However, sediments from a lake 10 km to the North West (Siqlukaq) produce CH4 of biogenic origin. Sukok and Siqlukaq were chosen among the four lakes surveyed to test the hypothesis that active CH4-producing organisms (methanogens) would reflect the distribution of CH4 gas levels in the sediments. We first examined the structure of the little known microbial community inhabiting the thaw bulb of arctic thermokarst lakes near Barrow, AK. Molecular approaches (PCR-DGGE and iTag sequencing) targeting the SSU rRNA gene and rRNA molecule were used to profile diversity, assemblage structure, and identify potentially active members of the microbial assemblages. Overall, the potentially active (rRNA dominant) fraction included taxa that have also been detected in other permafrost environments . In addition, Siqlukaq sediments were unique compared to the other sites, in that they harbored CH4-cycling organisms , as well as bacteria potentially involved in N cycling  whereas Sukok sediments were dominated by taxa typically involved in photosynthesis and biogeochemical sulfur (S) transformations. This study revealed a high degree of archaeal phylogenetic diversity in addition to CH4-producing archaea, which spanned nearly the phylogenetic extent of currently recognized Archaea phyla . Together these results shed light on expansive bacterial and archaeal diversity in Arctic thermokarst lakes and suggest important differences in biogeochemical potential in contrasting Arctic thermokarst lake sediment ecosystems.Developing a microbial ecological understanding of Arctic thermokarst lake sediments in a geochemical context is an essential first step toward comprehending the contributions of these systems to greenhouse gas emissions, and understanding how they may shift as a result of long term changes in climate. In light of this, we set out to study microbial diversity and structure in sediments from four shallow thermokarst lakes in the Arctic Coastal Plain of Alaska. Sediments from one of these lakes (Sukok) emit methane (CH Sukok is underlain by the Walakpa natural gas field and displays ebullition throughout the year, especially in the SukS area. Matheus Carnevali et al.  arctic thermokarst lakes on the north slope of Alaska were sampled during three different field campaigns .DNA extraction (i): DNA from 2011 sediments was used for Bacteria and Archaea SSU rRNA gene iTag sequencing and archaeal SSU rRNA gene clone libraries. We studied the upper sediment layers (0–30 cm) given observed differences in CH4 production in these layers : 2010 sediments were used for archaeal PCR-DGGE profiling and 2010 archaeal SSU rRNA gene clone libraries. Subsamples from 2010 cores  of the first 30 cm of the 2011 cores  following manufacturer's instructions using a SSU rRNA-specific directed primer  were sufficient to generate cDNA directly from the total RNA to be used in iTag sequencing. We then amplified the total RNA for direct comparison of the sequence-based identities of the potentially active bacteria and archaea across the set of 9 lake sediment samples. The MessageAmp II aRNA Amplification Kit (Life Technologies) was used according to manufacturer's instructions and final RNA products (aRNA) were quantified  on a fluorescence plate reader. Amplified RNA was then reverse transcribed to cDNA with primer 806R  and cDNA from both RNA (5 samples) and aRNA (9 samples) were sent to the Joint Genome Institute  for library preparation and paired-end (2 × 250 bp) MiSeq Illumina sequencing of the variable region 4 (V4) SSU rRNA using primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) following Caporaso et al. . Table 1Sequence processing included removal of PhiX contaminants and Illumina adapters, then quality was evaluated using the FastQC software (Babraham Bioinformatics). Trimmomatic  using R  ordinations of DNA and aRNA sequences were generated using the Bray-Curtis dissimilarity values calculated with 20 re-samplings for each sample. Pairwise differences in Bray-Curtis dissimilarity between sites were tested with the Mann-Whitney test on resampled communities. Significance thresholds were defined by the Benjamini-Hochberg procedure with a false discovery rate of 0.05. These statistical analyses were conducted in R using the vegan package as well as the p.adjust(method = “fdr”) commands included in the base stats package.P-values reflecting the significance of aRNA sequences being greater than DNA sequences, and thus “activity” was estimated by counting the number of times out of 100,000 the resampled aRNA sequences were less than the resampled DNA sequences. The Benjamini–Hochberg procedure was used to determine a p-value equivalent to false discovery rate (FDR) = 0.05 (p ≤ 0.00019). Thus, if the aRNA values were less than the DNA values less than 19 times out of the 100,000 resamplings, that OTU in that sample was classified as “active.” OTUs that may have been overrepresented as a result of RNA amplification were identified by plotting the five samples for which both RNA and aRNA data sets were available  than their DNA sequences (< 2), as well as OTUs with DNA sequences < 5, were not taken into consideration. OTUs with aRNA to DNA ratios >1 were broken into three groups as proxies to enable consistent comparisons of varying aRNA:DNA levels between sites. OTUs with ratios >2 and aRNA sequence occurrence levels over 500 were assigned to Group 1 (G1), those with ratios >2 and aRNA sequence levels between 250 and 500 were assigned to Group 2 (G2), and those with ratios between 1 and 2 with aRNA sequence levels ≥250 and DNA levels ≥250 were assigned to Group 3 (G3). OTUs with aRNA levels ≥1,000 or DNA levels ≤5 were not assigned a G-level given the potential for aRNA over-amplification and RNA to DNA ratio inflation with low DNA signals.We used both aRNA and DNA sequence types to understand the variance in composition of potentially active organisms between lake sediment sites. To identify OTUs that may have been potentially active in these lake sediments communities were resampled 100,000 times, at 75% of the smallest library size among DNA and aRNA samples . During each resampling, the resampled aRNA sequences were compared to resampled DNA sequences for each OTU. To survey the structure of the archaeal assemblages in the thermokarst lake sediments, core sections collected from all lakes sampled in 2010 . Given the short amplicon size of the archaeal iTag sequences, they were not used in tree reconstruction. However, we included SSU rRNA gene sequences of the closest neighbors (>90% identity) to the iTag sequences present in the Silva database (123) in the phylogenetic tree.To study the evolutionary relatedness between thermokarst sediment Archaea and known Archaea lineages, the SSU rRNA gene sequences were aligned  were revealed with non-metric multidimensional scaling (NMDS) ordination analysis of Bray-Curtis dissimilarity (20 in-silico re-samplings) of DNA and amplified RNA (aRNA) iTag occurrences from the three study sites , compared to the difference between Siq and SukB. Significant differences were also found between SukB and SukS . The NMDS analysis based on aRNA iTag sequences  by sequence type  forming an average of 1,397 ± 288 DNA OTUs, 1,397 ± 366 aRNA OTUs, and 1,447 ± 232 RNA OTUs; excluding doubletons and singletons Table . Patterns Figure  showed tWe observed a clear, positive relationship between aRNA and DNA iTag levels across the different taxa detected. The integrity of the relationship between the aRNA and RNA data sets showed best coherence for those sequences that were highly represented in the data set, while the relationship fell off at lower levels of detection Figure . 7 G1 OTOf the OTUs unique to Siq, one G1 OTU was affiliated with the Nitrospirae (OTU 4 in Siq-U), while two OTUs related to Methylomonaceae fell into G2 (OTUs 35) and G3 (OTU 21) Figure . OTUs afThe SukB data set had G1 OTUs affiliated with Cyanobacteria in the families Xenococcaceae and Leptolynbyaceae (OTUs 40 and 119), Intrasporangiaceae in the Actinobacteria (OTU 12), in addition to an unclassified Deltaproteobacteria (OTU 10) and unclassified Bacteria (OTU 322) Figure . SukB G2Figure 4C). G2 OTUs included Actinobacteria in the families Microbacteriaceae and Cellulomonadaceae (OTUs 65 and 79), uncultured Armatimonadetes (OTU 187), Xenococcaceae (OTU 40), Desulfarculaceae (OTU 23), Burkholderiaceae (OTU 9), Hydrogenophilaceae (OTU 34), and unclassified Bacteria (OTUs 148 and 370). An OTU (57) related to Desulfobulbaceae was the sole OTU in G3.Group 1 SukS OTUs were related to Intrasporangiaceae (OTU 12), Lentimicrobiaceae (OTU 13), and unclassified Deltaproteobacteria (OTU 10)  were observed, in addition to representatives of the Lokiarchaeota, Woesearchaeota, Thaumarchaeota and Bathyarchaeota. At SukS, Bathyarchaeota-, Thermoplasmatales-, and Thaumarchaeota-related OTUs were present. Additionally, three archaeal OTUs without close relatives  were present in SukB and SukS.Noteworthy distinctions among sites included OTUs affiliated with ; and only one iTag-related sequence was affiliated with Crenarchaeota. Methanogen representatives, Methanosarcinales, Methanomicrobiales, Methanocellales, and a new order of methanogens related to the Thermoplasmatales  indicated that thermokarst lake sediment Archaea from 2010 and 2011 samples span the a wide range of Archaea phyla recognized to date Figure . Only a 4 signature and higher levels of TOC and iron (Fe) compared to Sukok, which has a natural gas seep near SukS with a thermogenic CH4 signature, and higher levels of nitrate , we focused our attention on these contrasting coring sites. Thus, we hypothesized that the microbial assemblage composition, the potentially active fraction (indicated by high aRNA:DNA levels) in particular, differed following geochemistry.We set out to study the microbial assemblages that may be actively involved in geochemical processes relevant to thermokarst lake sediments. Siq has been reported to have a biogenic CHNMDS patterns based on DNA and aRNA suggested differences in microbial assemblage structure among the three sites that were consistent with geochemical profiling of sediment cores  in both Sukok sites, suggested different processes at each site.Diverse bacterial assemblages with taxa that may be actively involved in C, S, and N cycling were found at all three sediment coring sites. Differences in biogeochemical functional potential between the sites were revealed using rRNA-derived sequence analyses. Group 1 OTUs demonstrated this pattern, where N- or S-cycling Nitrospirae in Siqlukaq; photosynthetic, NMethylobacter and Crenothrix. Some Crenothrix can be responsible for the oxidation of dissolved iron (Fe2+) . The discrepancy in taxonomic affiliations are currently unresolved—regardless, both Crenothrix and Methylobacter are type I methanotrophs. M. tundripaludum is a psychrophile isolated from High Arctic wetland soil  oxidizers , Siq contained G1 Nitrospirae affiliated OTUs, not closely related to cultivated members of this group. Nitrospirae are metabolically versatile bacteria that include chemoautotrophic nitrite  and Siq G2), and are known to be associated with aerobic and anaerobic cellulose, chitin, and pectin degradation  in the lake sediments. The potential for reducing 2S) could be inferred from the presence of groups known to perform these functions, with high aRNA to DNA ratios at both Sukok coring sites. Conversely, at the Siq coring site, Syntrophaceae (G2 OTUs) and Hydrogenophilaceae (G2 OTUs) were the OTUs that could potentially be attributed to S cycling. The Hydrogenophilaceae OTU was affiliated with Thiobacillus, chemoautotrophic bacteria capable of coupling Fe or S oxidation with denitrification , is a diverse (76–87.7% intragroup similarity of the SSU rRNA gene) and ubiquitous group of Archaea , Woesearchaeota, and Lokiarchaeota, while OTUs in SukS were limited to Bathyarchaeota, Thaumarchaeota, and Thermoplasmatales (AMOS1A). The presence of OTUs affiliated with ANME-2d in SukB suggested the potential for anaerobic CH4 oxidation in the sediments, as observed in organisms (Candidatus Methanoperedens nitroreducens) that belong to this group , Thaumarchaeota (e.g., Archaeal phylogenetic analysis also revealed extensive diversity across several poorly characterized and recently recognized archaeal phyla. Here, we found sequences affiliated with recently proposed phyla Woesearchaeota and Pacearchaeota (Baker et al., 4 production and consumption, not to mention currently unrecognized roles of some pervasive new phyla now recognized in these sediments. Arctic thermokarst lakes are sensitive to climate warming (Osterkamp, In summary, our work revealed distinctive bacterial assemblages in the three sites studied, which are also implicated in different biogeochemical processes. Moreover, the high diversity of Archaea observed argues for further study to establish their roles in CHPM, CH, KH, JP, and AM contributed to the design of the study, analysis of the data, drafting of the manuscript, and approval of the content. All agree to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, tracking of spheroid growth and treatment-induced volume reduction has not been readily adopted. Here, squamous carcinoma cells were seeded at different starting cell numbers with growth and reduction kinetics monitored using live cell imaging. Following the initial growth phase, spheroids were treated with auristatin as small molecule (MMAE) or as antibody-drug conjugate containing non-cleavable auristatin drug payload (033-F). Compared to cells in monolayers, 033-F had notably weaker potency against spheroids despite potency levels of MMAE being similar against monolayers and spheroids. Accumulation of released payload from 033-F was reduced in higher volume spheroids, likely contributing to the potency differences. Despite lowered potency towards spheroids with 033-F, spheroid volume was still readily reduced by 033-F in a dose-dependent fashion, with >85% volume reductions at the highest concentrations for all spheroid sizes. Additionally, the core of the larger spheroids showed more resiliency towards microtubule inhibition. Overall, this work highlights how various  While many crucial molecular and cellular biology phenomena have been elucidated using cells cultivated on 2D surfaces, cell-to-cell communications, spatial orientation in the tissue, cell interactions with the surrounding matrix, and physiological signaling cannot be properly recreated1. Growing cells into three-dimensional (3D) spheroids can better capture aspects of tumor biology such as regions of high oxygen, nutrients, and subsequently proliferation, as well as regions of low nutrients and hypoxia that can lead to cell quiescence and ultimately necrosis2. A wide number of options for generating 3D spheroids has been put forth in the literature, including hanging drop, matrix-based methods, spinner flasks, and ultra-low adhesion plates, with each having its particular advantages and disadvantages3.In their native space, tumor cells experience interactions with neighboring cells over their entire surface area. This environment is vastly different than the conditions used for 4. More predictive models of drug efficacy and toxicity would help filter out candidates with a low chance of clinical success. Currently, many of the early-stage drug screens are performed with traditional 2D monolayers. Preclinical validation experiments typically lack many characteristics of the natural tumor milieu, instead presenting an environment that can be a stark departure from an in vivo tumor. Previous and current 2D screening paradigms may introduce significant biases during the initial selection stages of early drug discovery ‘hit-to-lead’ cellular screening approaches5, potentially advancing drug molecules with limited potential or even excluding promising drug molecules from moving further forward in drug development. Spheroids on the other hand have been successfully used to better recreate the gene expression patterns of in vivo tissues7 as well as predict in vivo tumor sensitivity to drug previously missed by 2D screens8.Due to approval rates under 10% for new therapeutics in oncology, improved methods of screening are needed to find better drugs to bring into clinical studies9. Antibody therapeutics typically only need to bind extracellular antigen to elicit an anti-cancer effect, whereas the majority of ADCs need to be brought into the cell in order to release the active drug payload. Having a preclinical evaluation system in place that incorporates aspects of tumor penetration, realistic antigen behavior, and small molecule-mediated cell killing over a relevant time period is important to fully evaluate whether the ADC is a viable drug candidate. For example, the number of antigens present on the cell surface becomes instrumental in dictating possible efficacy; levels of the frequently targeted antigens used in a number of ADCs  have been shown to change between 2D and 3D models10. Furthermore, certain spheroids can also have changed internalization biology, producing significantly altered rates of intracellular drug accumulation11.Different types of therapeutics pose additional new challenges. Protein therapeutics are significantly larger than small molecules in both molecular weight and physical radius. The ability to more closely replicate the natural physiological environment during screening may allow researchers to better understand key differences in how well a biologic can penetrate a tumor. Such considerations become especially pronounced for more complicated solid tumor therapeutics. For example, antibody-drug conjugates (ADCs) combine elements of receptor targeting via a specific antibody with small molecule inhibition in an effort to mediate greater efficacy than antibody or small molecule inhibition alone12 or efficacy determination challenges13. Because of the massive scale of many industrial drug development activities, reproducibility, cost, and ease of setup are paramount in the successful rollout of 3D technologies14. Successes have therefore focused on high-throughput methods providing both ease-of-use and compatibility with automation, which to-date has meant using ultra-low adhesion plates for spheroids formation and imaging systems or viability measurements for drug potency determination16. However, the current mainstream techniques do not capture time-based dynamics of spheroid size, missing valuable information regarding the rate and completeness of spheroid regression17. Limited data is also available regarding the influence of spheroid size on overall drug potency. Here, a high-content approach using low adhesion culture plates and live cell imaging technology was used to characterize the sensitivity of squamous carcinoma cell spheroids to ADCs and small molecules at varying plating densities. This proof-of-concept approach represents the first high-content analysis of spheroid volume over time following ADC treatment and sets the stage for more advanced future preclinical assessmentsWhile 3D platforms offer demonstrable benefits over standard 2D-based cell culture methods, the use of 3D technologies in drug discovery and screening efforts has been minimal, often due to throughput18. Here, A431 human squamous carcinoma cells expressing a fluorescent red nuclear protein were grown on round bottom ultra-low adhesion plates. The plated cells quickly came together into a spheroid, with a well-formed spherical shape observable in less than 12 hr  of epidermal growth factor receptor (EGFR) on the cell surface20. Our in-house produced version of cetuximab, Ab033 (same sequence but slightly different glycosylation pattern than cetuximab), was used to target EGFR22. Although cetuximab and Ab033 are cytotoxic to A431 cells as monoclonal antibodies24, Ab033 was additionally armed here with a non-cleavable payload featuring mono-methyl auristatin F attached to the reduced cysteines of Ab033 via maleimidocaproyl at an average drug-to-antibody ratio (DAR) of 4 (referred to here as 033-F). The intent of adding auristatin molecules to the 033 antibody was to investigate the platform as a potential system for assessing ADCs in vitro as well as further promote cell death through inhibition of microtubules by the cysteine-maleimidocaproyl-mono-methyl auristatin F (cys-F) payload that is released from 033-F following lysosomal degradation25. The non-cleavable, low permeability cys-F payload was selected to ensure any cell killing or spheroid reduction stemming from microtubule inhibition was a result of internalization and payload release within the cells and not bystander effect. Overall, 033-F has multiple mechanisms  that can result in cell killing, with these mechanisms perhaps even working synergistically.A431 squamous carcinoma cells have been shown to express high levels , A431 monolayers were treated with either an ADC, a drug linker, or a small molecule: the forms used were the 033-F ADC, the low permeability 033-F release product cys-F , or the permeable auristatin drug variant mono-methyl auristatin E (MMAE)ely Fig. . ConversTo compare potency with the spheroid format, A431 cells were next seeded at a middle range of cell concentrations, with 800 cells plated per well and allowed to form spheroids and grown for 9 days. The matured spheroids were then treated with 033-F and MMAE for 14 days. Dose-proportional responses were observed for both treatment types Fig. . IC50 va27. As spheroids and monolayers often have substantially different growth rates28, additional analyses were needed to determine if growth rate differences were influencing cellular potency IC50 values. By using the normalized growth rate inhibition (GR) values that have been introduced by Sorger’s group over the last few years29, the influence of division rates on potency metrics can be minimized29. GR values were therefore calculated for both spheroid and monolayer data , traditional potency metrics may not be suitable for comparing the sensitivity of the different growth conditions to each other31. For antigen levels above a sufficient amount, antibody cannot permeate across the entire spheroid due to the high levels of antigen acting as a sink and is instead confined to the outer edges of the spheroid. Limited tumor penetration of both small molecule and ADCs is also in line with observations of spheroid cell killing by both 033-F and MMAE occurring at the periphery and then slowly working inwards over time  and shapes (e.g. round versus oblong), making such generalizations of limited utility3. For studies considering cells that produce spheroids that are not well-defined in shape (and many cell types do produce irregular shaped 3D tumors)14, the general workflow used here would still work; however, special and perhaps more elaborate mathematical considerations would need to be undertaken to properly define the geometry of the spheroid.With these results in mind, any of the spheroid sizes used here could provide useful data for drug discovery efforts in the future, although the end objective and type of drug modality being used should guide study design. For example, if antibodies with different binding affinity are being compared, a larger spheroid model may be more insightful due to tumor penetration potentially affecting efficacy outcomes 42. Advancements in spheroid design have already been described in the literature, including angiogenesis models where endothelial networks are introduced into tumor spheroids to mimic the role that endothelial cells play in the tumor microenvironment43. Vascularization of the microtumors in an in vitro system has been demonstrated and could represent a major advance in the setup for in vitro experimentation44. Future studies could involve drug screening on intricately designed vascularized systems for more physiological relevance in vitro outcomes45. Lastly, great strides have been made in the areas of microfluidics46 and patient-derived spheroids47, so much so that one could envision future personalized clinical trials occurring in spheroids without the need for preclinical species48. In summary, the application of 3D cell culture and organs-on-a-chip for preclinical drug screening represents an exciting opportunity to accelerate the pace and success of drug discovery and development49.The work presented here showed that we were able to robustly and reproducibly grow spheroids in ultra-low adhesion plates and then perform long-term treatments of the spheroids with ADC and small molecule drug inhibitors. Through high-content imaging, large amounts of spheroid images could be taken over time and used to capture the spheroid volume kinetics. These data provide an informative look at the spheroid response to cytotoxic agents over time. Looking forward, this work could be readily expanded to high-throughput screening for potency in a variety of cancer cell models as well as included in preclinical safety testing on organoid models of normal tissues. To then further develop even more accurate representations of the natural cellular environment, co-cultures or organ-on-a-chip systems could be incorporated alongside live cell imaging16. Spheroids were formed by plating a defined number of cells onto clear round bottom ultra-low attachment 96 well plates (Corning) and centrifuging at 300 × g for 10 min. Spheroids were plated in the same manner regardless of initial starting cell numbers.A431 cells were obtained from American Type Culture Collection. Cells and spheroids were grown in RPMI-1640 media supplemented with 10% fetal bovine serum. The cells were transfected with lentivirus to stably express mKate2 nuclear red fluorescent protein through the constitutive promoter EF-1α . Cells were at roughly 25% confluency at the time of infection. A multiplicity of infection of 3 was used and the cells were incubated with the lentivirus for 24 hr. The media was changed to fresh growth media and the cells were expanded for 24 hr prior to selection with puromycin. The high expression of EGFR by A431 cells was confirmed using flow cytometry21. 033-F was produced by incubating Ab033 in DPBS with two equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) on ice for 5 hours and then incubating with cys-F at room temperature. Overall DAR was measured by hydrophobic interaction chromatography and confirmed to be an average of four.Ab033 was produced as described previouslyAn Incucyte Zoom system (Essen Bioscience) was used for live cell imaging of cells and spheroids. Images were taken of each spheroid either every hour or every other hour. The round bottom plates allowed the newly formed spheroids to settle into a predictable location at the center of the well such that imaging could be accomplished by the Incucyte on a single image per well. The spherical shape that A431 cells take when grown as spheroids permitted diameters of the spheroid to be defined through straightforward geometric calculations. The total red area per image was used in calculating spheroid diameter by solving for diameter of a circle when the area of a circle is known. To obtain the total area of the red signal, a fixed threshold of 10 mean image fluorescence (RCU was used to differentiate spheroid from background). This value was higher than the typical signal a single cell would produce, which allowed for any single cells on the outer spheroid perimeter, as well as dead cells, to be excluded during spheroid area calculations. Occasionally an Incucyte image was found to be out of focus, with the out of focus image leading to a large discrepancy in values between adjacent time points. When this occurred, the unfocused image was excluded from the data set.3 cells expressing red fluorescent nuclear protein on 96 well cell culture-treated plates. The cells adhered overnight prior to addition of drug and transfer to the live cell imager. As with the spheroids, the total red area signal was used to determine growth inhibition. A fixed threshold of 1.5 RCU was used to differentiate cells from background signal or debris.Drug potency determination in monolayers was performed by plating 3 × 102 column was used with a 0.9 min LC gradient. Mobile Phase A was composed of HPLC grade water with 0.1% Formic Acid. Mobile Phase B consisted of HPLC grade acetonitrile with 0.1% Formic Acid. The selective reaction monitoring (SRM) transition used for cysteine-maleimidocaproyl-monomethyl auristatin F was set to 1046.6 m/z for the precursor mass and 428.1 m/z for the fragment mass. Standard curves were run alongside samples for conversion to drug concentrations.For each replicate and spheroid size, 12 spheroids were collected from individual wells and pooled together. The spheroids were centrifuged, and media supernatant was aspirated. The collection of spheroids was then washed once with phosphate-buffered saline, centrifuged, and supernatant aspirated. Cold organic solvent composed of 95:5 acetonitrile and methanol with 50 nM carbutamide was added to the spheroid pellet at a volume of 90 µL and vortexed. The mixture was centrifuged at 3500 RPM to pellet precipitated matter and the supernatant transferred to a plate containing both water and dimethyl sulfoxide (DMSO) for mass spectrometry analysis at a final composition of 18:2:1 organic solvent, water, and DMSO, respectively. A Sciex 5500 triple quadrupole mass spectrometer coupled to an Agilent 1290 liquid chromatography system with a CTC PAL autosampler was used for the liquid-chromatography tandem mass spectrometry (LC-MS/MS) analysis of free drug. A Kinetex 5 µm 100 A C18, 30 × 2.1 mm50.Values from live cell imaging experiments were imported into Prism 5 . Least squares curve fits were obtained for potency calculations by inputting log-transformed data into the nonlinear regression function for log(agonist) versus response with variable slope. Statistical significance was calculated in Prism as well. All GR-related values (GR50 and Emax) were calculated using an online tool named GRcalculatorSupplementary informationSupplementary information2Supplementary information3Supplementary information4Supplementary information5Supplementary information6


Heterosis, a multigenic complex trait extrapolated as sum total of many phenotypic features, is widely utilized phenomenon in agricultural crops for about a century. It is mainly focused on establishing vigorous cultivars with the fact that its deployment in crops necessitates the perspective of genomic impressions on prior selection for metric traits. In spite of extensive investigations, the actual mysterious genetic basis of heterosis is yet to unravel. Contemporary crop breeding is aimed at enhanced crop production overcoming former achievements. Leading cotton improvement programs remained handicapped to attain significant accomplishments.1 hybrids derived from Line × Tester mating design were evaluated under 10 diverse environments. Heterosis, GCA and SCA were estimated from morphological and fiber quality traits by L × T analysis. For the exploration of elite marker alleles related to heterosis and to provide the material carrying such multiple alleles the mentioned three dependent variables along with trait phenotype values were executed for association study aided by microsatellites in mixed linear model based on population structure and linkage disequilibrium analysis. Highly significant 46 microsatellites were discovered in association with the fiber and yield related traits under study. It was observed that two-thirds of the highly significant associated microsatellites related to fiber quality were distributed on D sub-genome, including some with pleiotropic effect. Newly discovered 32 hQTLs related to fiber quality traits are one of prominent findings from current study. A set of 96 exclusively favorable alleles were discovered and C tester (A971Bt) posited a major contributor of these alleles primarily associated with fiber quality.In mentioned context, a comprehensive project was designed involving a large collection of cotton accessions including 284 lines, 5 testers along with their respective FHence, to uncover hidden facts lying within heterosis phenomenon, discovery of additional hQTLs is required to improve fibre quality. To grab prominent improvement in influenced fiber quality and yield traits, we suggest the A971 Bt cotton cultivar as fundamental element in advance breeding programs as a parent of choice.The online version of this article (10.1186/s12864-018-5129-4) contains supplementary material, which is available to authorized users. Gossypium covers economically sustainable and diverse amount of diploid as well as tetraploid cotton species grown in most of the regions worldwide  obtained via STRUCTURE with ΔK (ad hoc statistic), K value was precisely estimated . On the 2 of each pair of microsatellites was calculated using the software package TASSEL 2.1 based on rapid permutations in 1000 shuffles with rare alleles  treated as missing data [2 distribution for unlinked markers, which determined whether LD is due to physical linkage, was treated as the background LD level [2 values of each pair of microsatellites were plotted against map distance (Mbp), and LD decay was estimated. By utilizing Sigmaplot version 12.5 an inner fitted trend line i.e., nonlinear logarithmic regression curve was sketched in order to elaborate the affiliation between r2 and Mbp of microsatellites prevailing on single chromosome.To estimate LD pattern in Upland cotton genome, the weighted average of squared correlation coefficient ring data . Every lLD level . The r2 2 values of each significant association. The detail of genotypic and phenotypic data combinations used in the TASSEL analysis is given in Table Mixed linear model (MLM) was used to construct markers-fiber quality trait association tests using the TASSEL 2.0.1 software package . For theSignificantly, associated loci were further scrutinized for determining the favorable alleles respective of their targeted traits on the basis of association results previously obtained. This phenotypic effect value was calculated through comparison between the average phenotypic value over genotypes with specified allele and that of all genotypes:However,ai: phenotypic effect of the ith alleleij: phenotypic value over the jth accession with the ith allelexni: number of accessions with the ith alleleNk: phenotypic value over all accessionsnk: number of accessionsai came larger than zero then allele was considered with a positive effect, otherwise with negative effect.If value for Additional file 1:1s. (DOCX 19 kb)Estimates of the weighting coefficient (Eigen vector) associated with the principal components and different characters of Parents and FAdditional file 2:Summary of significant associations between markers and Phenotypic traits. (XLS 44 kb)Additional file 3:Association of fiber quality and agronomic traits with microsatellites (XLS 56 kb)Additional file 4:Association table displaying 46 microsatellites significantly (log10 > 3) associated with fiber quality and agronomic traits. (XLS 110 kb)


SCGN gene causing Mendelian early-onset ulcerative colitis. SCGN encodes a calcium sensor that is exclusively expressed in neuroendocrine lineages, including enteroendocrine cells and gut neurons. SCGN interacts with the SNARE complex, which is required for vesicle fusion with the plasma membrane. We show that the SCGN mutation identified impacted the localization of the SNARE complex partner, SNAP25, leading to impaired hormone release. Finally, we show that mouse models of Scgn deficiency recapitulate impaired hormone release and susceptibility to DSS-induced colitis. Altogether, these studies demonstrate that functional deficiency in SCGN can result in intestinal inflammation and implicates the neuroendocrine cellular compartment in IBD.Inflammatory bowel disease (IBD) affects 1.5–3.0 million people in the United States. IBD is genetically determined and many common risk alleles have been identified. Yet, a large proportion of genetic predisposition remains unexplained. In this study, we report the identification of an ultra rare missense variant  in the  Inflammatory bowel disease (IBD) is a condition that results from both genetic predisposition and environmental exposures, which has become increasingly prevalent with the advent of industrialization around the world . Twin coThe genes implicated in both GWAS and familial studies are enriched for pathways that are critical in host-microbiome interactions. This is likely the case because the luminal aspect of the intestine represents the largest epithelial surface in the body  and housNEUROG3, the transcription factor required for EEC differentiation, have been described in patients with chronic diarrhea secondary to malabsorption without an inflammatory component  that was shared by the three probands and not their unaffected siblings. This variant mapped to one of the segments of shared LOH on chromosome 6. To ensure additional candidate variants were not missed in areas of low WES coverage within the proband-shared LOH regions, we performed whole-genome sequencing (WGS) of two of the affected probands (P1 and P2). WGS confirmed that the SCGN variant is the only rare homozygous nonsynonymous candidate variant in the segments of shared LOH among these probands. Finally, familial segregation of the SCGN p.R77H mutation was confirmed by Sanger sequencing, showing that both parents were heterozygote carriers, and the unaffected siblings were either a heterozygote carrier or had reference alleles, as per WES results  and copy number variants . Next, w results .The allelic frequency for the variant found in affected individuals in this family is very rare . As Hispanics are generally underrepresented among public genomic databases, we genotyped 2,000 Hispanic individuals (mostly of Mexican origin) from the Dallas Heart study . The mutSCGN reported in ClinVar are large copy number variants involving dozens to hundreds of genes, with phenotypes associated with congenital anomalies and intellectual disabilities .Further screening of different databases  indicates that there are no individuals identified thus far with predicted homozygous essential loss of function variant (stop codon/frameshift). The only potentially deleterious homozygous variant found was a nucleotide deletion (c.83-1delG) predicted to affect a splice acceptor site 5’ upstream of exon 2 (rs60502981). This allele is enriched among individuals of African descent, with an allele frequency of 2.8% in this population: four homozygous individuals  are reported in ExAC . Importantly, ClinVar does not include this variant and validation of its predicted effect on splicing or gene function is currently lacking. In fact, all variants of SCGN gene, is a calcium sensing protein predicted to have 6 EF–hand domains  clones that displayed loss of protein expression  or the p.R77H variant  mouse model by targeting the third exon of murine Scgn using CRISPR/Cas9 technology. Multiple pronuclear injections of Cas9 mRNA and custom synthetic gRNA led to numerous independent C57BL/6J Scgn targeted founder mice harboring different indels within the third exon of Scgn. Two independent founder lineages were selected (Secret1 and Secret2), each with Scgn deletions that could be easily detected by PCR-based genotyping (Secret1 and Secret2 mice expressed a truncated transcript skipping exon 3 of the gene (Given the identification of notyping . Founderthe gene . Immunofthe gene .Scgn deficient mice were generated and cohoused in a specific-pathogen-free (SPF) environment. At baseline, Scgn deficient mice (Secret1 and Secret2) did not develop any overt phenotype, including no obvious architectural abnormality in the colonic epithelium as seen in HE or alcian blue staining  and staining , and no staining . Furtheret1 mice .Scgn-deficient mice to DSS-induced colitis, a widely used model of acute colitis in mice. Importantly, most genetic defects thought to lead to increased IBD susceptibility in humans also display colitis sensitivity in this model  colon explants, but not from colons of EEC ablated (Neurog3ΔCol) littermates. In addition, GLP-1 and GLP-2 release in response to DHA was completely blunted in colon explants from Secret1 homozygous animals, consistent with the role of SCGN in this response , our patients did not display any other clinically overt phenotypes outside of colitis. Interestingly, mutations in other regulators of the SNARE complex, namely STXBP2, can lead to altered NK cell degranulation, hemophagocytic lymphohistiocytosis and colitis . Intriguent mice , the fasficiency . While tSCGN deficiency.Unlike STXBP2, SCGN protein is absent from the immune system, pointing specifically to neuroendocrine cells as the likely source of pathology in these patients. It is important to note that both the EEC and neuronal compartments have been shown to be highly interconnected, with inputs reaching the central nervous system . EECs plDNA was extracted from 5 mL of whole blood collected in EDTA coated tubes using an automated QIAGEN AUTOPURE LS – large sample nucleic acid purification instrument. DNA concentration and quality was assessed on a nanodrop instrument. DNA was aliquoted and stored at −20°C until use. DNA samples from probands and their unaffected siblings were genotyped for common variants along the genome using the Infinium HumanCoreExome Beadchip SNP array (Illumina), which includes 264,909 tag SNP markers . Genotyping was performed by the McDermott Center for Human Genetics according to the manufacturer’s standard procedures. Areas of loss of heterozygosity that were shared by probands and not their unaffected siblings were identified as potential regions harboring a recessive allele responsible for disease linkage.Three micrograms of genomic DNA isolated from peripheral blood were used for whole-exome sequencing. The exome was captured using the Agilent SureSelectXT Human All Exon V4 Kit and sequenced on the Illumina HiSeq 2500 generating 100 bp paired-end reads. Variant calls were filtered to only include exonic, non-synonymous changes with minor allele frequencies < 1% in ExAC, 1000 genomes and TOPMED. We filtered further to include variants that were exclusively shared by all probands in a homozygous manner and present at most in a heterozygote manner in unaffected siblings. The analysis was conducted both at the genome-wide level and subsequently, the variants were reanalyzed focusing on areas of LOH identified by genome-wide SNP arrays as noted above. Because of gaps in WES capture affecting the areas of LOH, DNA samples from two probands were subsequently subjected to whole genome sequencing (Complete Genomics) and repeat analysis for rare variants (MAF <1%) in the segments of LOH that were shared between the two probands was performed. Furthermore, using WGS data, we remapped LOH segments. Heterozygous and homozygous variants were identified and assigned into 100 kb windows throughout the 6 p and 12q chromosome arms. Average heterozygosity was calculated for each window and plotted along the chromosome arm.Samples from 2000 Hispanic individuals enrolled in the Dallas Heart study were used for direct genotyping for rs376721140 using TaqMan probes flanking the area of interest. The assays were performed by the McDermott Center for Human Genetics using an Applied Biosystems 7900HT real time PCR instrument.SCGN cDNA was purchased from Open Biosystems (MHS6278-202826064). Forward and reverse primers containing sites for BamHI and NotI restriction enzymes respectively, were used to PCR amplify the coding sequence for SCGN. The product was digested with BamHI and NotI and ligated into the multicloning site of the pEBB vector. The inserted sequence (SCGN) was validated by Sanger sequencing. SCGN R77H was generated using a site-directed mutagenesis kit (Agilent), and the mutation was confirmed by Sanger sequencing. The coding sequences for SCGN (WT or R77H) were subcloned into the FG9 lentiviral expression system containing a N-terminus 2xHA tag, as described before , lentiviral CRISPR constructs (Lenti CRISPR system) were generated, as previously reported (STC-1 cells were obtained from ATCC (Cat No. CRL-3254) and grown in culture using DMEM supplemented with 10% FBS. Identity testing was not performed as these cells were directly obtained from the vendor. Cells were tested and found to be Mycoplasma-free at time of experiments. To target the reported . STC-1 cImmunofluorescence staining of STC-1 cells was carried out as previously described . Briefly2, 1.2 mM MgCl2, 20 mM Dextrose) before adding 400 µL of DHA-containing media. To prepare DHA-containing media, stock DHA (nu-chek U-84-A) solutions were prepared in 100% ethanol at a concentration of 10 mg/mL and frozen at −20°C until used. DHA-containing media at a concentration of 100 µM was prepared on the day of experiment by addition to a HEPES buffer, which was sonicated for 5 min prior to addition to cells. Cell supernatants were collected after 15 min and GLP-1 concentrations were determined using an ELISA kit . To account for possible differences in cellular density, ELISA results were normalized to total protein content (measured by Bradford assay) after cellular lysis of the corresponding wells.All cells were counted and seeded on 6-well plates at equal density. Once cells were confluent they were placed in serum free media for a 2 hr period and washed twice with HEPES buffer  into 1 cell stage blastocysts, together with a tp53 MO to avoid the non-specific effects from injection (scgn MO was coinjected with mRNAs encoding either human SCGN wild-type or R77H into blastocysts of one-cell stage. Protein re-expression was confirmed by western blot (data not shown). In situ hybridization was carried out as previously described  and Cas9 mRNA (Sigma). Mice were generated on a C57BL/6 background and resulting progeny was characterized for potential targeting of the Scgn allele by PCR amplification of a 214 bp region flanking the site of sgRNA cleavage. The amplified product was subjected to Sanger sequencing. Two founder mice with Scgn mutations resulting in significant deletions at this site, referred to here as Secret1 and Secret2 (Mice were kept in a specific pathogen free (SPF) barrier facility, in a standard 12 hour day cycle and fed standard irradiated chow.  Secret2 , were se Secret2 .Neurog3 floxed mice, a kind gift from Andrew Leiter with authorization from Gérard Gradwohl.Colonic EEC deficient animals were generated by mating the CDX2 Cre driver line (Jax 009350) against Acute colitis was induced by providing a 6 day course of DSS (Alfa aesar J63606-22) in drinking water at a concentration of 1%, 2% or 3% . DSS solution was replaced every three days. On 10 day long DSS experiments, drinking water was replaced with autoclaved water as indicated on experimental figures. Mice of different genotypes were cohoused at the time of DSS experiments. Weight was recorded on a daily basis. Disease activity index (DAI) was calculated on day seven in a blinded fashion by adding individual scores from stool consistency, presence of blood in stool and weight change, as previously described  Cooper .Tissue analysis from patient samples involved archived intestinal biopsy tissue blocks from control and affected individuals. Tissues from experimental animals were freshly isolated after euthanasia and fixed overnight in freshly prepared 4% PFA, to be later embedded in paraffin. For all staining, 4 µm sections were utilized. Hematoxylin and eosin (HE) staining was performed by standard methods. For immunostaining, patient slides were stained using the Dako Omnis automated system, whereas slides obtained from experimental animals were manually stained. Briefly, the sections were first deparaffinized and rehydrated by serial xylene and decreasing alcohol concentration immersion. Antigen retrieval was performed by heat-induced epitope retrieval and citrate solution. Slides were incubated in blocking buffer for 45 min (PBS and 3% goat serum – vector labs), and then incubated overnight at 4°C in primary antibodies diluted in blocking buffer. Primary antibodies used for tissue immunostaining included the following: Secretagogin (Santa Cruz biotechnology sc-374355), chromogranin A (Abcam ab15160), chromogranin B (Abcam ab12242), 5-HT (Immunostar 20080), SNAP25 (Abcam ac5666), GCG (Santa Cruz biotechnology sc-514592), Tuj1 (Biolegend 801213) and synaptophysin (Abcam ab32127). Slides were washed in PBS three times (5 min each), and then incubated for 30 min in secondary antibodies in blocking buffer. Secondary antibodies used included goat anti-mouse alexa fluor 488 (Invitrogen) and goat anti-rabbit alexa fluor 555 (invitrogen). Slides were then washed in PBS on two occasions and then incubated for 10 min in PBS and Hoechst at 1:10,000 dilution. Slides were washed in PBS on three additional occasions prior to mounting using gold antifade reagent (Invitrogen).For DSS colitis histological severity scoring, HE slides were reviewed by a GI pathologist in a blinded fashion. Tissues were scored using a modified scoring system  generate2, 4.6 mM KCl, 120 mM NaCl, 0.7 mM Na2HPO4, 1.5 mM NaH2PO4, 15 mM NaHCO3). The intestinal segments were then filled with 1 mL KRB buffer containing 10 mM EDTA and 1 mM DTT and tied off on either end. These segments were placed in a sealed 50 mL conical tube containing 30 mL of KRB buffer and placed in a rocking deck incubator at 220 RPM for 40 min at 37°C. The luminal contents were collected into 1.5 mL microfuge tubes and washed three times with KRB buffer prior to storing in RNAlater solution at −80°C. RNA was then extracted following instructions by the manufacturer.For DSS experiments, cecal tissue was collected at the end of the experiments and immediately stored in RNA later solution (QIAGEN), following the manufacturer’s instructions. RNA stabilized tissue was later thawed and 20 mg of tissue was disrupted using a glass tissue douncer and homogenized using QIAshredder columns prior to RNA extraction using RNeasy spin columns (QIAGEN). For preparation of small bowel and colonic epithelial isolates, the entire small bowel or colon were carefully excised and intestinal contents rinsed with KRB buffer . We carried out quantitative real time PCR using SYBR green mix. Primers used for RNA Seq was performed as previously reported . BrieflySecret1) were euthanized and the colon was immediately excised. Intestinal contents rinsed with ice-cold incubation buffer , as previously described  using an Illumina MiSeq platform. Sequencing data were then subjected to standard QIIME2 pipeline workflow  cells were isolated as described before . In briet test was utilized to parametric variables between two groups. The log rank test was calculated for statistical analysis of survival rates. Statistical analysis of 16S sequencing was performed as described above. For zebrafish experiments statistical analyses were performed using one-way ANOVA, Tukey's multiple comparisons test incorporated in Prism 7 (GraphPad Software).The unpaired Student’s All human studies were carried out in accordance with UT Southwestern Medical Center institutional review board guidelines under an approved protocol (STU 112010–130). All subjects agreed to participation and written informed consent was obtained from all participants or legal guardians. Assent was obtained from individuals older than 10 years of age at time of enrollment. Murine studies were approved by the UT Southwestern Institutional Animal Care and Use Committee under study number APN 102011. All zebrafish (Danio rerio) experiments were performed according to standard procedures, and were performed in accordance with the guidelines of the animal ethical committee of Sichuan University. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.Acceptance summary:We are grateful that you have chosen to publish your data with us, showing that a rare secretagogin variant  variant is linked to colitis and proctitis in sib offspring from consanguineous partners. Your investigations with SCGN variants and lentiviral complementation in a cell line that secretes GLP-1 has shown the that the variant has hypomorphic activity and your genetic survey shows that it affects an EF hand motif, consistent with calcium-dependent signaling. Cellular staining and immunoprecipitation results show the defective association with SNAP25 and cellular localization, especially on the plasma membrane. Your in vivo work demonstrates that the SCGN enteroendocrine cells are predominantly in the large intestine, and that murine SCGN deletion increases the susceptibility to dextran sulfate sodium-induced colonic injury and inflammation.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Wendy Garrett as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Holm H Uhlig (Reviewer #3).Thank you for submitting your article "SCGN deficiency results in colitis susceptibility" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This study shows that a rare secretagogin (SCGN) variant is linked to colitis and proctitis in three sib offspring from consanguineous partners. Further work with SCGN variants and lentiviral complementation in a cell line the secretes GLP-1 has shown the hypomorphism of the variant and a genetic survey shows that this is placed in a EF hand motif, consistent with calcium-dependent signaling. Cellular staining and immunoprecipitation results demonstrate the defective association with SNAP25 and cellular localization, especially on the plasma membrane.The SCGN enteroendocrine cells are shown to be predominantly in the large intestine, and that murine SCGN deletion increases the susceptibility to DSS colitis although deficiency of murine colonic enteroendocrine cells were unable to phenocopy the susceptibility to colitis, so the mechanisms of pro-inflammatory functional SCGN-deficiency and specifically whether neuronal specific SCGN-deficiency promotes colitis in vivo remain unclear.Essential revisions:The reviewers have reached the conclusion that in vivo studies of neuronal specific SCGN deficiency would be beyond the scope of the current manuscript, although any further progress in this direction would be especially welcomed.1) The Abstract should be revised since this is not just a susceptibility gene but a causative monogenic defect with Mendelian inheritance. Also, the Abstract currently says that the SCGN deficiency recapitulates 'colitis susceptibility'. This is strictly true, but the fact that DSS needs to be used as a trigger is very relevant to a correct impression of the result.2) Table 1. shows areas of shared loss-of-heterozygosity (LOH) among affected probands as defined by SNP array – the filter strategy should be provided in more detail and variants listed.3) The authors should screen the different databases whether any other predicted homozygous essential loss of function variant (stop codon/frameshift) is present in public databases. Are any deletions described with neurodevelopment defects in databases such as Clinvar?4) It would be helpful to have some more patient details with the follow-up. Since the phenotype can apparently present with proctitis, it would be important to understand the severity and trajectory of the condition.5) Immunoprecipitation of SCGN in STC-1 cells expressing either wild type or the R77H variants show comparable co-precipitation of endogenous SNAP25 whereas recombinant human SCGNR77H expressed in bacteria displayed reduced SNAP25 in vitro binding – what is the mechanistic basis for this difference? Differential glycosylation?6) Since GLP-2 has been more widely reported to have anti-inflammatory immune properties, could this be the explanation of the SCGN effect (Frontiers in Immunology 2017 01734)? Essential revisions:The reviewers have reached the conclusion that in vivo studies of neuronal specific SCGN deficiency would be beyond the scope of the current manuscript, although any further progress in this direction would be especially welcomed.1) The Abstract should be revised since this is not just a susceptibility gene but a causative monogenic defect with Mendelian inheritance. Also, the Abstract currently says that the SCGN deficiency recapitulates 'colitis susceptibility'. This is strictly true, but the fact that DSS needs to be used as a trigger is very relevant to a correct impression of the result.We have amended the Abstract to reflect the comments above. Please note the changes in the revised manuscript (highlighted in gray).2) Table 1. shows areas of shared loss-of-heterozygosity (LOH) among affected probands as defined by SNP array – the filter strategy should be provided in more detail and variants listed.We have added a table (Supplementary file 1) that lists all copy number variants and LOH regions identified in all 5 individuals tested . Further, we have explained in more detail in the Results section how this data was filtered in order to identify areas of shared LOH among probands . We have also included an analysis for LOH obtained from WGS data from P1 and P2 that confirmed the LOH segments noted by SNP arrays .3) The authors should screen the different databases whether any other predicted homozygous essential loss of function variant (stop codon/frameshift) is present in public databases. Are any deletions described with neurodevelopment defects in databases such as Clinvar?We have performed this suggested analysis and include it now in the paper. For ease of review, we copy that content here:“Further screening of different databases  indicates that there are no individuals identified thus far with predicted homozygous essential loss of function variant (stop codon/frameshift). […] In fact, all variants of SCGN reported in ClinVar are large copy number variants involving dozens to hundreds of genes, with phenotypes involving congenital anomalies and intellectual disabilities .”4) It would be helpful to have some more patient details with the follow-up. Since the phenotype can apparently present with proctitis, it would be important to understand the severity and trajectory of the condition.We have added an additional figure that summarizes the clinical trajectory and course of the disease over time in all affected probands .5) Immunoprecipitation of SCGN in STC-1 cells expressing either wild type or the R77H variants show comparable co-precipitation of endogenous SNAP25 whereas recombinant human SCGNR77H expressed in bacteria displayed reduced SNAP25 in vitro binding – what is the mechanistic basis for this difference? Differential glycosylation?E. coli-generated recombinant protein which binds poorly to SNAP-25, HEK293T-generated protein has minimal to no change in binding ability in pulldown assays using STC-1 cellular lysates as a source of SNAP-25 . Unlike  SNAP-25 .R77H is currently hard to explain structurally. After consideration of the various results, we have decided that for this paper it would be most appropriate to present the clear mislocalization phenotype and leave the in vitro binding data out of this paper until it can be more clearly interpreted.It therefore suggests that there is likely a mammalian modification that may be at play as suggested by the reviewer. Additional work in collaboration with Dr. Da Jia (co-author in this paper) has led to the crystal structure of the SCGN-SNAP25 complex, which is the subject of a separate report. This structure now indicates that the interaction between these proteins is based on a different domain of SCGN, and not EF hand 2 where the R77 residue is located. Therefore, on balance, the decreased binding of recombinant, bacterially made SCGN6) Since GLP-2 has been more widely reported to have anti-inflammatory immune properties, could this be the explanation of the SCGN effect (Frontiers in Immunology 2017 01734)?Scgn deficient animals. Importantly, deletion of all EECs in the colon of Neurog3ΔCol animals leads to as much or more significant defects in GLP-1 and GLP-2 secretion. Since these EEC depleted mice do not have increased sensitivity to DSS colitis, we conclude that defective GLP-2 secretion cannot explain the colitis sensitivity of the Scgn knockout.We appreciate this remark and have performed additional colonic ex-vivo GLP-2 secretion assays to answer this question. Our new data  indicate that GLP-1 and GLP-2 secretion are both limited in


South Africa (SA) has expanded efforts to reduce mother-to-child transmission of HIV (MTCT) to less than 2% at six weeks after birth and to less than 5% at 18 months postpartum by 2016. Despite improved antiretroviral regimens and coverage between 2001 and 2016, there is little data on infant HIV drug resistance. This paper tracks the prevalence of HIV drug resistance patterns amongst HIV infected infants from three nationally representative studies that assessed the effectiveness of national programs to prevent MTCT (PMTCT). The first study was conducted in 2010 , the second from 2011 to 12 (PMTCT Option A policy) and the third from 2012 to 13 (PMTCT Option A policy). From 2010 to 2013, infant non-nucleoside reverse transcriptase inhibitor (NNRTI) exposure increased from single dose to daily throughout breastfeeding; maternal nucleoside reverse transcriptase inhibitor (NRTI) and NNRTI exposure increased with initiation of NNRTI-and NRTI- containing triple antiretroviral therapy (ART) earlier in gestation and at higher CD4 cell counts.Three nationally representative surveys were conducted in 2010, 2011–12 and 2012–13. During the surveys, mothers with known, unknown, or no exposure to antiretrovirals for PMTCT and their infants were included, and MTCT was measured. For this paper, infant dried blood spots (iDBS) from HIV PCR positive infants aged 4–8 weeks, with consent for additional iDBS testing, were analysed for HIV drug resistance at the National Institute of Communicable Diseases (NICD), SA, using an in-house assay validated by the Centers for Disease Control and Prevention (CDC). Total viral nucleic acid was extracted from 2 spots and amplified by nested PCR to generate a ~ 1 kb amplicon that was sequenced using Sanger sequencing technologies. Sequence assembly and editing was performed using RECall v3.p < 0.0001), particularly to the NNRTI drug class. Pooled analyses across all three surveys demonstrated that infants whose mothers received ART showed the highest prevalence of resistance (74%); 26% (21/82) of HIV PCR positive infants with no or undocumented antiretroviral drug (ARV) exposure harboured NNRTI resistance.Overall, HIV-1 drug resistance was detected in 51%  [45–58%]) of HIV PCR positive infants, 37% (95% CI [28–47%]) in 2010, 64% (95% CI [53–74%]) in 2011 and 63% (95% CI [47–77%]) in 2012 (These data demonstrate increasing NNRTI resistance amongst newly-diagnosed infants in a high HIV prevalence setting where maternal ART coverage increased across the years, starting earlier in gestation and at higher CD4 cell counts. This is worrying as lifelong maternal ART coverage for HIV positive pregnant and lactating women is increasing. Also of concern is that resistant virus was detected in HIV positive infants whose mothers were not exposed to ARVs, raising questions about circulating resistant virus. Numbers in this group were too small to assess trends over the three years. From April 2010, Option A was introduced, wherein lifelong maternal ART, using tenofovir (TDF) with 3TC or emtricitabine (FTC) and NVP (or AZT with 3TC and NVP) was initiated if CD4 cell count was < 350 cells/mm3, or AZT prophylaxis offered from 14 weeks gestation with sdNVP and 3 hourly AZT during labour and single dose TDF/FTC postpartum; infants received daily NVP for six weeks (if not breastfed) or till one week post breastfeeding cessation. Under Option A, all HIV-exposed infants received daily nevirapine (NVP) for six weeks if not breastfeeding or until one week following cessation of breastfeeding. This study reports data collected during the implementation of Option A in South Africa. In April 2013, national PMTCT policy transitioned to WHO PMTCT Option B, and in 2015 to PMTCT Option B+; the latter recommends initiation and lifelong continuation of ART amongst all pregnant and lactating women, regardless of their CD4 cell count. Since 2010, antiretroviral treatment guidelines recommended that HIV-infected infants and children less than 3 years of age should be initiated on a protease-inhibitor (PI) based regimen immediately after diagnosis ) in 2010, 64% (95% CI [53–74%]) in 2011 and 63% (95% CI [48–77%]) in 2012 (p = 0.268) but was associated with year of specimen collection . Genotyping PCR amplification in 2012 was 31% lower than previous years, but was consistent across all exposure categories. Rates of NNRTI resistance detection were not affected by changes in any ARV exposure categories over the years .Non-amplification of 47 specimens was not associated with infant ART exposure status (p = 0.034), compared with other PMTCT regimens, Table Analysis of infant resistance in relation to antiretroviral drug exposure demonstrated that NNRTI resistance was detected in 74% (95% CI [56–87%]) of infants exposed to maternal ART plus infant ARV (daily NVP ± AZT), and in 68% (95% CI [55–79%]) of infants exposed to maternal ARV prophylaxis plus infant ARV, Table 3 or ARV prophylaxis from 28 weeks with single dose NVP to one that recommended maternal ART from as soon as possible after diagnosis for women with higher CD4 cell counts (≤350 cells/mm3) with six weeks infant NVP or maternal ARV (AZT) prophylaxis from 14 weeks, and daily infant NVP throughout breastfeeding (Option A). However, prevalence of resistance increased to 64 and 63% in 2011 and 2012 when the national policy had fully transitioned to PMTCT Option A, with increased infant NNRTI post-exposure prophylaxis (six weeks or more compared with sdNVP in the previous policy). Resistance to the NNRTI drug class only, which was part of all PMTCT policies in place during these surveys, with increasing infant NNRTI exposure between 2010 and 2012, was most commonly detected, whilst dual NRTI-NNRTI resistance was low (~ 5%) of specimens overall. The major NNRTI mutations Y181C/I and K103N were the most commonly detected mutations. Resistance to PIs was negligible. Paediatric antiretroviral treatment policies recommend abacavir (NNRTI), 3TC (NNRTI) and Kaletra (PI) in children < 3 years, or abacavir (NNRTI), 3TC (NNRTI) and efavirenz (NNRT) in children > 3 years, therefore we postulate that the increased NNRTI resistance would not impact the efficacy of the PI-containing treatment regimens used to treat early (< 3 years of age) paediatric management within the country. Whilst the NEVEREST study demonstrated that NNRTI resistance could re-emerge after a wash-out period, following re-exposure to the selective pressure of the NNRTI, we do not know what impact this has at programmatic level, in the current setting where children between 42 weeks gestation and 3 months of age are often treated with NVP-containing regimens, before switching to PI- (lopinavir/ritonavir)-containing regimens. None of the infants in our setting were on treatment at the time of HIV diagnosis.We analysed infant HIV drug resistance using specimens from the 2010, 2011–12 and 2012–13 nationally representative South African PMTCT surveys. These measured 3.5, 2.7 and 2.6% MTCT at 4–8 weeks postpartum respectively [Detection of resistance in infants was more common amongst those with documented exposure to maternal and/or infant PMTCT prophylaxis, in that more than two thirds of infants exposed to PMTCT harboured resistance. However, among infants with no documented exposure to PMTCT interventions resistance was detected in 26% of infants.These findings are similar to those reported from other studies performed in the southern African region over the same time frame. NNRTI resistance was detected in 57% PMTCT-exposed HIV-infected infants aged ≤2 years (median age 19 weeks) in the Johannesburg area in 2011 . NationaDrug resistance was further detected in a quarter of specimens with no or undocumented exposure to PMTCT interventions, again similar to previous reports, wherein HIVDR was detected in 9–50% of infants with undocumented PMTCT exposure , 19, posThe low rate of specimen amplification in the 2012 specimen set relative to those collected during the other surveys is concerning, but there does not appear to be significant changes in infant exposure status over the years, nor did this lower rate appear to have had an effect on resistance prevalence estimates. However, the actual numbers tested for resistance in each exposure category by year were small, limiting modelling efforts to determine associations between years and exposure categories.The study is limited by reliance on self-reporting of maternal and infant PMTCT prophylaxis during the time of data collection for the current pregnancy, and lack of information on possible PMTCT exposure during any prior pregnancies. The study further analyses data from infants predominantly exposed to dual prophylaxis or option A and therefore does not provide an understanding of the impact of prolonged daily NVP exposure or option B+ on the prevalence of HIV-1 drug resistance in infants beyond 8 weeks of age. In addition, conventional specimen sequencing strategies were employed to detect resistance mutations in the iDBS specimens. It can be expected that more sensitive technologies such as next generation sequencing will increase the frequency of drug resistant variant detection in these studies.Data from these national surveys illustrate the complex drug-resistance challenges that arise as more efficacious antiretroviral therapy is introduced and at earlier time points. With the policy change to PMTCT Option B+ , efforts to administer treatment early, achieve optimal adherence and monitor virological suppression regularly during pregnancy and lactation are needed to facilitate the elimination of MTCT. In the event of vertical HIV transmission such efforts may prevent maternally-acquired drug resistance or the development of infant HIV drug resistance following ARV exposure.Whilst PMTCT programs have achieved significant reductions in levels of infant HIV infection, these programs should strive to provide optimal care, adherence counselling and virological suppression to pregnant women to further reduce the risk of HIV transmission to infants, and especially the transmission of resistant virus. The high rate of resistance in infants with no or unknown exposure further confirms that history of PMTCT is not the only predictor of resistance. Additionally, the low frequencies of PI resistance, supports the recommendation to treat all infants infected with HIV with PI-based regimens at the outset; however, the poor palatability of PI’s, and questionable safety of PIs in children under the age of 2-weeks to three months and in sick and premature infants, preclude their unconditional use. In the context of PMTCT Option B+, more data are needed on the relationship between maternal ART duration, maternal ART adherence and the transmission of resistant virus to the infant, versus transformation of virus into a resistant form due to infant drug exposure.


The earliest evidence for cheese production in the Mediterranean is revealed by stable carbon isotope analyses of individual fatty acids in pottery residues from the Dalmatian coast of Croatia. Lipid residue data indicate the presence of milk in the earliest pottery, Impressed Ware, by 5700 cal. BCE (7700 BP). In contrast, by 5200 cal BCE (7200 BP), milk was common in refined Figulina pottery, meat was mostly associated with Danilo ware, cheese occurred in Rhyta, and sieves contained fermented dairy, representing strong links between specific function and stylistically distinctive pottery vessels. Genetic data indicate the prevalence of lactose intolerance among early farming populations. However, young children are lactase persistent until after weaning and could consume milk as a relatively pathogen-free and nutrient rich food source, enhancing their chances of survival into adulthood. Fermentation of milk into yogurt and cheese decreases lactose content. The evidence for fermented dairy products by 5200 cal BCE indicates a larger proportion of the population was able to consume dairy products and benefit from their significant nutritional advantages. We suggest that milk and cheese production among Europe’s early farmers reduced infant mortality and helped stimulate demographic shifts that propelled farming communities to expand to northern latitudes. Current data on the transition to agriculture in Europe indicates an episodic spread of farming over ca. 3 millennia, starting about 7000 cal BCE. This expansion has been linked with the availability of milk and milk products as a calorie rich, potentially storable food source . Milk prHere we report the earliest evidence for cheese production in the Adriatic through residue analysis of pottery sherds from two Neolithic village sites . Lipids Archaeological data from northern Dalmatia indicate that farming with a full commitment to plant and animal husbandry as a subsistence system and sizable villages that persisted for centuries were already in place beginning 8000 years ago e.g., –16). Ong. Ong16])Two villages, Pokrovnik and Danilo Bitinj, were occupied during the Early and Middle Neolithic . This pottery is defined by impressed decorations on hand built, globular vessels with rounded bases and range from <5 cm diameter cups to large cooking or storage vessels exceeding diameters of 20 cm . ImpressWithin a few centuries, Middle Neolithic potters began making a new suite of pottery styles with different decorative and technological methods. The presence of increasing technological refinement in Danilo ceramics defines Middle Neolithic (5400–4900 BCE) pottery in Dalmatia . TypicalThree subtypes of pottery are found in the Middle Neolithic. First, figulina, a less common and more refined pottery type, represents up to 5% of pottery assemblages. This ware was crafted by careful processing of local clays to create fine-grained textures. Vessels were fired at high temperatures in oxidizing atmospheres to produce buff-colored wares that were often slipped and painted in decoration . Second,The shifts in pottery form and refinement though time may have been accompanied by either significant changes or specialization in how the types of ware were used. The newly collected and predominantly unwashed potsherds from two Neolithic sites with long occupations prove a unique means to evaluate the co-development of form and function. Here, residue analyses from the different pottery styles sampled across the occupation series are used to elucidate the emerging importance of dairying by early farmers in Dalmatia. This progression suggests shifting dietary factors may have fostered the spread of farming from the Balkans into central Europe.Potsherds from Pokrovnik (n = 27) and Danilo Bitinj (n = 20) consisted of Impressed ware (n = 10), figulina (n = 9), typical Danilo ware (n = 20), rhyta (n = 4), and sieves (n = 4). All samples were collected during recent excavations –16. The AMS radiocarbon dates were determined using bone and carbonized seeds from Pokrovnik and Danilo Bitinj in order to constrain the ages of associated pottery samples. Domestic animal bone samples (n = 8) were prepared in The Pennsylvania State University’s Isotope and Human Palaeoecology laboratory following methods outlined in McClure et al. ,29 and Kδ13C value quadrangles defined by δ13C18:0 versus δ13C16:0 values, as well as the isotopic difference between the two, ) and DCM (unsaturated fatty acids). Because milk fat is rich in acylglycerides, aliquouts of the methanol fractions (underivatized) furthermore were reacted with methanolic sodium hydroxide solution (5% v/v) for about 60 min at 70°C. Upon acidification with hydrochloric acid, hydrolyzed lipids were extracted as ‘free’ fatty acids into DCM before evaporated to dryness under nitrogen and derivatization with BSTFA. Note silylation was used to derivatize oxygen-containing groups (as opposed to methylation) because associated lipids also will be used for molecular radiocarbon analysis, which means a removable derivatizing group is preferable to a more stable derivatizing group  because (1) we still lack comprehensive radiocarbon analyses of the derivatizing groups in either case and (2) this approach circumvents any potential issues with transesterification  and IAEA-CH-7 [polyethylene foil]). Isotopic corrections for carbons added by BSTFA were made via measurements of the δ13C of benzene-1,2-dicarboxylic acid [commonly called phthalic acid (Schimmelmann standards)] after derivatization and then correcting for the mass-balanced δ13C value of derivatization carbon molecules.Within-run precision (1σ) and accuracy were determined by coinjected internal standards and are equal, respectively, to 0.10‰ and 0.11‰ (S1 Table(PDF)Click here for additional data file.S2 TableEN = Early Neolithic; MN = Middle Neolithic. Note: Pokrovnik Trench D level 2 and 3 are from one chronological stratum; Pokrovnik Trench D levels 5, 6, 7, and 8 are all from one chronological stratum . Radioca(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.