Mytilus californianus, to explore how simultaneous transcript and protein abundance patterns may diverge under different environmental scenarios. Field-acclimatized mussels were sampled from two disparate intertidal sites; individuals from one site were subjected to three further treatments  that vary in temperature and feeding time. Assessing 1519 genes shared between the two datasets revealed that both transcript and protein expression patterns differentiated the treatments at a global level, despite numerous underlying discrepancies. There were far more instances of differential expression between treatments in transcript only (1451) or protein only (226) than of the two levels shifting expression concordantly (68 instances). Upregulated expression of cilium-associated transcripts (likely related to feeding) was associated with relatively benign field treatments. In the most stressful treatment, transcripts, but not proteins, for several molecular chaperones (including heat shock proteins and endoplasmic reticulum chaperones) were more abundant, consistent with a threshold model for induction of translation of constitutively available mRNAs. Overall, these results suggest that the relative importance of transcript- and protein-level regulation (translation and/or turnover) differs among cellular functions and across specific microhabitats or environmental contexts. Furthermore, the degree of concordance between transcript and protein expression can vary across benign versus acutely stressful environmental conditions.Organismal responses to stressful environments are influenced by numerous transcript- and protein-level mechanisms, and the relationships between expression changes at these levels are not always straightforward. Here, we used paired transcriptomic and proteomic datasets from two previous studies from gill of the California mussel,  Summary: The importance of transcript- and protein-level regulation differs across cellular functions and environmental contexts, and transcript and protein expression have higher concordance in stressful conditions. Plasticity in the molecular phenotype, such as changes in transcript and protein abundance, can facilitate tolerance to climate change-induced environmental changes . Yet, moMytilus californianus) is one such sessile invertebrate with a long planktonic larval duration, high gene flow and relatively high genetic homogeneity across its range  can global transcriptome and/or proteome profiles differentiate ecologically relevant treatment conditions; (2) are treatment differences in the transcriptome consistently reflected in the proteome, or is protein expression more tightly coupled to transcript expression for particular cellular functions or in certain conditions; and (3) which components of the transcriptome and/or proteome are most correlated with physiological measurements on the same tissue?This objective of this study was to investigate the effects of acclimatization to distinct intertidal habitats on transcript and protein expression in The treatments and individual mussel samples for this study are the same ‘baseline’ samples as those described in detail in Mytilus californianus Conrad 1837 mussels (60–70 mm in shell height) were sampled from five field and lab treatments in 2014 (n=9) and a warm wave-protected site (n=9) at Hopkins Marine Station in Pacific Grove, CA, USA . These field-acclimatized mussels were compared between the two field sites. For this study, only the three subsequent treatments pertaining to mussels collected from the wave-protected site were used, because their physiological differences across treatments were more robust (n=5) and kept constantly submerged in 13.5±1°C seawater supplied from Monterey Bay. Mussels were fed daily with a commercial bivalve diet  to supplement the natural food in the seawater. Some individuals were sampled at the end of this common garden period, and two other treatment groups were placed back into the field (i.e. they were outplanted) at either a cool low intertidal site (n=8) or a hot high intertidal site (n=10) for an additional month before they were sampled. All individuals were sampled at a comparable point in the tidal cycle, just as tides were receding from the field sites, to avoid confounding factors from rhythmic gene expression  and quality  using CLC Genomics Workbench 9.0. For PE150 sequencing, reads shorter than 60 bp were discarded; for PE50 sequencing, reads shorter than 30 bp were discarded. PE150 reads were used to construct a assembly . Using tassembly  were usede novo M. californianus transcriptome described above was translated to amino acid sequences, and these sequences were used as a reference database for matching proteins to MS/MS spectra. Protein IDs obtained from the bioinformatic search engines Mascot 2.2.7, X!Tandem Alanine, PEAKSC and Byonic 2.12 were considered valid if: (1) matches were based on at least two unique peptides, (2) matches met a protein false-discovery rate (FDR) of 1%, and (3) matches met a peptide FDR of 0.1%. The PCAmethods package in the R Bioconductor suite  EFA versus PFA (origin site effect), (2) PFA versus PCG (response to a benign environment with continuous immersion), (3) PCG versus POL , (4) PCG versus POH  and (5) POL versus POH . We excluded all comparisons of exposed field acclimatized (EFA) versus protected common garden (PCG), protected outplant low (POL) and protected outplant high (POH) groups, because they involved both a difference in treatment and mussels from different origin sites. We also excluded protected field acclimatized (PFA) versus POL and POH treatments because they were not ‘sequential’ according to the experimental design (e.g. PFA groups were put in a common garden first before outplanting).www.CRAN.R-project.org) according to default parameters. Briefly, trimmed mean of M-values (TMM) normalization was performed to eliminate composition biases between treatment group libraries .In addition to examining which contigs were differentially expressed at the transcript and protein level, results were further examined to determine whether contigs that were DE changed expression levels in the same direction . To determine whether genes with certain functions were more commonly found to mirror expression changes at both the RNA and protein level, four different groups of genes were identified: (1) those that are DE in both datasets and share the same direction of expression change; (2) those that are DE at both levels but change expression in opposite directions; (3) those that are DE at the protein but not the transcript level; and (4) those that are DE at the transcript but not the protein level. These four gene lists were generated for each of the five treatment pair comparisons. Functional enrichment analyses were then performed on each of the 20 resulting gene lists.We gained further insight into transcript and protein expression patterns and how they correlate with three physiological measures of antioxidant capacity in gill tissue from the same individuals  using thhttps://horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/) to identify genes most significantly correlated with the trait of interest (gene significance >0.4) that also have high module membership (module membership >0.8). These genes are the most influential, ‘central’ elements of the modules associated with each physiological trait.Modules of genes that were significantly correlated with mussel gill catalase enzyme activity against hydrogen peroxide [international units (IU) per gram fresh tissue mass (g FM)] and antioxidant activities against peroxyl  and hydroxyl radicals [arbitrary units (au) per gram of tissue] in RNA expression and protein expression data were identified using the module eigenvalues. Blast2GO enrichment analyses were used to detect any functional GO categories that were overrepresented in these modules that had a significant correlation with physiological metrics. Further intramodular analysis was performed following conservative WGCNA recommendations (n=446) were functionally enriched for unfolded protein binding, protein catabolic processes and response to heat; genes negatively correlated with PC1 (n=403) were functionally enriched for cell projections, cilium organization and assembly, and microtubule-based processes  EFA versus PFA, (2) PFA versus PCG, (3) PCG versus POL, (4) PCG versus POH and (5) POL versus POH, respectively (5% FDR correction). The two comparisons including the stressful POH treatment revealed the highest numbers of DE transcripts . For thePatterns of DE in the transcriptome were not consistently mirrored in the proteome. Overall, 29, 33, 49, 77 and 132 DE proteins were found in the five treatment comparisons (5% FDR correction). Except for the EFA versus PFA comparison, the number of DE proteins was much lower than the number of DE transcripts. As above for the DE transcripts, the two comparisons including the stressful POH treatment revealed the highest number of DE proteins . No funcP<0.0001 in each case).For each of the five treatment comparisons, the total number of transcripts and proteins whose expression differed was significantly different  56 unique genes  were DE at both levels and shared the same direction of expression change, (2) 25 unique genes (28 instances) were DE at both levels but changed expression in opposite directions, (3) 177 unique genes (226 instances) were DE at the protein but not the transcript level, and (4) 853 unique genes (1451 instances) were DE at the transcript but not the protein level ], the directions of expression changes matched between the two levels . CompariA larger group of 177 unique genes fell into the third set and were regulated at the protein but not the transcript level in at least one treatment comparison. Although no GO terms were significantly enriched in any treatment comparison, many genes in this group are involved in maintenance functions. Four cell division proteins (two contigs from the PCG versus POH comparison plus two contigs from the POL versus POH comparison) are involved in the mitotic cell cycle and mitotic spindle/sister chromatid organization. Immunity-related proteins include six C1q domain containing contigs  and four myticin c contigs (one from the PCG versus POH comparison and three from POL versus POH comparison). Lastly, proteins involved in ribosome formation and translation include three 60S ribosomal proteins (one from the PCG versus POH comparison and two from the POL versus POH comparison) and eight 40S ribosomal proteins (four from the PCG versus POH comparison and four from the POL versus POH comparison).Patterns of functional enrichment were more complex for the much larger set of genes whose transcript levels changed significantly without a corresponding change in protein abundance. Only one unannotated gene met the criteria for the EFA versus PFA comparison, and no functionally enriched GO categories were identified for the PFA versus PCG comparison (184 genes). For the PCG versus POL comparison, the 146 genes in set 4 had overrepresentation of nine biological process GO terms related to regulation of biosynthetic and metabolic processes . These gA total of 541 genes out of the 1519 examined (35.6%) remained stably expressed at the transcript and protein level across all five treatments (i.e. no DE). This set of genes was not functionally enriched for any GO terms, but 34 genes were identified as structural constituents of the ribosome  and 20 were identified as integral components of the membrane . We identified 46 genes with high gene significance (>0.4) and high TR2 module membership (>0.8) , and the TR9 module contained 13 contigs with high gene significance and high module membership. These central module contigs represented functions such as calcium ion binding  were significantly positively correlated with catalase activity, while the TR1 and TR7 modules were negatively correlated with catalase. The TR2 module contains 205 genes, and average expression of genes in this module was highest in the PFA and EFA groups and lowest in the POH group. Contigs in the TR2 module had overrepresented GO terms related to the microtubule cytoskeleton, cilium assembly and movement . There wp (>0.8) : six of R=0.65, P<2.2e-16). Forty-eight genes in this module had high gene significance and high module membership, including HSP10, HSP30, HSP70, two dnaj homologs and two 78 kDa glucose-regulated proteins . Twelve central module contigs with high gene significance and high module membership included membrane-related proteins such as vesicle-associated membrane protein-associated protein A, charged multivesicular body protein 4b, erlin-2 and Saposin-related isoform A (Two other transcript modules (TR1 and TR4) were significantly positively correlated with anti-peroxyl radical capacity. The TR1 module contained 355 genes, was functionally enriched for GO terms related to unfolded protein binding and protein folding, and had highest average expression in the POH treatment. There is a significant correlation between a gene's significance for anti-peroxyl radical capacity and its membership in the TR1 module . Both the PR5 and PR8 modules had a significant correlation between module membership and gene significance for anti-peroxyl radical capacity . Finally, the PR11 protein module was the only module across transcript and protein significantly positively correlated with anti-hydroxyl radical capacity, and it was functionally enriched for structural constituents of the ribosome and translation GO terms.Although one or more protein modules were correlated with each of the physiological metrics, none of the genes in these protein modules had module memberships above 0.8 to meet criteria as important module nodes . SpecifiM. californianus. Our results clearly indicate plasticity in the molecular phenotype of adult mussels moved between environmental contexts. This plasticity was observed at the global level using PCA, within correlated modules of genes, and in differential expression of specific gene products. Both transcriptomic and proteomic data similarly differentiated our treatments, mirroring the separation of treatments observed in multivariate space for nine physiological metrics of antioxidant capacity from the same individuals  are downregulated in POH relative to all other treatment groups. Although a decrease in ribosomal density could prevent the high number of stress-response transcripts from being translated before they are required , it is lM. californianus  treatment group showed a clear separation from all other treatments in multivariate space, which is likely driven at least in part by the large differences in maximum temperatures between the POH and outplant low (POL) sites . MoreoveM. galloprovincialis, in which different HSP contigs are upregulated under unique conditions , HSP70, HSP75 and HSP90] are expressed differently across microhabitats. Notably, different isoforms seem to be used in microhabitats that vary in the degree of temperature stress. For example, two different HSP90 and dnaj subfamily member B isoforms were significantly differentially expressed between the POL and the POH treatment groups. However, for both genes, one of these isoforms was more highly expressed in the POL group, and the other was more highly expressed in the POH group. Similar results have been found in other marine invertebrates such as sea anemones and ons e.g. .Our results also illustrate the importance of the endoplasmic reticulum (ER) chaperone system in coping with stressful microhabitats. This stress pathway performs quality control for the folding of newly synthesized proteins translocated into the ER . For exaIn general, major disparities in the transcriptome and proteome were identified in these expression datasets, mirroring previous findings . AlthougHowever, employing a proteomics approach does entail challenges. Proteomics methods are noisier than transcriptomic methods , and thiTaken together with those caveats, the present results suggest the possibility of ‘false positives’ when attempting to infer from RNA-seq transcript expression levels to protein abundance, with a comparatively small set of ‘false negatives’. However, this ratio appears to be treatment dependent. For example, 348 unique transcripts were shared in set 4 (genes DE at the transcript but not protein level) between the comparisons involving the POH treatment; this list includes numerous HSPs and other genes involved in the cellular stress response. In contrast, only 13 unique proteins were DE without a corresponding change in transcript expression (set 3) in the same comparisons involving POH. Contrasting these numbers with those for the EFA versus PFA comparison  clearly illustrates the shifting state of transcriptional and translational regulation across treatments. For example, the higher number of DE proteins versus transcripts in the EFA versus PFA comparison suggests that acclimatization to temperature variation caused by presence or absence of wave splash involves post-transcriptional mechanisms. Additional data are needed to clarify these patterns and to examine their generality across species and experimental conditions.The stronger relationship between transcript and protein expression in the POL and POH treatments could be related to preferential translation of proteins needed under stressful conditions  or a higAs for the univariate analyses, the WGCNA approach identified unique co-expression patterns within the transcriptomic and proteomic datasets. Overall, we identified transcript and protein co-expression modules that were correlated with physiological data from the same individuals. However, important disparities exist between the transcript and protein expression data. For example, the PR11 protein module that was significantly enriched for ribosome and translation GO terms was significantly correlated with anti-hydroxyl radical capacity, but no transcript modules were correlated with this physiological trait. In addition, two transcript modules (TR1 and TR4) and two protein modules (PR5 and PR8) were significantly positively correlated with anti-peroxyl radical capacity. The transcript modules are enriched for unfolded protein binding and Ras protein signal transduction, respectively; however, the protein modules that correlated with this same physiological metric are composed of fewer genes, have no enriched GO terms, and exhibit lower module membership values. Similar patterns emerge for transcript and protein module correlations with catalase enzyme activity. These results further demonstrate that the transcriptome and proteome datasets supplement each other and together identify candidate genes related to physiological metrics that would be missed if only one of the two datasets was used.M. californianus to distinct microhabitats. Specifically, acclimatization to the outplant intertidal sites involves a higher number of genes with strong correlation between transcript and protein expression, along with constitutive transcript (but not protein) expression of select molecular chaperones involved in the unfolded protein response. Variations in temperature and feeding time affect transcript expression patterns. Genes required for general maintenance functions such as immunity and cell division seem to be regulated at the protein level. Co-expression modules of transcript and protein are correlated with physiological metrics, but the co-expression structure differs markedly between transcriptome and proteome. Overall, our results provide further insight into how intertidal invertebrates survive in their extremely variable environment and illustrate the additional information gained by conducting studies assessing multiple levels of gene expression regulation.This study suggests that transcriptional and protein-level regulation play unique roles in the acclimatization of the intertidal mussel 10.1242/jexbio.245962_sup1Supplementary informationClick here for additional data file.Table S1. The contig identity and Gene Ontology (GO) category or categories of all genes with a significant correlation between their normalized expression values and Dimension 1 or 2 of the transcript and protein PCAs in Fig. 1.Click here for additional data file.Table S2. Overrepresented Gene Ontology (GO) functions and corresponding normalized expression values in each treatment (average±b 1 SEM) for differentially expressed transcripts in the (i) PCG v. POL, (ii) PCG vs. POH, and (iii) POL vs. POH treatment comparisons. No overrepresented functions were identified for the EFA vs. PFA and PFA vs. PCG comparisons and thus no genes from those comparisons are presented here. The treatment group written first in each comparison is the reference, and asterisks on individual gene names indicate transcripts that showed significantly higher expression  in the reference group.Click here for additional data file.Table S3. List of gene names and associated Gene Ontology (GO) terms for those contigs that were 1) differentially expressed (DE) at both the RNA and protein level and share the same direction of expression change (aka match); 2) those that are DE at both levels but change expression in opposite directions (aka mismatch); 3) those that are DE at the transcript but not the protein level; 4) those that are DE at the protein but not the transcript level; and 5) stably expressed at both the RNA and protein level (no DE) in each of the five treatment groups. The treatment group written first in each comparison is the reference. Upregulated = higher expression in the reference group; downregulated = lower expression in the reference groupClick here for additional data file.Table S4. Contigs in WGCNA transcript and protein modules that are significantly correlated with catalase enzyme activity at the transcript (TR2 and TR9) or protein level (PR6), with anti-peroxyl radical capacity at the RNA transcript (TR1 and TR4) or protein level (PR5 and PR8), and with anti-hydroxyl radical capacity at the protein level (PR11). Annotation information, gene significance with each respective physiological metric, and module membership values are provided for each contig.Click here for additional data file.


Here, we provide a summary and review of the use of molecular response for the prediction of outcomes in patients with advanced cancer, including the current state of science, its application in clinic, and next steps for the development of this predictive tool.The therapeutic landscape for patients with advanced malignancies has changed dramatically over the last twenty years. The growing number of targeted therapies and immunotherapeutic options available have improved response rates and survival for a subset of patients, however determining which patients will experience clinical benefit from these therapies in order to avoid potential toxicities and reduce healthcare costs remains a clinical challenge. Cell-free circulating tumor DNA (ctDNA) is shed by tumor cells into systemic circulation and is already an integral part of routine clinical practice for the non-invasive tumor genotyping in advanced non-small cell lung cancer as well as other malignancies. The short half-life of ctDNA offers a unique opportunity to utilize early on-treatment changes in ctDNA for real-time assessment of therapeutic response and outcome, termed  Based upon an improved understanding of the biology of cancer, we have witnessed a paradigm change in the management of patients with advanced malignancies. Treatment options including targeted therapies for oncogene-addicted tumors and checkpoint inhibitors have contributed to significant improvements in quality of life, toxicity and overall survival outcomes for patients. Despite these improvements, responses are often limited to a subset of patients, and additionally, the efficacy of targeted therapies is limited by the emergence of resistant populations of tumor cells , 2. Detemolecular response. The use of molecular response is a promising approach to guide therapeutic decisions early in the course of treatment and as an emerging biomarker with broad implications for integration in the clinic and design of clinical trials. Here we provide a summary and review of molecular response for the prediction of outcomes in patients with advanced cancer, including the current state of science, its application in clinic, and next steps for the development of this predictive tool.Cell-free circulating tumor DNA (ctDNA) is shed by tumor cells into systemic circulation. With advancements in sequencing technologies offering the ability to detect mutations, plasma genotyping of ctDNA is increasingly being utilized to identify driver mutations and match patients to the appropriate personalized therapies. The use of ctDNA offers the advantage of being minimally invasive, can be repeated sequentially, and may provide a more systemic view of tumor clonal evolution over time . Plasma Plasma cell-free DNA (cfDNA) consists of short DNA fragments (about 150–200 base pairs) secreted or released into the bloodstream through apoptosis or necrosis with a half-life of approximately one to two hours in circulation . The majAssessment of molecular response involves the measurement of ctDNA kinetics between baseline ctDNA VAF and a prespecified early on-treatment timepoint to identify therapeutic response and predict patient outcomes Table . While tEGFR L858R or EML4-ALK fusion). Measurement of ctDNA clearance is easy to calculate, and has been shown to effectively predict outcomes in a number of studies across multiple tumor types including NSCLC, breast cancer, and others [Various methods of measuring ctDNA molecular response have been published and can be grouped into three main categories: 1) ctDNA clearance, 2) delta VAF, and 3) proportion or ratio VAF methods. Clearance of ctDNA is a simple binary assessment of the presence or absence of detectable ctDNA at the early on-treatment timepoint in patients with detectable ctDNA at baseline. It may refer to the reduction in all baseline somatic variants below the limit of detection at an early on-treatment timepoint, or in the setting of receipt of targeted therapy, will refer to the clearance of the specific driver mutation(s) being targeted , which is measured as an increase or decrease in ctDNA VAF between baseline and an on-treatment timepoint has also been used, and demonstrated utility across several studies as a predictor of outcomes –26. TherThe proportional change, percent change, ratio VAF methods can help account for both the relative change in ctDNA as well as the amount of residual ctDNA remaining at the on-treatment timepoint , 29, 30.EGFR mutant NSCLC in trials of gefitinib, afatinib, osimertinib, and multiple other EGFR TKIs [EGFR mutant NSCLC. In this study, patients with clearance of the respective EGFR exon 19 deletion or L858R mutation at three weeks on therapy had a longer PFS than those without clearance of the EGFR mutation  compared to those without ctDNA clearance [ALK-positive NSCLC [ALK variants detected by NGS at week six in a cohort of 57 patients receiving second-line lorlatinib therapy [p = 0.0011), while patients with stable or progressive disease did not. In addition, patients with dVAF < 0 had better PFS  and OS  compared to those with dVAF ≥ 0 [RET altered NSCLC treated on the phase 1 trial of selpercatinib, 44% of patients had clearance of the RET driver alteration detected by NGS at cycle one (day 15) and 79% had at least a 50% decrease in ctDNA, supporting the clinical activity of the drug [MET exon 14 skipping driver mutation at six weeks on therapy with tepotinib was assessed in 51 participants of the VISION trial using NGS [MET VAF from baseline) had a complete/partial radiographic response and six patients had stable disease resulting in a disease control rate of 88% [Accurate therapeutic response assessment using these methods has also been observed in studies of other targeted therapies in NSCLC. In 51 patients with learance . Soo andve NSCLC . In both therapy . PatientdVAF ≥ 0 . In 34 pthe drug . Percent one day  and 79%  one day  and 79% p < 0.001) and improved PFS  and OS  compared to molecular non-responders) [p = 0.013) [Molecular response assessed by NGS has also demonstrated an association with radiographic response and clinical outcomes in NSCLC patients receiving immune checkpoint inhibitors (ICI) , 41–44 A= 0.013) . In an a= 0.013) . A recen= 0.013) .p = 0.0486), PFS 123 versus 66 days (p = 0.029) [HER2-positive gastric cancer with clearance of ctDNA assessed by NGS at nine weeks on combination pembrolizumab and trastuzumab had improved PFS compared to those without ctDNA clearance  [n = 23) assessed by NGS had superior OS than those with <50% decline in max VAF  [There have been a number of studies published supporting the ability of ctDNA molecular response assessment to predict clinical outcomes in patients with metastatic gastrointestinal cancer treated with immunotherapy or targeted agents Table . In a st= 0.029) . Maron e = 0.02) . In anot0.1–0.8) .KRAS mutated metastatic colorectal cancer (mCRC), patients with clearance of the KRAS mutation detected by ddPCR at four weeks on treatment with chemotherapy and bevacizumab were found to have improved overall survival compared to those without ctDNA clearance on therapy  [BRAF V600E mutant mCRC treated with vemurafenib, cetuximab, and irinotecan, clearance or near clearance (>90% decrease) of the BRAF mutation detected by ddPCR or NGS at six weeks on therapy correlated with RECIST response [BRAF V600E mutant mCRC, 87% of patients in the vemurafenib arm had a reduction in the BRAF V600E VAF (percent change) detected by NGS, while none of the patients in the control arm  had a ctDNA decrease. Disease control in the vemurafenib arm was significantly longer compared to the control arm 65% vs 21%, p < 0.01), respectively [p < 0.0001) and patients with >30% decrease in ctDNA had a longer PFS  [HER2 amplified mCRC, a decrease in the ctDNA fraction at 3 weeks was associated with superior PFS  and OS  [In 35 patients with < 0.001) . In 12 presponse . Similarectively . Parikh  0.0001) . In an a12–0.82) .PIK3CA mutation level was assessed using ddPCR; PALOMA-3 assessed cycle 2 day 1 while BEECH tested multiple time points, determining day 28 was optimal. Both studies found significantly improved PFS in patients with a reduction in PIK3CA ctDNA levels on treatment [p = 0.025) than molecular non-responders  [A growing number of studies have found an association between molecular response and outcomes in patients with metastatic breast cancer (mBC) treated with various therapies that are either investigational or are now standard of care. In an early proof of concept study involving 30 patients with metastatic breast cancer, ctDNA was detected at baseline in 97% of patients and ctDNA dynamics correlated with changes in tumor burden and outcome . Since treatment , 51. RecBRAF V600 mutated metastatic melanoma, an early study correlated change in ctDNA copies per milliliter at four to eight weeks on treatment with response to MAPK inhibition  or immunotherapy  [p = 0.0071). One of the two non-responders also had a 10-fold reduction in ctDNA and had stable disease for >6 months. Conversely, only four of 15 patients treated with immunotherapy responded and there was very little decrease in ctDNA observed in these patients [p < 0.01) and best overall response p = 0.024 [In lizumab) . Eight opatients . Two othpatients , 54. A s = 0.024 . More re = 0.024 .Multiple studies have also correlated early on-treatment ctDNA changes with response to various immunotherapies across several advanced solid tumors , 57, 58.STK11 or KEAP1 mutations, to tumor mutation burden (TMB) improves prediction of response to pembrolizumab [Studies published to date highlight the potential use of ctDNA molecular response to predict treatment response and long term outcomes to both targeted agents and immunotherapies across a variety of tumor types. This promising tool may facilitate early re-stratification of patients at high-risk of treatment failure to other effective therapies and at the same time, avoid the potential toxicity of treatment in patients exhibiting a molecular response Fig. . Such anolizumab . Such anIn summary, ctDNA molecular response assessment has been shown to predict outcomes in a number of studies across solid tumors and in patients receiving a broad spectrum of therapeutic strategies. This technology has great potential for use to inform more timely treatment decisions in clinical care and facilitate more efficient clinical trials as a shorter term endpoint, however further validation of the use of molecular response to predict response and outcomes is necessary prior to its integration into clinical practice. Further, prospective trials employing this tool to trigger intervention are needed to establish the clinical utility of molecular response in these scenarios. With these efforts, the promise of ctDNA molecular response as an early on-treatment biomarker may be fully realized.


Staphylococcus aureus (MRSA). The extracellular matrix of MRSA biofilms contains significant amounts of double-stranded DNA. MRSA cells also secrete micrococcal nuclease (Nuc1) which degrades double-stranded DNA. In this study we used a nuc1 mutant strain to investigate the role of Nuc1 in MRSA biofilm formation and dispersal. Biofilm was quantitated in microplates using a crystal violet binding assay. Extracellular DNA (eDNA) was isolated from colony biofilms and analyzed by agarose gel electrophoresis. In some experiments, broth or agar was supplemented with sub-MIC amoxicillin to induce biofilm formation. Biofilm erosion was quantitated by culturing biofilms on rods, transferring the rods to fresh broth, and enumerating CFUs that detached from the rods. Biofilm sloughing was investigated by culturing biofilms in glass tubes perfused with broth and measuring the sizes of the detached cell aggregates. We found that a nuc1 mutant strain produced significantly more biofilm and more eDNA than a wild-type strain in both the absence and presence of sub-MIC amoxicillin, nuc1 mutant biofilms grown on rods detached significantly less than wild-type biofilms. Detachment was restored by exogenous DNase or a wild-type nuc1 gene on a plasmid. In the sloughing assay, nuc1 mutant biofilms released cell aggregates that were significantly larger than those released by wild-type biofilms. Our results suggest that Nuc1 modulates biofilm formation, biofilm detachment, and the sizes of detached cell aggregates. These processes may play a role in the spread and subsequent survival of MRSA biofilms during biofilm-related infections.Biofilm formation is an important virulence factor for methicillin-resistant  Staphylococcus aureus is a major public health burden in both community and hospital settings . In hospitals, S. aureus is a major cause of infections associated with implanted medical devices because of the ability of most S. aureus strains to form biofilms on biomaterial surfaces . S. aureus biofilms on implanted devices can act as a nidus of infection when cells detach from the biofilm and seed other parts of the body such as the blood, lung, or bladder , a multidrug-resistant strain that further complicates treatment of these common nosocomial infections . eDNA consists of genomic DNA released by lysis of a subpopulation of cells within the biofilm (nuc1 mutant strains contain more eDNA than wild-type biofilms (nuc1 mutant strains (nuc1 is repressed during biofilm formation (Extracellular DNA (eDNA) is the most common component of biofilms produced by most MRSA strains . Bacterial inocula were prepared in sterile broth from 24-h-old agar colonies and then passed through a 5-μm pore-size syringe filter to enrich for single cells as previously described  were transferred to the wells of a 6-well microtiter plate  , and the plate was incubated statically under conditions of low vibration as previously described . After 1Aliquots of inocula  were transferred to the wells of a 96-well microtiter plate (Corning #3599) containing 20 μl of antibiotic dissolved in water at a concentration equal to 10 times the desired final concentration. Control wells were filled with 180 μl of inoculum and 20 μl of water, or 180 of sterile broth and 20 μl of water. The plates were incubated for 18 h. The amount of biofilm biomass in each well was quantitated by rinsing the wells with water and staining for 1 min with 200 μl of Gram’s crystal violet. The wells were then rinsed with water and dried. The bound crystal violet was dissolved in 200 μl of 33% acetic acid and quantitated by measuring the absorbance of the wells at 595 nm.Extracellular DNA was isolated from lawns of bacteria cultured on agar, also known as colony biofilms, using the method described by An apparatus to measure biofilm erosion under static conditions  was consAn apparatus to measure biofilm sloughing under flow conditions  was consBiofilm erosion assays were performed in triplicate tubes and were repeated 2–6 times. The significance of differences between percent CFUs in broth values was calculated using a 2-tailed Student’s t-test for pairwise comparisons, and a one-way ANOVA with Tukey’s post hoc analysis for comparison of more than two groups. A P value of <0.05 was considered significant. Biofilm erosion assays were performed in duplicate reactors and were repeated twice. The significance of differences between particle size distributions in the sloughing assay was calculated using a two-way Chi squared test.nuc1−, nuc2− and nuc1−/nuc2− strains were cultured in broth in 6-well microtiter plates at low densities and under conditions of low vibration. After 18 h, the wells were photographed  than the wild-type and nuc2− strains. All four strains produced high molecular weight DNA in the presence of low-dose amoxicillin (To inves)gate the role of Nuc1 and Nuc2 in USA300 eDNA production, we isolated eDNA from colony biofilms of wild-type, phoresis . The nucxicillin , which sxicillin , but posxicillin  and antixicillin .nuc1− biofilms on polystyrene or glass rods, transferring the rods to fresh broth, and enumerating the CFUs that detached from the rods after 2 h (nuc1− mutant biofilms (68% of CFUs in broth for wild-type versus 38% for nuc1− mutant). The same erosion phenotype was observed when biofilms were cultured on glass rods (76% CFUs in broth for wild-type versus 38% for nuc1−)  . Greaterth DNase , or whenplasmid. . These fnuc1− mutant biofilms in glass tubes perfused with fresh broth and measured the number of CFUs and sizes of detached cell aggregates in the eluate /(broth CFUs + rod CFUs) × 100. us DNase  or a wil plasmid , confirmnuc1 mutant strain produced significantly more biofilm than the wild-type strain as evidenced by a greater amount of crystal violet-stained material in the silicone tubes leading into the reactors , and a significantly greater number of CFUs in the eluate over time . nuc1 mutant biofilms released cell aggregates that were significantly larger than those released by wildtype biofilms , and capable of initiating colonization of endothelial cell layers under flow .Our results demonstrate that Nuc1 modulates biofilm erosion and sloughing in fluenzae . Since efluenzae , it is p


Aerosolized lung surfactant therapy during nasal continuous positive airway pressure (CPAP) support avoids intubation but is highly complex, with reported poor nebulizer efficiency and low pulmonary deposition. The study objective was to evaluate particle size, operational compatibility, and drug delivery efficiency with various nasal CPAP interfaces and gas humidity levels of a synthetic dry powder (DP) surfactant aerosol delivered by a low-flow aerosol chamber (LFAC) inhaler combined with bubble nasal CPAP (bCPAP). A particle impactor characterized DP surfactant aerosol particle size. Lung pressures and volumes were measured in a preterm infant nasal airway and lung model using LFAC flow injection into the bCPAP system with different nasal prongs. The LFAC was combined with bCPAP and a non-heated passover humidifier. DP surfactant mass deposition within the nasal airway and lung was quantified for different interfaces. Finally, surfactant aerosol therapy was investigated using select interfaces and bCPAP gas humidification by active heating. Surfactant aerosol particle size was 3.68 µm. Lung pressures and volumes were within an acceptable range for lung protection with LFAC actuation and bCPAP. Aerosol delivery of DP surfactant resulted in variable nasal airway (0–20%) and lung (0–40%) deposition. DP lung surfactant aerosols agglomerated in the prongs and nasal airways with significant reductions in lung delivery during active humidification of bCPAP gas. Our findings show high-efficiency delivery of small, synthetic DP surfactant particles without increasing the potential risk for lung injury during concurrent aerosol delivery and bCPAP with passive humidification. Specialized prongs adapted to minimize extrapulmonary aerosol losses and nasal deposition showed the greatest lung deposition. The use of heated, humidified bCPAP gases compromised drug delivery and safety. Safety and efficacy of DP aerosol delivery in preterm infants supported with bCPAP requires more research. Each year 1 million preterm infants die from respiratory distress syndrome (RDS) in low- and middle-income countries (LMICs) due to a lack of respiratory support devices and surfactant therapy . SurfactAerosolized surfactant with small, inhaled particles generated by a nebulizer has been shown to produce more uniformly distributed medication deposition deep into the lungs  and feweDry powder (DP) surfactant aerosol could provide higher pulmonary aerosol concentrations over a shorter dosing period than nebulized liquid surfactant. DP surfactant aerosol combined with an inhaler in-line with a manual resuscitator and ventilator was reported over 40 years ago in preterm infants with RDS by Morley et al. ,13. HoweWe conducted pre-clinical in vitro studies with the DP surfactant aerosol delivery system (LFAC) described by Walther et al.  to evalu® pulmonary DP technology to formulate the B-YL: Trehalose surfactant by adding 49 weight% of DPPC and 21 weight% of POPG-Na , 25 weight% of the excipient Trehalose , 3 weight% of B-YL, and 2 weight% of NaCl (Sigma-Aldrich) to the organic solvent used for spray-drying. Micronized surfactant particles were produced using GEA Niro PSD-1 or  or Buchi B-290 mini  spray-dryers. After spray-drying, the DP B-YL: Trehalose surfactant was loaded into size 00 capsules (30 mg per capsule) and packaged in heat-sealable pouches with desiccant. The surface activity of the B-YL: Trehalose surfactant was quantified using captive bubble surfactometry. All reagents were stored at 7 °C and 40% humidity and kept in blister packaging before testing.B-YL, a 41-amino acid peptide mimic of surfactant protein B (SP-B) , was synThe B-YL: Trehalose surfactant was aerosolized with a low-flow aerosolization chamber (LFAC) designed by Acorda Therapeutics Inc., based on simplicity of design and use, minimum number of parts, and low cost of goods and manufacturing. The LFAC is a cylindrical chamber/inhaler with several holes at one end that accommodates a perforated DP capsule and dispenses aerosol into the inhalation pathway via the nasal prong interface . The LFA® silicone spray, dried, and weighed with an AX205 Delta Range Lab balance . A vacuum and frit resistor  was used to maintain nominal impactor flow (15 L/min) and confirmed with a TSI 5200 Flow and Pressure Analyzer . An NGI leak test was performed per the manufacturer’s specifications before testing. The LFAC was pre-loaded with a punctured B-YL: Trehalose capsule (30 mg), and the bellows was actuated 50 times. Aerosol was dispersed from the LFAC outlet adaptor and injector tubing into the NGI inlet. Following nebulization runs (n = 6), the aerosol drug mass (µg) deposited on the staged filters was weighed with a balance. MMAD and geometric standard deviation (GSD) were calculated based on gravimetric changes in filter mass (µg) delivered to the different NGI stages following nebulization. The fine particle fraction (FPF) was the proportional aerosol mass delivered to the NGI with an MMAD <5.4 µm. Particle size analysis was performed with Inhalytics software version 1.0 , which is 21 CFR Part 11 compliant and meets the requirements of United States Pharmacopoeia (USP) 43 and European Pharmacopoeia (Ph Eur) 10.0.A multi-staged next-generation impactor  was used to characterize aerodynamic particle size distribution (APSD) and classify the DP surfactant into respirable size fractions. The impactor uses seven individually staged gravimetric particle trays that are recessed to accommodate a 45 mm glass fiber (GF) filter substrate  for aerosol collection. GF filters were pre-conditioned with Molykote2O, airway resistance of 100 cm H2O/L/s, respiratory rate of 50/min, and VT of 8 mL (~7 mL/kg) [A neonatal nasal airway and lung model was configured to generate the realistic, spontaneous breathing parameters of a 1200 g preterm infant affixed with a realistic nasal airway cast, bCPAP, surfactant delivery system, and different nasal CPAP interfaces see . A high-7 mL/kg) ,19. A 3D2O, and system flow was adjusted (6–8 L/min) to maintain constant bubbling throughout the respiratory cycle. Before testing, bCPAP system pressures and flows were confirmed with a calibrated analyzer . Bubble CPAP is widely used in LMICs, and pressure is created with the underwater seal . The bCPn = 3), and cannula prototypes (n = 2) specifically designed for DP aerosol delivery. The RAM Cannula  and Hudson Prongs  were adapted to streamline aerosol delivery with bCPAP. The RAM cannula  with an internal channel designed to separate the fluidic paths of aerosol from CPAP flow at the nasal interface [We selected nasal prong interfaces commonly used to deliver CPAP  [2O above CPAP [T, and mean airway pressure  were measured within the ASL 500 lung model during simulated bCPAP of 6 cm H2O and the various interfaces (n = 50) with manual bellows deflation timed to coincide with the lung model chest-rise analog inflation. Descriptive data were obtained from within the ASL test lung and reported as mean ± SEM.Experiments were performed to evaluate operational feasibility and compatibility between the LFAC delivery and bCPAP. In consideration of the LFAC flow output (~8 L/min) generated by the bellows for inspiratory drug delivery, we sought to determine whether additive flow combined with bCPAP and superimposed on spontaneous breathing potentially increases the risk for pulmonary overdistention or volutrauma. Acceptability criteria were established a priori to identify pressure and volume limits commonly associated with increased risk for pulmonary injury and inflammation in preterm infants. Testing conditions that resulted in a peak inspiratory pressure (PIP) >25 cm H0 mL/kg) ,24,25, aove CPAP  during aterfaces  without ®, dried, and weighed prior to nebulization. The lung model filter was placed within a low dead space filter housing and attached to the nasal airway. A series of one-way valves was placed between the filter housing and lung model to prevent inhaled aerosol on the GF filter from recirculating back into the bCPAP system . Each of the CPAP interfaces (n = 5) was tested over six runs (n = 6) for a total of 30 measurements for the passive humidification condition. The difference in the DP capsules’ gravimetric mass (µg) between pre- and post-nebulization and following the cleaning of the capsule was used to determine the nominal, residual, and emitted DP surfactant dose from the LFAC nebulizer. Following aerosol delivery, the nasal airway and lung model filters were reweighed and the changes in mass delivered at each location represented the depositional loss to the upper airway and estimated inhaled lung dose, respectively. Each was referenced to the total emitted dose of the LFAC for each run. Depositional losses to the LFAC, interfaces, and bCPAP system could not be measured directly and were estimated based on the mass balance differences between the capsule loading and residual doses and cumulative mass deposited in the nasal and lung model. All devices were cleaned with sterile water and dried in a vacuum following nebulization.A 30 mg capsule of DP B-YL: Trehalose surfactant was punctured and placed into the LFAC chamber. Aerosol was generated with bellows deflation timed to coincide with the inflation of the lung model chest-rise analog (r = 0.94).The deposited mass within the nasal airway and GF filter represented the entirety of the solid mass of the active B-YL: Trehalose surfactant used in our gravimetric assay. Prior to testing, the gravimetric assay was validated by comparing the mass recorded by the balance in 30 mg capsules (49 weight%) with the phosphatidylcholine (PC) content using a biochemical PC assay . The colorimetric mass values were measured at OD450 nm with wavelength correction set to 540 nm with a multi-mode microplate reader . There was a high correlation between gravimetric and biochemical PC assay methods (n = 5) using the same methods described above. Gravimetric analysis resulted in instability due to the high moisture content. Following nebulization, the nasal airway and lung filters were eluted with 10 mL of 0.1 M NaCl and 0.1 M NaHCO3. The recovered mass was quantified using PC assay and referenced to the emitted and total PC mass (49 weight%) of the capsules. The final experiments were designed to evaluate aerosol delivery efficiency with active heated and humidified bCPAP gases with select nasal interfaces. The MR 850 humidifier reservoir was filled with sterile water with the heater in the “ON” position to obtain “active” heated humidification with a relative humidity of 100% and temperature of 39 °C (confirmed with the hygrometer). The lung model chamber was heated to 37 °C with an internal heat controller to heat exhaled gas through the nasal airway and prevent condensation from forming within the lung cylinder. The internal components of the bCPAP system, lung model, and interfaces were exposed to “active” heated humidity for 10 min prior to nebulization. Nebulization was performed with Neotech prototype 2 and Ginevri (Afectair) prong interfaces  for statistical analysis. Measurements of particle size and the operational compatibility of DP surfactant delivery were analyzed using description statistics. The mean pressure and volume delivery outcomes were compared against the acceptability limits established a priori.The primary outcome of the study was to evaluate nasal airway deposition and lung dosage of DP surfactant aerosol during bCPAP among five different nasal interfaces. Reported values were summarized as means ± standard deviation (SD) in text and figures. Comparison of multiple groups during delivered dose studies was evaluated by one-way ANOVA with Tukey’s post-hoc test. Data for humidity testing were carried out by comparing passive humidity to active humidity conditions and analyzed with a two-tailed unpaired The DP surfactant aerosol particles generated at the LFAC outlet had a MMAD of 3.68 µm and GSD of 1.78. The FPF (<5.4 µm) was 71% and 41% based on the cumulative mass deposited in the NGI and the capsule loading dose, respectively. T limit. The Neotech (Prototype 2) and all other interfaces delivered VT and pressure measurements that were well within the established clinical limits when the bellows flow was superimposed on spontaneous breathing with bCPAP.The effects of peak pressure and volume delivery in the lung model are shown at baseline (no bellows) and with bellows actuation with different nasal prong interfaces and bCPAP in p < 0.05)  . p < 0.001) and a higher delivered lung dose than the RAM or Hudson interfaces (p < 0.001)  interface resulted in greater nasal deposition than any of the other interfaces (< 0.001) . The RAM< 0.001) .p < 0.001) (During active humidification of bCPAP gases (99% RH), the emitted dose and residual DP drug mass was not impacted (<10%) . However< 0.001) . The dep< 0.001) . The red< 0.001) A–C. OnlyOur findings indicate that a high dose of DP surfactant particles can be delivered beyond the upper airway to the lung without increasing the potential risk for lung injury during concurrent aerosol and bCPAP treatment in a preterm infant model. However, delivery efficiency was highly dependent on the type of nasal prongs and the gas humidity levels provided with bCPAP. Specialized prongs designed to disperse aerosol proximal to the nasal airways resulted in the lowest nasal airway deposition and greatest inspired drug dose (30–40%) of surfactant. The use of a heated humidified bCPAP gas source resulted in notable DP surfactant adhesion and agglomeration within the prongs and nasal airways and compromised drug delivery and safety, raising significant safety concerns for preterm infants.2O (2O) was attenuated by the LFAC chamber and outlet (10 cm H2O). The inflation pressure was further attenuated by the CPAP interfaces, breathing circuits, and release from the water column during bCPAP. The bCPAP water-seal is considered a “pure threshold resistor” [2O) were diverted to the CPAP hoses and water column and released to the atmosphere. The natural pressure release or “pop-off” with bCPAP is less likely to occur with LFAC when applied with other forms of CPAP that employ resistive valves to maintain CPAP.A recent publication by the World Health Organization stated that “aerosolized surfactant and its delivery system should be as safe and efficacious as conventional surfactant formulations” for use in preterm infants in LMICs . In vitr2O . This reesistor” . The LFAThe LFAC inhaler system applies aerosol to the nasal prongs by manually deflating a 60 mL bellows bottle. This simplistic method for generating aerosol for inhalation extends on the practice of applying positive pressure with a manual resuscitator (bag) to preterm infants, which is commonly performed in newborns with weak or ineffective inspiratory efforts in LMICs. In contrast to manual ventilation, the infant remains on bCPAP and nasal aerosol delivery can be provided through prongs without increasing the positive pressure to the infant. Of course, aerosol delivery efficiency relies on coordinated efforts to appropriately synchronize aerosol dispersion with the infant’s inspiration. This may be difficult as the respiratory rate of newborn infants is about 40/min and increases during respiratory distress.We found that DP surfactant aerosols generated with a LFAC have a MMAD of ~3.8 µm. A small particle size is desirable to prevent nasal impaction and optimize pulmonary delivery in preterm infants. However, there is little data about the optimal particle size for surfactant aerosols in preterm infants that enhances deposition in the acinar lung regions without being exhaled. Using a 30-week gestational age preterm infant model, Clark  reportedLung deposition of aerosols is challenged by their particle size, nebulizer type, and nebulizer placement location within the nasal CPAP system. Nebulizers that generate aerosol throughout inspiration and expiration result in high expiratory losses to the upper nasopharyngeal airways and CPAP system. We found that small, inhaled synthetic DP surfactant particles can be generated with a simple low-cost inhaler chamber and bellows bottle and applied to nasal prongs and bCPAP without increasing the risk for lung injury. This resulted in low nasal airway deposition and high inhaled lung dose (30–40%) with passive humidification, but only when therapy was applied with prongs that were specifically designed to separate the bCPAP flow from the aerosol flow. 2O) that overcome dead space and facilitate aerosol movement and penetration into the distal airways. We observed small increases in airway pressure and lung volume during DP delivery with LFAC that may have resulted in minimizing aerosol losses by enhancing particle movement into the distal airways during inhalation. Amirav et al. have previously shown that small increases in tidal volume result in large increases in the lung delivery of aerosols in infants’ nasal airway models . The higWe found large differences in aerosol efficiency between the different CPAP prong interfaces configured for drug delivery. Using off-the-shelf interfaces (Hudson and RAM) with a “mainstream” aerosol administration delivered a negligible inhaled mass (<2%) to the lung model. The lack of aerosol deposition in the nasal and lung filters and the low residual drug remaining in the capsule (~10%) with RAM and Hudson prongs indicate that the entirety of the emitted aerosol was lost in the nasal prongs and bCPAP system flow. Interestingly, we observed fine aerosol particles exiting the bCPAP water-seal and exhaust port or depositing in the water column, creating foam. DP aerosol delivery was most efficient when using interfaces that emit aerosol proximal to the nasal openings with specialized aerosol flow channels that mitigate cannula losses and dilution of particles with the bCPAP source gas. The Neotech prototype prongs, designed with specialized channels that effectively decouple aerosol flow from bCPAP flow and provide a more direct route for nasal delivery, enhanced the delivered lung dose to 30–40% with only modest nasal airway losses (<10%). Based on comparisons with previous bench studies of nebulized surfactants, this is the highest efficiency published to date for inhaled dose. Interfaces that circumvent the effects of CPAP system flow with a “sidestream” aerosol delivery method may reduce the residence time of the aerosol from being exposed to humidity, limit the hygroscopic growth of aerosol particles, and prevent the entrainment of aerosol into the CPAP flow and prevent expiratory drug loss.Experimental nebulizers designed for liquid aerosol of surfactant delivery typically generate a constant output of the drug during the respiratory cycle that may be less efficient for pulmonary drug delivery than breath-synchronized nebulizers designed to time aerosol delivery with the patient’s inspiratory phase ,32,33. MDP aerosol surfactant delivery with an active heated humidified bCPAP gas source resulted in the notable adhesion and agglomeration of surfactant in the nasal prongs and airways, reduced lung delivery efficiency, and significant clinical safety concerns for preterm infants at risk for airflow obstruction during bCPAP. There are concerns about hygroscopicity, solid state stability, and aerosol efficiency when applying spray-dried inhalation powders into a heated/humidified bCPAP system. The exposure of emitted DP aerosol particles to condensed water vapor within internal plastic surfaces  can result in agglomeration and high depositional loss of aerosol in systems with high RH from active humidifiers. Thus, the hygroscopic growth of aerosol particles in ventilator circuits can reduce medication delivery by as much as 40%. In some cases, this resulted in partial or total nasal airway occlusion of with pasty glue-like material in the 3D printed model . There iWe used a single preterm infant nasal airway and lung model as we were limited in our ability to acquire models that would span the individual neonatal sizes from the child cohort that is being treated with surfactant for RDS in an intensive care setting. A single operator manually activated the bellows based on the visual chest rise and fall using an infant chest-rise analog. The delivery efficiency of DP surfactant aerosols may be heavily influenced by the operator’s ability to observe chest rise and appropriately time LFAC actuation with inhalation, especially in tachypneic newborns with severe RDS. Efforts should be made in the future to fully automate and synchronize inspiratory delivery of DP surfactant aerosols so that infants can receive more effective dosing. Our results with active humidity should be approached with trepidation because they are limited to the use of non-heated anatomic airway models which are more prone to condensation and “rain-out” and powder agglomeration than newborn human infant airways. Evaluation of the hygroscopic growth and sedimentation of DP aerosol particles should be explored in greater detail before this therapy can be implemented in the clinical setting. The filter technique employed to measure inhaled lung dose only estimates the total surfactant dose delivered to the lungs and allows no conclusion to be reached about the amount that reaches the lower airways where it is active.Future testing is required to address particle size related to hygroscopic particle growth when using a heated/humidified gas source or when capsules are exposed to different environmental factors. Next steps should also include testing in the lung model of higher fill doses (50 instead of 30 mg) of B-YL: Trehalose surfactant to determine efficiency and nasal deposition. A pre-clinical study in surfactant-deficient rabbits would be useful for evaluating dosing responses with 50 mg capsules and the potential risk using selected aerosol flow-generating procedures, nasal interfaces, and bCPAP systems prior to being used in humans.


Structurally complex natural productswere used as testbeds to showcase the late-stage tagging methodologyof marine natural products to enable liquid chromatography–massspectrometry (LC–MS) detection. This enabling methodology thereforeprovides a general method for the environmentally friendly and biocompatiblederivatization of sp3 alkyl halide bonds.Aqueous transformations confer many advantages, includingdecreasedenvironmental impact and increased opportunity for biomolecule modulation.Although several studies have been conducted to enable the cross-couplingof aryl halides in aqueous conditions, until now a process for thecross-coupling of primary alkyl halides in aqueous conditions wasmissing from the catalytic toolbox and considered impossible. Alkylhalide coupling in water suffers from severe problems. The reasonsfor this include the strong propensity for β-hydride elimination,the need for highly air- and water-sensitive catalysts and reagents,and the intolerance of many hydrophilic groups to cross-coupling conditions.Here, we report a broadly applicable and readily accessible processfor the cross-coupling of water-soluble alkyl halides in water andair by using simple and commercially available bench-stable reagents.The trisulfonated aryl phosphine TXPTS in combination with a water-solublepalladium salt Na Drug candidates thathave escaped from the medicinal chemistry flatlands of aryl and heteroarylsystems, adopting the more three-dimensional structures that highersp3 bond counts and chiral centers exhibit, have met withclinical success4 and have driven boththe need and desire for new chemistries that can be used to accessor modulate these systems. Novel methodologies enabling sp3–sp2 or sp3–sp3 bondformation have adapted palladium –X bonds,while placing steric bulk around the metal center to avoid β-hydrideelimination reactions and the formation of unwanted alkene side products.17 For secondary alkyl halides, the catalytic systemassists in the stabilization of radical intermediates.18The metal-catalyzed cross-couplingof carbon–halogen bondswith organometallic reagents, one of the most important reactionsof the 20th century, was recognized through the award of the NobelPrize in 2010.alladium 1a,5−8 nimistries1b.15,16 in vivo natural product derivatization,20 site-selective protein and biomolecule labeling,25 prodrug activation,28 and one-pot C–Hactivation combining bio- and metal catalysis.30 These aqueous reactions have also been considered as a step forwardto more green processes of cross-coupling, moving away from conditionsdependent on toxic and unsustainable organic solvents.31 Although there are a growing number of proceduresenabling the cross-coupling of alkyl halides in organic solvents,32 there are no general methods for the cross-couplingof alkyl halides in water, as current methods often require the rigorousexclusion of both air and moisture and are unable to tolerate hydrophilicfunctionalities with free protons. As the initial investigations intowater-active catalysts for aryl halide coupling led to a plethoraof further applications, it is obvious that there is a need for thecornerstones of aqueous alkyl halide coupling to be set, enablingaccess to more 3D and sensitive molecules under mild and green chemistry.33The scope of the research into aqueous cross-couplingcontinuesto expand rapidly, with one of the preliminary driving forces foraqueous cross-coupling being the crucial foundation of robust andmild conditions in bioorthogonal settings. Great successes in thisarea have enabled insitu organozinc formation have been demonstratedusing surfactants to enable micellar reactivity using palladium catalysis–X alky halidecross-coupling that could be used when modulating compounds containingsensitive functional groups or even biomolecules, we explored variouscatalysts and ligands. We demonstrate that Na2PdCl4 and TXPTS could be employed in a fully aqueous reaction,even in the presence of air, to enable the cross-coupling of primaryalkyl halides with aryl boronic species  was explored in the first instance (para-methoxyphenyl boronic acid (2a), using 10% Pd(OAc)2 and 5 equiv of NaOH.Aliphatic aminoacids are highly challenging testbed substrates for aqueous Suzuki–Miyauracoupling, with low solubility in organic solvents and modest solubilityin water. Both the amine and carboxy groups have a propensity to coordinateto palladium, often competing with the active catalyst ligand, andfree protons often interfere with more sensitive catalysts and reagents.instance 1. Aryl b37 and this provided one starting point for our explorations into determiningand developing a methodology for the aqueous cross-coupling of alkylhalides. Using the same catalysts and ligands, we explored the toleranceof the cross-coupling reaction to high concentrations of water. Pd(OAc)2 and [HPCy3][BF4] or [HPtBu3][BF4] were explored as ligands in the 80%aqueous reaction of 1a and 2a; however,only unreacted starting material could be observed  –X aryl halidesprovided a parallel starting point in our search for conditions toestablish and develop the aqueous cross-coupling of C(sp3)–X alkyl halides. Both tetramethylguanidine (TMG) and X-Phosare well known and highly useful ligands for the aqueous Suzuki–Miyauracoupling of aryl halides;19 however, whenexplored in the context of alkyl halides, no conversion to the coupledproduct was observed ; however, the employment of sSPhosalso resulted in considerable β-hydride elimination and thegeneration of alkene side products (44% by NMR). Gratifyingly, TXPTS,a simple sulphonated triarylphosphine,38 led to minimal alkene formation. A combination of high steric bulk,electron richness at the phosphine center, and water solubility ledTXPTS to enable high conversions in aqueous conditions and, underthese conditions, prohibit β-hydride elimination . Generally, as anticipated, lower yieldswere observed when stronger bases were used, presumably a consequenceof the SN2 hydrolysis of the alkyl bromide to the primary alcohol.Less nucleophilic and milder bases reduced this side reaction andresulted in higher yields. It was found that the employment of inorganicbases, in particular, sodium, potassium, and cesium carbonate, couldenhance yields. NMR yields of 22% could be observed when K3PO4 was used; this is promising and affords future opportunitiesfor the employment of phosphates as both biological buffers and basesin potential biorthogonal cross-coupling reactions. For the purposeof developing a robust, yet mild, set of conditions, potassium carbonatewas taken forward as the best candidate, enabling yields of 68%. Increasingthe temperature to 45 °C and reaction time to 24 h led to a goodNMR yield of 84% (isolated yield 71%) for this challenging brominatedamino acid substrate.With 61% yield using TXPTS as the ligand,we observed full consumptionof the starting material. We identified alcohol 2.21a-OH were observed as theprominent undesired products. In control experiments , and the yield of the reaction was notreduced, indicating the lack of radical formation within species I oxidative addition. This lack of radical reactivity is thoughtto be the reason for the limited success of palladium catalysis towardsecondary alkyl halide cross-coupling. Most secondary halide couplingsinvolve metals that have access to a greater number of stable oxidationstates, such as nickel, enabling radical-type mechanisms. When thesecondary halide bromocyclohexane 1y was subjected toour cross-coupling conditions, we observed no conversion to product 2y or alkene, indicating again a lack of radical oxidativeaddition within our mechanism and that our conditions can be usedto regioselectively react primary alkyl halides.Whereas an SN2-type oxidative addition is generallythe acceptedmechanism for the Suzuki–Miyaura couplings of primary alkylhalides, to rule out a radical mechanism, the standard reaction conditionswere doped with radical trap TEMPO. If II–V).40 However itis worth noting that the majority of the Suzuki–Miyaura mechanisticstudies have been performed on the cross-coupling of aryl halidesand boronic acids in purely organic or biphasic systems,41 very different to this alkyl halide aqueoussystem. Water has been shown to have a profound effect on couplingreactions43 and yet is partitioned away from the organiccatalytic cycle in previous proposed mechanisms; because of the hydrophobicnature of aryl halides and boronic acids, the Suzuki reactions inpure water are still considered biphasic.45 A full mechanistic investigation of this unique system of water-solublecatalyst and alkyl halide substrates would be highly complex, andas such is outside the scope of this project.We proposea mechanism here involving palladium-coordinated hydroxidefrom basic aqueous media involving oxo-palladium transmetalation , free acid (1c),and Boc (1d), in similarly good yields (1e), and notably enabled the reaction with an alkyl chloride (1f). Often, much more forcing conditions are required forthe coupling of alkyl chlorides, a much cheaper and more stable reagentthan alkyl bromides. Simply extending the reaction time of this moreunreactive chloride enabled high yields akin to that of bromides withminimal conversion to hydroxide.Using the conditions developedon testbed compound d yields 3. PleasiS)-tyrosine (1g) derivative was synthesized. The reactionof the enantiopure compound 1g afforded good conversionwhile gratifyingly retaining the stereochemical integrity at the α-carbonin these basic aqueous conditions. Furthermore, medicinally relevantprivileged heterocyclic moieties, often found in drug-like molecules,were successfully derivatized. Heterocycles, including brominatedindole, (1h) coumarin (1i), and isatin (1j), were afforded in good yields under these conditions.The low yield of 1j, potentially due to the low watersolubility, could be almost doubled by doubling the catalyst loading.As an example of a more complex halogenated biomolecule, a dipeptide(1m) containing the unnatural brominated amino acid wassynthesized and cross-coupled with para-methoxyphenylboronic acid in good yields. The use of the Suzuki–Miyaurareactions to site-selectively derivatize amino acids and peptidesin biorthogonal (or aqueous conditions) reactions has been establishedas an important reaction in the derivatization of peptides, synthesisof peptide-based drugs, or even protein tagging.25 Our coupling of 1m represents the first suchexample of this being translated to alkyl halide amino acids and peptides.Avoiding epimerization is often a challenge associatedwith cross-coupling,particularly when organic solvents necessitate the use of strongerbases. To determine whether our conditions would be sufficiently mildto avoid epimerization, an enantiopure unnatural  and then coupled with boronic acid to generate a metolachloranalogue, providing a potential two-step method for the diversificationof less reactive alkyl halide agents in water. As practicality andscalability are often important considerations in the developmentof novel methodologies, the unnatural free amino acid 1n was synthesized and coupled at a 1 mmol scale with a very good,isolated yield of 69%. High levels of conversion meant that columnchromatography was not required during purification, with the productbeing precipitated as a hydrochloride salt. By simply varying theboronic acid, this process could be used for the effective large-scalesynthesis of amino acid derivatives.Metolachlor is a racemic alkyl chloride used as a herbicide, particularlyagainst grass.1a as the testbed (2m). Reactivity was generally determined by the electron richness ofthe carbon–boron bond rather than solubility, with para- and ortho-methoxyphenyl boronic acids(2a and 2b) giving excellent yields andthe electron-poor hydrophilic 2i giving a much loweryield than the electron-rich hydrophilic 2h. Curiously, ortho-tolyl (2d) gave much higher yields than para-tolyl boronic acid (2c). Ortho-substituted boronic acids are often resistant to palladium-basedcoupling, with the steric bulk inhibiting the transmetalation steponto palladium. This organometallic intermediate is less stericallyhindered in reactions with terminal alkyl halides when compared toaryl halides, and thus, conversion is enabled. With the addition of10% acetonitrile cosolvent, the yield of 2c couplingcould be increased to 46%, indicating that the low solubility of unfunctionalizedboronic acids under aqueous conditions is likely to impact yields;however, this seems to be less impactful than the aryl group electronics.Boronic acids containing a fluorine (2k) and a dansyl(2m) group could be reacted with 1a, indicatingthat this chemistry has high practical use in the tagging of halogenatedbiomolecules with either a 19F or a fluorescent probe.A BPin ester (2o) and borofluorate salt (2n) could also be reacted, meaning that this system is not entirelydependent on the boronic acid species. It is worth noting that theisolation of modified amino acids proved nontrivial on a small scale,leading to relatively low isolated yields. While attempts were madeto derivatize these amino acids to their Boc carbamates for easierpurification, this method did not improve isolated yields.Having demonstrated theability to couple a range of challengingalkyl halides, we next set out to explore how different boronic acidswould perform under these mild, aqueous conditions, again using brominatedamino acid  testbed 4. All co2.447 Produced in the oceans, where they could be readily washedaway, certain metabolites are often highly lipophilic,49 potentially promoting their uptake and retention within variousbiological systems. Their interplay between organic and aqueous environmentsis critical, and the possibility to tag and track or immobilize thesemetabolites has the potential to open new horizons in the chemicalecology. The natural marine product, bromosphaerol (5a), produced by the red alga Sphaerococcus coronopifolius,50 is shown to have marine antifoulingactivity. Semisynthetic generation of analogues to explore their functionand tune their activity has been explored successfully;51 however, this requires a very selective andmild chemistry on the native molecule, which is inherently prone tosubstitution, elimination, and rearrangement reactions. By using theprimary alkyl bromide as an orthogonal handle, we have been able toselectively install aryl groups to generate analogues 5b, a primary amine tag to enable LC–MS visualization, and 5c, a fluorescent tag, in 100% water on an analytical scale(6a) is found in the deep-sea soft coral Alcyonium roseum;52 subjectingthis inherently less reactive organochloride to our coupling conditionsenabled the detection of the derivative by LC–MS by virtueof the readily ionized primary amine tag –X bonds in molecules containing highly sensitive functionalities.We designed our approach to be accessible using commercially availablecatalysts and ligands. We demonstrated that the reaction can be carriedout on the bench with no need for air exclusion and is tolerant toa range of challenging functional groups as well as applied to twonatural products. Potential practical uses of this reaction are evident,such as bioorthogonal peptide or amino acid diversification, naturalproduct analogue generation, and enabling the orthogonal tagging ofmetabolites for analysis or even discovery. The mild conditions andlow temperature open up the possibility of merging aqueous alkyl halidecross-coupling with bioenzymatic alkyl halide generation. This expandsour groups’ “GenoChemetic toolbox” of aqueouscoupling reactions, this being the first example on alkyl halide substrates,paving the way to potentially modulate and tune alkyl halides in thepresence of living systems.19The discovery and developmentof aqueous reaction conditions isan important but challenging pursuit that can not only open a gatewayto more sustainable synthetic procedures and increase compatibilitywith sensitive functional groups but also provide new tools for chemicalbiology or even enable synbio–synchem compatibilities. In thisstudy, we have shown the first general method for the fully aqueousSuzuki–Miyaura coupling of unactivated primary alkyl halideswith boronic acids, allowing for the diversification of C(sp


Chronic inflammation causes bone destruction in middle ear cholesteatomas (MECs). However, the causes of their neoplastic features remain unknown. The present study demonstrated for the first time that neoplastic features of MEC are based on proto-oncogene mutations.NOTCH1 and MYC) variants and the presence of bone destruction and granulation tissue formation. MYC and NOTCH1 variants were detected in two and five of the 22 samples, respectively. Two of the NOTCH1 variants were located in its specific functional domain, one was truncating and the other was a splice donor site variant. Mutations of the two genes in attic cholesteatomas (n = 14) were significantly related with bone destruction (p = 0.0148) but not with granulation tissue formation (p = 0.399).DNA was extracted from MEC and blood samples of five patients to detect somatic mutations using depth-depth exome sequencing. Exons with somatic variants were analyzed using an additional 17 MEC/blood test pairs. Variants detected in MECs but not in blood were considered pathogenic variant candidates. We analyzed the correlation between proto-oncogene (This is the first study to demonstrate a relationship between neoplastic features of MEC and proto-oncogene mutations.The online version contains supplementary material available at 10.1186/s12920-023-01640-6. Cholesteatomas are benign, squamous epithelial hyperproliferative conditions of the tympanic cavity associated with keratin debris accumulation . They caVarious hypotheses regarding the origin and pathogenesis of cholesteatomas have been proposed , 4. CholIn the present study, we analyzed the pathological genetic variants that may be related to cholesteatomas and investigated whether somatic mutations were associated with bone destruction and inflammatory reactions, such as granulation tissue formation.MYC and NOTCH1 variants of interest were detected in two and five samples, respectively. Variants in LY75-CD302, EFCAB6, HRASLS, and UBP with approximately 50% variant allele frequencies (VAF) could be somatic variants acquired before cholesteatoma development. All of these variants are registered in the dbSNP database and may not be pathogenic. Meanwhile, the VAF for NOTCH1 was 2–7%, indicating that somatic mutations accounted for a small portion of cholesteatomas. One of the variants (p.I471T) was located in the 11th and 12th epidermal growth factor (EGF) repeat domain, while another (p.C1505R) was located in the LIN-12/NOTCH repeats (LNR) domain. EGF repeat and LNR domains are functionally significant in NOTCH1 [NOTCH1 function  but not with granulation tissue formation (p = 0.399)  were identified  analysis was performed on the first five specimens, and the presence of gene mutations in previously known neoplastic variants could be precisely evaluated at the whole-gene level. However, the remaining 17 samples were evaluated with target capture sequencing; these samples may have other mutations in protooncogenes, which are not included in the 24 specific genes already detected in the present study. Moreover, mutations of either gene in attic cholesteatomas were significantly related to bone destruction (p = 0.0148) but not with granulation tissue formation(p = 0.399).Acquired cholesteatomas are considered non-neoplastic pathologies with epithelial keratinizing lesions, which may lead to invasion and/or destruction of the temporal bone –4. IntraMYC and NOTCH1 [A review of the potential proto-oncogenic modifications in cholesteatomas failed to provide sufficient genetic evidence to support neoplastic features . Howeverd NOTCH1 –15. BaseNOTCH1 [NOTCH1 is expressed on the cell surface as a heterodimer composed of non-covalently associated extracellular (NEC) and transmembrane subunits. The NEC subunit consists of 36 iterated EGF-like repeats that include the binding region and three LNRs [NOTCH1 and neoplastic features of cholesteatomas [NOTCH1 expression in cholesteatoma epithelium compared to auditory canal skin epithelium, suggesting that NOTCH1 may alter the balance from cellular differentiation to hyperproliferation and subsequently contribute to neoplastic features of the pathology [NOTCH1 was performed in a surgical sample  that are frequently mutated in head and neck tumors were sequenced in another 17 cholesteatoma-blood pair test samples. Bait oligos  were used to capture the exons in target genes. Sequencing was performed using the MiSeq platform and the variants were detected using MuTect2. Except for attic cholesteatomas (n = 14), there were less than three cases of all other types of the pathology . DNA was extracted from cholesteatoma using the QIAamp DNA Mini kit  and from blood using the QIAamp DNA Maxi kit (QIAGEN) according to the manufacturer’s protocols. Coding exons were captured using the SureSelect XT AUTO HUMAN ALL Exon V5 kit  and sequenced using the HiSeq2500 system  in 101 bp paired-end reading mode. The reads were aligned to GRCh37/hg19 using Novoalign  and duplicate reads that were excluded from the analysis were marked using the Novosort software (Novocraft Technologies). Local realignment and variant calling were performed using the HaplotypeCaller in the Genome Analysis Toolkit  to generate variant call format (VCF) files. Variants found in the cholesteatoma but not in the blood of a participant were considered cholesteatoma-specific variants (CSVs). Variants found in any of the five blood samples were excluded as noise, even if they were CSVs in pair comparisons. CSVs were also identified using MuTect2 and were confirmed using MiSeq after specific polymerase chain reaction amplification. CSVs and six additional genes (Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


South Africa is faced with a high HIV and STI prevalence and incidence, respectively, with pockets of high burden areas driving these diseases. Localised monitoring of the HIV epidemic and STI endemic would enable more effective targeted prevention strategies. We assessed spatial variations in curable STI incidence among a cohort of women enrolled in HIV prevention clinical trials between 2002 and 2012. STI incidence rates from 7557 South African women enrolled in five HIV prevention trials were geo-mapped using participant household GPS coordinates. Age and period standardised incidence rates were calculated for 43 recruitment areas and Bayesian conditional autoregressive areal spatial regression (CAR) was used to identify significant patterns and spatial patterns of STI infections in recruitment communities. Overall age and period standardised STI incidence rate were estimated as 15 per 100 PY and ranged from 6 to 24 per 100 PY. We identified five significant STI high risk areas with higher-than-expected incidence of STIs located centrally (three-locations) and southern neighbouring areas of Durban (two-locations). Younger age (<25), not married/cohabitating, parity <3 and poor education were all significant correlates of high STI communities. Findings demonstrate sustained STI incidence rates across the greater Durban area. The role of STI incidence in HIV acquisition in high HIV endemic areas need to be revisited as current highly effective PrEP interventions do not protect from STI acquisition. In these settings there is an urgent need for integrative HIV and STI prevention and treatment services. South Africa accounted for 200,000 new human immunodeficiency virus (HIV) infections in 2019 UNAIDS, . High HIThe HIV Prevention Research Unit (HPRU) of the South African Medical Research Council (SAMRC) participated in five multi-centre HIV prevention trials at six sites across greater Durban in the province of KwaZulu Natal, from 2002–2012 , Neisseria gonorrhoea (NG), syphilis and Trichomonas vaginalis (TV)) as observed in these trials over a period of 10 years. We also used Bayesian conditional autoregressive areal spatial regression (CAR) in order to identify significant geographical patterns of STIs. Bayesian hierarchical models are one of the main statistical techniques to investigate geographical variations. These models have been employed previously to provide fine-scale estimates of HIV prevalence in Sub-Saharan Africa and high-risk areas of TB/HIV coinfection in Kenya  and rural areas. A comprehensive description of the study population and procedures are described elsewhere  except for one trial which enrolled women aged 16 and older. Median age across the trials varied marginally between 24 and 28 years. The average screening to enrolment ratio was 47% in this combined cohort. Other eligibility criteria were broadly similar for all studies. At each visit, participants received HIV risk reduction counselling, STI testing and treatment and had access to male and/or female condoms. Women who tested positive for an STI were provided with treatment according to the respective study protocols and local South African guidelines. Women who HIV seroconverted during the trial remained in the study and received ongoing safe sex counselling, STI testing and treatment, and condom provision. All participants provided written informed consent to participate in the studies.HIV diagnostic testing was performed using two rapid tests on whole blood sourced from either finger-prick or venepuncture . The Abbot IMX ELISA test , in combination with the Vironostika HIV1/2 ELISA was used on whole blood sourced from venepuncture for discordant/unequivocal results.The following methods were employed for STI detection depending on the clinical trial: (1) CT and GC was either detected by DNA PCR , PCR  or the BDProbe Tec ET assay  using a urine sample, (2) TV was identified by PCR  or by wet mount microscopy and (3) syphilis was detected by rapid plasma re-agin (RPR) and confirmatory Treponema pallidum particle agglutination assay (TPHA)  using whole blood sample.The following characteristics were collected and combined across the five clinical trials: age, contraceptive use, STI at screening  and condom use at last sex act. Marital status/cohabiting and parity data were collected in four of the clinical trials. The number of sex partners in the last three months were collected in three of the five clinical trials.STI incidence rates were calculated in each of 15 strata (5 age groups for 3 time periods) in every recruitment area and mapped. Participant data were classified into five age categories  across three-time periods . We employed direct age-time period standardisation to obtain standardised STI incidence estimates per area to facilitate legitimate geographical comparison .n = 7557) following exclusion of a) outlying participants not resident within core recruitment areas (n = 194), or residing in areas with less than 50 total PY (n = 207), (b) participants for whom no GPS data had been collected (n = 1054) and (c) participants who did not attend any follow up visits post enrolment (n = 133)  were included in STI incidence rate calculations (n = 133) . The cruThe characteristics of the study population were described and formally compared across pre-defined STI incidence rate categories . This analysis utilised data at the individual-level within this ordinal variable (i.e. categories of STI incidence rate). Age, marriage/cohabitation, type of contraception used, STI at baseline,p < 0.10) was used to construct the final multivariable model. Adjusted odds ratios (aOR) and 95% confidence intervals (CI’s) were presented in number of sexual partners in the last three months and parity data were included as the explanatory variables at the individual level. Univariable and multivariable ordered logistic regression, with recruitment area (community or group of communities) robust standard errors, were used to identify prominent factors associated with higher incidence. Stepwise forward selection regression  model proposed by Besag, York and Molliè (BYM) formulated as follows to assess incidence risk across the 43 areas  is based on the sum of the weighted neighbourhood values and utilises an adjacency matrix of common neighbours (or shared boundaries) of a given area. For the unstructured recruitment area level random effect, we assumed an independent normal distribution ϵi ∼ N  with variance σ2 ϵ. Non-informative gamma priors were used for both variance parameters, namely Gamma . We assumed vague normal distributions for the fixed effects coefficients (β). We estimated exceedance probability that risk ratio in a given recruitment area was significantly >1 and also utilised 95% uncertainty intervals around RRs. The model was fitted using Markov chain Monte Carlo simulation .A total of 1253 incident STI infections observed in the total 8646 PY, corresponding to an overall crude incidence rate of 14.5 (13.7–15.3). Overall age and period standardised STI incidence rate were estimated as 15 per 100 PY and ranged from 6 Risecliff/Montford/Arena Park/Croftdene/to 24 per 100 PY in the Chesterville/Cato Manor/Mayville/Ridgeview. All crude and adjusted incidence rates are presented in Supplementary Table 1.p < 0.001). There was an increasing trend between the proportion of women who used injectables as a family planning method and STI incidence categories. Compared with women who were living in the areas with relatively lower STI incidence rates, those who resided where STI incidence rates were 15+, reported higher usage of injectables . These areas had also significantly higher proportion of women with <3 children . In addition, 60% of the women who lived in the high STI incidence areas also reported that they don’t know whether or not their partner had another partner, compared to 52% and 54% where the incidence rates were <10 and 10–14 per 100 PY, respectively (p < 0.001). Further, a vast majority of the women were also reported to be unmarried and/or not cohabiting with their partners . Women who lived in the highest HIV incidence rates were also correlated with the areas with high STI incidence rates.Characteristics of areas were compared across increasing levels of STI incidence rates: <10 per 100 PY, 10–14 per 100 PY, 15+ per 100 PY . There wp < 0.001). Using non-barrier family planning methods such as injectables and oral contraceptives (i.e. pills) were also significantly more prevalent areas in with high STI incidence rates . Women who reported that they know their partner has another partner were also more common in STI high risk areas with aOR: 1.38 . In comparison, those who did not know whether their partner had another partner or not were also more significantly more prevalent in areas where incidence STI was estimated to be more than 15 per 100 PY . Women who lived in these high STI incidence areas also had significantly low education . Although it was not statistically significant, we included baseline diagnosis with STI in the model because of its potentially confounding impact. Finally, we have observed significant correlation between the women who lived in high STI infections areas and high HIV prevalence areas. For example compared to women who lived in the regions with HIV incidence rates of <5, those who lived areas with 5–9 per 100 PY were 81% more likely to be the regions with highest STI infection rates ; while women were more than four times more likely to be living in the high STI areas if they were coming from the regions where HIV incidence rate was estimated to be higher than 10 per 100 person year .In the combined study population, overall age and period standardised STI incidence rate was estimated as 15 per 100 PY and ranged from 6 to 24 per 100 PY. Adjusted odds ratios (aORs) and 95% confidence intervals (CIs) were presented for the risk factors measured consistently across the trial populations . In multRelative risks (RRs) from the Bayesian CAR model across the 43 communities are shown in We observed a persistently high STI burden in the greater Durban area over a ten-year period; with STI incidence rates ranging from 6 to 24 per 100 PY. Similarly, a individual participant data (IPD) meta-analysis based on 18 prospective HIV prevention studies identified a higher STI prevalence in clinic/community-based populations in South Africa relative to homogenous populations elsewhere in Southern/Eastern Africa  and not being married/cohabitating were associated with high HIV and STI incidence rates  . Further, young women in this region are predominately at high risk of both HIV and STI acquisition. Identification of these ‘hot-spots’ is critical for targeted biomedical, behavioural and structural interventions to reduce the burden of HIV/STIs especially among the young women in these communities. Globally, there is an urgent need to address the high burden of STIs and its impact on HIV incidence and reproductive health.