 3 , whereas resazurin is weakly fluorescent. Resazurin-based cell viability protocols have proven simple, accurate, and reproducible methods to quantify and assess the metabolic activity of organisms, particularly bacteria70. In this work, normalized emission of resorufin was measured and used to produce a normalized growth (Ng) value elucidating the degree of bacterial proliferation, and thereby the antibiotic-induced bacterial growth inhibition, in each solution72.The ability of the developed 3D Ng values for solutions containing µ-drug cocktails from 100% antibiotic to 100% buffer are plotted from outlets along one edge of the device, i.e., outlets 1, 3, 5, 7, 8, and 13. The MIC value of the antibiotic was determined with the first apparent stepwise reduction in mean Ng value, i.e., enhancement in growth inhibition. The diagram in Supplementary Material Fig. Experimental single-antibiotic MIC testing results are presented in Fig. 73. The tetracycline-buffer-buffer gradient . This specific chemistry should therefore be avoided in treating an infection caused by this specific strain of bacteria. Furthermore, the gradient between amikacin and ciprofloxacin , and 8 ; these specific chemistries are therefore be highly recommended in treating such an infection. Moreover, the gradient between tetracycline and amikacin , amikacin and ciprofloxacin , and ciprofloxacin and tetracycline , in addition to the implementation of a three-antibiotic Ng data from the three-antibiotic CDS experiments  demonstrated synergism compared to the 100% solutions of amikacin and ciprofloxacin and an additive effect compared to the 100% solutions of amikacin and tetracycline, as well as ciprofloxacin and tetracycline. In this demonstration, the µ-drug cocktails from outlets 2, 4, 5, 6, 8, and 13 exhibited more effective inhibition of bacterial proliferation than those of the three-antibiotic µ-drug cocktail and would, therefore, represent more effective doses for treatment. Regardless, the three-antibiotic µ-drug cocktail is the direct result of the unique 3D fluidic routing capability demonstrated by the 3D µ-CGG microchannel network, which is otherwise impossible using planar fluidic routing processes.The mean nts Fig.  revealedµ-CGG devices is fundamentally limited by the inability of such systems to generate symmetric concentration gradients of more than two antimicrobial solutions at a time. In this work, we developed a 3D µ-CGG prototype employing a truly 3D microchannel network through the use of an additive manufacturing approach to accomplish fluidic routing in three dimensions to generate symmetric three-fluid concentration gradients.The throughput of multidrug AST applications using conventional µ-mixing structures to produce 13 distinct output µ-drug cocktail solutions for bacteriological studies. Experimental characterizations validated the generated concentrations to within 10% of the predicted values, justifying the use of the proposed 3D µ-CGG system for multidrug AST evaluations.Analytical modeling and theoretical simulations were used to design and optimize the microchannel network via the inclusion of embedded 3D E. coli bacteria. The MIC values of the individual antibiotics were characterized and were in agreement with the documented ranges for each compound. Furthermore, the known synergistic, additive, and antagonistic effects of each combination of antibiotics were experimentally observed through individual pairwise CDS studies.As a proof-of-concept, the fabricated prototype was used to evaluate the efficacy of three clinically relevant antibiotic compounds against model antibiotic-resistant µ-drug cocktail containing all three antibiotic species, in 5 h from a single operation for enhanced throughput over conventional multiantimicrobial CDS approaches.As such, the unique 3D fluidic routing capabilities enabled a three-antibiotic CDS study to simultaneously generate three distinct pairwise antibiotic concentration gradients, including one µ-CGG designs to produce tailored concentration gradient characteristics. Such devices can be rapidly prototyped and fabricated in clinical point-of-care settings using commercially available 3D printers to reduce the time-to-deployment and manufacturing costs. Ongoing technological advances in additive manufacturing resolution, material variety, and scalability will enable engineering of evermore advanced 3D µ-CGG designs that can be incorporated into more complex micrototal analytical systems to significantly increase the throughput of AST to combat emerging antibiotic-resistant bacterial infections in clinical and drug development settings.Moving forward, additive manufacturing permits straightforward and on-demand modification of 3D All microfluidic designs were modeled using Solidworks computer-aided design software . All inlet and outlet geometries enabled device-to-world interfacing via standard 20-gauge stainless-steel catheter couples . All microchannels, bulbs, and solid bodies were saved as individual part files then arranged in a single assembly file to produce the positive-feature network Fig.  for use 82 (3D Systems) and Visijet S100 hydroxylated wax83 (3D Systems). Both materials  attached to a 20-Gauge Luer stub  until all of the interior support material was removed. Finally, the process was repeated using an aqueous soap solution and potable water to remove any residual mineral oil  Fig. . The matoil Fig. . For furN) of the species from one inlet (C1) at each outlet for a range of input fluid flow rates was calculated as the average N value over the entire microchannel outlet face  simulations were performed using COMSOL Multiphysics  finite element analysis software to determine the theoretical performance of all 3D models. The theoretically predicted normalized concentration (Escherichia coli (E. coli) bacteria were procured from Agilent Technologies, CA, USA86, in 2-mL cryovials as glycerol stock and stored at −80 °C. A scraping from the glycerol stock was added to 10 mL of Lysogeny Broth (LB media), a bacteria-specific nutrient-rich solution, in a 25-mL T25 flask with a breathable filter cap  and enriched in a bacteria incubator at 37 °C and 4% CO2 overnight.Ampicillin-resistant BL21(DE3) Gram-negative 10, which was stored at −4 °C for use for up to 1 month. Before each day of bacteriological experiments, a fresh bacterial inoculation was created by adding a single bacterial colony harvested from the agar plate to 10 mL of LB media and incubating overnight at 37 °C. The cell density of the solution was measured the next day using a UV-VIS spectrophotometer  following the OD600 method87; then, a serial dilution was performed to create an inoculation with an initial cell density of ~5 × 105 cfu/mL following cell viability90 and AST91 conventions. LB media was prepared by dissolving LB powder  in deionized (DI) water to make a 25 g/L solution in a 1000-mL autoclave bottle (ThermoFisher Scientific), followed by autoclave sterilization at 120 °C for 25 min. For further details, see Supplementary Material Sec. The following day, ~10 µL of the solution was transferred to agar media to create a solid bacteria colony plateE. coli. For all concentrations of antibiotic stock solutions used throughout this work, see Supplementary Material Fig. Whenever solids were added to liquids or multiple solutions were combined to create new solutions, sterile 50-mL conical polypropylene centrifuge tubes  were used, aggravated using a vortex mixer  for ~30 s to ensure a homogeneous solution, and then wrapped in aluminum foil and stored at −4 °C for up to 1 week of use. Rhodamine fluorescent dye solution was created by adding 50 drops of 0.04% fluorescent red dye Rhodamine B solution  to 50 mL of DI water. Resazurin metabolic indicator solution was prepared to a concentration of ~4.4 mM by adding ~5 mg of resazurin salt powder (Sigma-Aldrich) to 50 mL of sterile LB media. Antibiotic solutions were prepared by combining the required mass of antibiotic powder (each acquired from Sigma-Aldrich) to 500 mL of sterile LB media to create the desired antibiotic stock solution concentration, roughly twice the approximate average of published MIC values for a specific antibiotic against µ-CGG prototype were collected using the experimental setup conceptually illustrated in Fig. Discrete fluidic outputs from the fabricated 3D µ-CGG device was used to generate gradients between one rhodamine fluorescent dye solution and two DI water inputs, and 90 µL of output solution was collected on a 96-well plate. Briefly, using the fluorescence imaging setup  µ-drug cocktail solutions were collected on a 96-well plate, then 30 µL of each resazurin metabolic indicator solution and E. coli inoculation were pipetted into each well. A 0% antibiotic (fully uninhibited proliferation) control consisting of 30 µL each of bacterial inoculation, resazurin solution, and LB media was pipetted into one well, and the no-proliferation control, consisting of 30 µL of resazurin solution and 60 µL of LB media, was pipetted into another well. For further details, see Supplementary Material 69 using a 585-nm optical filter 93. The fluorescence emission of resorufin in each solution was quantified following the image analysis protocol detailed in the Supplementary Material Sec. For each experiment, the 3D Supplementary Materials: 3D Microfluidic Concentration Gradient Generator for Combination Antimicrobial Susceptibility Testing


Resistance to antibiotics is a worldwide concern and community pharmacies can play a strategic role in controlling this issue through rationalizing antibiotic consumption. Considering that dispensing any type of antibiotics without a prescription is prohibited according to Iran’s regulations, this study was conducted to quantify the rate of antibiotic dispensing without a prescription by pharmacists in Tehran, Iran. A descriptive cross-sectional study was conducted from September 2016 through May 2017. Two scenarios of common infectious symptoms including sore throat and dysuria were simulated by pharmacy student in three different regions of Tehran. Each scenario was performed in three levels of demand including requesting for any medicine, asking for a stronger medicine, and direct request for an antibiotic. A total of 388 pharmacy visits were acceptable including 195 and 193 pharmacies for dysuria and sore throat, respectively. Antibiotics were provided in 39.9% of dysuria (67.5% in the first level of demand) and in 52.3% of sore throat (49% in the first level of demand) simulations. The time devoted by the pharmacists to each case was less than 60 second in more than 90% of the cases. The completion of the course of antibiotic therapy was emphasized by pharmacists in only 18% of cases in both scenarios. Our findings revealed that antibiotic dispensing without a prescription is a routine practice in community pharmacies in Tehran, Iran. Unfortunately, patient assessment and evaluation of the symptoms are not performed properly by pharmacists as well.  Since their discovery, antibiotics have been one of the most important groups of medicines and have revolutionized human confrontation with infections. Recent medical achievements, such as organ transplantation and cancer chemotherapy are beholden to the discovery of antibiotics. Besides the benefits of these agents in the treatment of infectious diseases, resistance to existing antibiotics has become a global challenge in recent decades. Unfortunately, the rate of resistance to antibiotics has exceeded the rate of developing new antibiotics , 2. Widespread use of antibiotics leads to microbial resistance and many first-line antibiotics are not effective anymore at usual doses. The results of previous studies suggest that antibiotic resistance leads to prolonged of hospital stay and increases morbidity and mortality in patients with infectious diseases. Moreover, antibiotic resistance imposes a significant financial burden to the population that is exposed .The emergence of antibiotic resistance in Iran has been accelerated by misuse and overuse of antibiotics, which exposes the health care system to serious problems . Antibioet al., in 1976. In the mentioned study, the simulated patient method was used to evaluate drug dispensing without prescription on sore throat scenario in only 30 pharmacies .p-values < c 0.05 were considered as statistically significant. Data analysis was performed using SPSS statistics software version 21.0 .The results are reported as numbers and frequencies. Chi-square test was used to compare the frequency of antibiotic dispensing and frequency of referral to a physician between the two scenarios and evaluate the association between geographic regions and the frequency of antibiotic dispensing. In all analyses, A total of 405 pharmacies were approached in this study. Seventeen pharmacies were excluded as the simulated patient was recognized by the pharmacist or staff. One hundred and ninety-five pharmacies were evaluated using the sore throat scenario and 193 pharmacies using the dysuria scenario. p = 0.008). The distribution of approached pharmacists based on gender was 189 (48.7%) females and 199 (51.3%) males. The estimated age of pharmacists based on appearance was ≥ 30 years in 101 (26%) cases and < 30 years in 287 (74%) pharmacies. Considering all of the 388 pharmacies, the rate of antibiotic dispensing was not significantly different according to gender , but it was considerably different according to two age categories .Antibiotics were sold without a prescription in 102 (52.3%) pharmacies where the sore throat was simulated and in 77 (39.9%) pharmacies where dysuria was simulated. Compared to the sore throat scenario, the frequency of antibiotic dispensing by the pharmacists was significantly lower in the dysuria scenario .Forty-four of 195 (22.8%) and 122 of 193 (62.8%) cases were referred to a physician in sore throat and dysuria scenarios, respectively. Our findings revealed that the rate of referral to a physician was considerably higher in the dysuria scenario compared to sore throat . The comparison of antibiotic dispensing in three different levels of demand between the two scenarios is shown in (The rate of dispensing antibiotics at the first level of demand was similar in both scenarios . At the shown in .p = 0.030). The community pharmacies in each region and the frequency of antibiotic dispensing are shown in The rate of antibiotic dispensing in the five geographical regions was significantly different in the sore throat scenario indicating an increasing trend from the northern to southern regions  was the most frequently sold antibiotic followed by azithromycin 250 mg  and cefixime 400 mg . Of 77 pharmacies that sold an antibiotic for dysuria, ciproﬂoxacin 500 mg  and co-trimoxazole 400/80 mg  were the most frequently sold antibiotics and nitrofurantoin was only suggested in one case. Clindamycin vaginal cream 1% and fluconazole capsule 150 mg were each dispensed in one case without any question about the history of sexually transmitted diseases or vaginal discharge.In the sore throat scenario, 122 (62.6%) pharmacies committed to dispensing medicines including antibiotics and non-prescription products. In 102 (52.3%) cases, antibiotics and non-prescription medicines were dispensed simultaneously. Cold candy , diphenhydramine elixir , and herbal products  were the most common non-antibiotic prescription medicines recommended by the pharmacists in sore throat scenario. Taking phenazopyridine or drinking lots of water  were recommended for the alleviation of dysuria by pharmacists who did not dispense antibiotics and did not refer the simulated patient to a physician. In addition, 92 pharmacies (79.3%) did not sell any drugs. p = 0.001). The onset of symptoms, the other therapeutic measures, taken to eliminate the symptoms, the other drugs used by the patient, the history of drug allergy, and the pregnancy/breastfeeding were only asked in less than 5% of the cases. In dysuria, the simulations for which antibiotics were dispensed, supplementary questions such as the presence of fever, as well as hematuria and back pain for distinguishing complicated from uncomplicated UTI were not asked. The dose of antibiotics was mentioned in 69 (89.6%) cases of dysuria and 70 (68.6%) cases of sore throat. Moreover, the duration of treatment was only mentioned by 14 (18.2%) and 19 (18.6%) cases for dysuria and sore throat, respectively. In more than 90% of the cases (374 0f 388), the time devoted to counseling by the pharmacist was less than 60 sec. It also should be noted that only 26 (32.5%) pharmacists in the dysuria and 64 (62.7%) pharmacists in the sore throat scenario that sold an antibiotic asked about other symptoms, which were significantly higher in the sore throat scenario (Concerns about the rate of antibiotic resistance were stated in only 4 cases (1.9%) in whom the pharmacist refused to dispense antibiotics.The most remarkable result of the current study was that nearly half of the pharmacists dispensed antibiotic without prescription in both scenarios (179 out of 388 cases). The findings showed that antibiotics were dispensed and could be easily purchased without a prescription in community pharmacies of Iran, especially for treatment of suspected bacterial symptoms.The simulated patient strategy used in the current study can closely mimic what happens in the real-world situation. Therefore, the results may reliably be extrapolated to the real setting.Similar studies were also conducted in other countries when the global attention about antibiotic resistance had been intensified -18. Despet al. That showed that male staff were more likely to provide antibiotics without a prescription  (We found that pharmacists were less likely to dispense antibiotics in a case of dysuria compared to sore throat. This finding was not in line with the results of a study by Lior s 79.7%) . Althougp = 0.001). Finally, the results of the current study indicated less antibiotic dispensing in the dysuria scenario in parallel with further referral to a physician. These results may be explained to some extent by the complexity of underlying causes of dysuria, leading to the lower tendency of pharmacists to antibiotic dispensing and raise the rate of referral to a physician. The results of the current study revealed that about half of antibiotic dispensing for sore throat and two-third of dispensing for dysuria happened in the first level of demand without any insistence. Even a higher rate of antibiotic dispensing (95%) in the absence of any patient demand, was reported in the study conducted in Saudi Arabia . MoreoveThe findings of the present study suggest that the rate of antibiotic dispensing in the sore throat scenario was considerably affected by geographical region. Perhaps socio-economic differences were involved in its creation. The lower socio-economic level of the southern regions . led to et al., to assess the effect of culture on the pattern of antibiotic use in a city in Belgium and a city in the Netherlands. The result of this study showed that in the Netherlands, upper respiratory symptoms were often considered as a result of the common cold or flu. However, in Belgium, these symptoms were mostly regarded as bronchitis and antibiotics were used to treat them. (The influence of socioeconomic status, culture, and education on the pattern of antibiotic use without a prescription has addressed in various studies , 23. TheIn line with similar studies, Amoxicillin was the most common dispensed antibiotic when a sore throat scenario simulated . SurprisIn more than 90% of the cases, the estimated allocated time to each case by the pharmacist was less than 60 sec and antibiotics were dispensed without sufficient initial assessment. Patient assessment did not follow a correct pattern, so questions about the history of allergy, pregnancy status, onset of symptoms, and patient’s other medications were asked in only less than 5% of the cases. On the other hand, if the simulated patient was properly evaluated, all dysuria cases should have been referred based on the scenario. This was in contrast to the sore throat scenario in which there was no need for referring the patient to a physician and symptom alleviation could be obtained by offering non-prescription medicines. Lack of a systematic approach to the patient to ascertain the need for referral was observed in this study. et al., found that important information such as food-drug interactions, management of missed doses, and contraindications were ignored in more than 50% of the cases (In addition, the type of the provided information for dispensed antibiotics mainly focused on dosing while parameters like the duration and directions for use, precautions, and contraindications and adverse reactions were poorly addressed. Moreover, in both scenarios, completion of the treatment course was emphasized by pharmacists in only 18% of the cases for those who received antibiotics. Similarly, Surur he cases . This caPharmacists participating in the face-to-face interview in Saudi Arabia stated the ease of access to pharmacists compared to other health care provider, patients’ trust in pharmacists, the lack of strong regulatory enforcement, customer pressure, and concerns about the financial survival of the pharmacy are the most common reasons for dispensing antibiotics without prescription through the pharmacies . On the According to the results of this study, it can be claimed that pharmacists are not sufficiently aware of their vital role in reducing the rate of antibiotic resistance by limiting unnecessary uses of antibiotics. It seemed that they are desensitized to dispensing antibiotics and its correlation with increasing the rate of antibiotic resistance. It might be possible to perform knowledge, attitude, and practice studies in future investigations to determine current pitfalls in non-prescription antibiotic dispensing by pharmacists.The results of the current study showed that antibiotics were dispensed easily without a prescription in the community pharmacies in Tehran, capital city of Iran. Reinforcement of existing regulations regarding antibiotics dispensing by regulatory organizations seems necessary in the current situation. It seems that pharmacists need complementary educational courses to execute their crucial role in rationalization of antibiotic use. Sholeh Ebrahimpour contributed in literature search, data analysis, and manuscript revision. Delaram Soltani, Seyed, Hossein Hajimiri, and Zahra Jahangard-Rafsanjani were involved in study design, data collection, and writing manuscript. Aarefeh Jafarzadeh Kohneloo participated in designing and data analysis. All authors read and approved the manuscript.


As acquired immunodeficiency syndrome (AIDS) becomes more widespread, there will be an increasing need for diagnostic AIDS-related neurological syndromes. AIDS-related myelitis is easy to be ignored, and AIDS-related longitudinal myelitis has not yet been reported.A 45-year-old male patient was admitted to our hospital after 3 days of progressive slurred speech and limb weakness. Neurologic examination revealed near-complete four-limb paralysis with dyspnea, dysarthria, and neck rigidity. Contrast-enhanced T2-weighted magnetic resonance imaging showed hyperintensities within the entire spinal cord. Cerebrospinal fluid analysis showed elevated white blood cell count and protein level. He was administered high-dose immunoglobulin and methylprednisolone. There was rapid regression in his symptoms after a month of therapy.This unique presentation of AIDS with longitudinal myelitis involving the entire spinal cord enriches our understanding of the clinical spectrum of this condition. Our case provides essential information for the diagnosis and treatment of longitudinal myelitis in AIDS patients. A 45-year-old man was admitted to our hospital after 3 days of progressive slurred speech and limb weakness accompanied by constipation and urinary retention. He was diagnosed with acquired immunodeficiency syndrome (AIDS) 3 years before presentation by an infectious disease specialist according to the WHO diagnostic criteria . He was 6/L), protein level (65.18 g/L), and glucose level (6.26 mmol/L). His CSF and blood cultures yielded no bacterial, anaerobic, or fungal growth. There were no immunoglobulin M antibodies in the CSF for toxoplasma, rubella virus, cytomegalovirus, or herpes simplex virus. Oligoclonal bands are detected by separation of CSF proteins while not demonstrable in the serum. Magnetic resonance imaging showed hyperintense lesions in the central medulla oblongata , erythrocyte sedimentation rate (79 mm/h), and elevated levels of C-reactive protein (129.53 mg/L), serum aspergillus antigen (1.72), β-D glucan (247.65 pg/mL), and anti-SS-antigen-A/Ro52KD antibodies (173 AU/mL). Serum T-lymphocyte subset analysis showed that T lymphocyte (33.8%), T-helper lymphocyte (7.5%), and CD3 + CD4 + /CD3 + CD8 + (0.33) levels were decreased. Toluidine red unheated serum and cerebrospinal fluid (CSF) tests were positive. Syphilis antibodies were found in serum and CSF samples. Quantification of serum human immunodeficiency virus type 1 RNA using polymerase chain reaction showed an increased RNA level ; however, CSF human immunodeficiency virus RNA was undetectable. CSF analysis showed elevated leukocyte count  involves more than four spinal cord segments and is a rare comorbidity of AIDS. LM is likely to result in severe and disabling sequelae. LM may be caused by systemic lupus erythematosus , JapanesThe findings extend the neuroimaging spectrum of AIDS-related LM. The patient’s symptoms improved rapidly after active immunotherapy. We considered that AIDS-related LM might be related to dysimmunity. There are no reports on AIDS-related LM treatment protocols and regimens. With our experience and the related reports –4, we reThis case enriches our understanding of the treatment and neuroimaging spectrum of LM and AIDS-related central nerve injuries, specifically AIDS-related LM. A more comprehensive understanding of the etiology of LM is needed.