L. pneumophila density, respectively. To elucidate the impact of phosphate on NTMaggregation, a comparison of planktonic and aggregate fractions ofNTM cultures grown at varying phosphate concentrations was conducted.Aggregation assay results suggested that higher phosphate concentrationscause more disaggregation, and the interaction between phosphate andNTM is species specific. This work reveals new insight into the consequencesof orthophosphate application on the DWDS microbiome and highlightsthe importance of proactively monitoring the DWDS for DWPIs.Increases in phosphateavailability in drinking water distributionsystems (DWDSs) from the use of phosphate-based corrosion controlstrategies may result in nutrient and microbial community compositionshifts in the DWDS. This study assessed the year-long impacts of full-scaleDWDS orthophosphate addition on both the microbial ecology and densityof drinking-water-associated pathogens that infect the immunocompromised(DWPIs). Using 16S rRNA gene amplicon sequencing and droplet digitalPCR, drinking water microbial community composition and DWPI densitywere examined. Microbial community composition analysis suggestedsignificant compositional changes after the orthophosphate addition.Significant increases in total bacterial density were observed afterorthophosphate addition, likely driven by a 2 log 10 increase in nontuberculousmycobacteria (NTM). Linear effect models confirmed the importanceof phosphate addition with phosphorus concentration explaining 17%and 12% of the variance in NTM and  Limited work has explored the impactsof full-scale distributionsystem orthophosphate lead corrosion control on drinking water microbiomesand pathogen abundance. This study reports changes in drinking waterpathogen abundance and organism impacts after full-scale orthophosphateaddition. Alongside pH and alkalinity adjustments,PO43– is among the most widely used approachesfor corrosion control due to its versatility in waters with variablewater quality parameters, such as pH, alkalinity, and dissolved inorganiccarbon.6 However, theaddition of excess PO43– into the drinkingwater distribution system  may pose an unexpectedchallenge of increased microbial growth due to abundant nutrient availability.Lead contaminationin drinking water supplies in the United Statesis a major concern, as millions of lead service lines remain in use.9 Furthermore,DWDSs are oligotrophic environments in which diverse microbial communitiescompete for limited nutrient availability. Thus, the introductionof excess bioavailable PO43– into DWDSswhich are typically PO43– limited mayaid microbial growth and cause changes in microbial community compositionin the DWDS.11 For example, recent metagenomicsstudies in a United Kingdom DWDS found an increase in microorganismsthat can more readily metabolize phosphate 13 after increased PO43– addition. Likewise,PO43– has been observed to be importantfor the growth of drinking-water-associated pathogens that cause infectionsin the immunocompromised (DWPI) such as Legionellapneumophila, Pseudomonas aeruginosa, and nontuberculous mycobacteria (NTM).17DWDSs host a diverse aquatic ecosystem that can be altered by severalfactors including source water quality, temperature, treatment processes,disinfection methods, and piping materials.18 whereby diseaseincidence exceeds that of traditionally monitored fecal-borne pathogens.22 DWPIs are often found in complex biofilm communities within theDWDS that can be affected by nutrient fluctuations and availability.24 As such, it is important to understand the potential impacts fromthe addition of nutrient-based corrosion inhibitors  on both the DWPI densityand biofilm formation (or aggregation) potential. From a public healthstandpoint, it is estimated that pulmonary infections associated withDWPIs annually cause >145000 infections25 and cost the economy >$62 billion in losses due to deaths alonebased on a value of statistical life calculation using the Departmentof Health and Human Services central value.26DWPIs are typically definedas organisms that pose no threat tohealthy individuals but can cause infection in immunocompromised people. DWPIs are not currently regulated by theU.S. EPA despite the fact they are a leading cause of morbidity andmortality in the U.S.28 we still knowrelatively little about the impacts that large-scale changes in operation have on the drinking water microbiome and microorganismsrelevant to public health. As such, it is imperative that any operationalchanges in the DWDS also involve proactive DWPI monitoring. This studyaimed to assess the impacts of PO43– corrosioncontrol addition on the microbiome and DWPI density across multiplesites in a DWDS. It was hypothesized that the addition of excess PO43– into a phosphorus-limited DWDS wouldalter both the microbial community structure and density of DWPIs.Additional work was done to examine the impacts of PO43– corrosion control on NTM aggregation potential toobserve potential impacts on biofilm formation.Despite the vast improvement in the understanding of the drinkingwater microbiome in the past decade,Figure A1 in the Supporting Information).This plant treats surface water by using coagulation, sedimentation,filtration, and disinfection by chlorination. After disinfection,the treated water is pumped to a storage reservoir and then treatedat a smaller treatment plant before transport through the DWDS. The seven sites are in six differentpressure districts representing residence times ranging from 59 to229 h as estimated by a tracer study.29From February 2019 toMarch 2020, samples were collected from sevenroutine monitoring sites in a DWDS in Pittsburgh, PA, USA, all ofwhich received water from the same drinking water treatment plant.Prior to April 2019, soda ash was used as the corrosion controlagent in the DWDS. After a year-long model pipe loop study conductedwith the Pennsylvania Department of Environmental Protection, thedrinking water utility decided to switch their corrosion control overto PO30 Allsamples were filtered within 1 h of collection through a 0.2 μmpolycarbonate filter , and the resulting filters were stored at −20 °Cfor DNA extraction. In reviewing the preliminary results, we observeda significant 2 log 10 increase in NTM density and determined thatfurther testing would need to be performed to understand what causedthis increase. As such, 12 L water samples were collected at the treatmentplant and used for our NTM bench-scale reactor assays. Two separatesampling bottles  were filled with 6 L of waterpre and 6 L post PO43– addition and storedon ice after collection and their chlorine residual was quenched usingsodium thiosulfate before use.1 L water samples from the sevendistribution system monitoring sites were collected after flushingthe faucet for at least 5 min and waiting for both the temperatureand chlorine residual to stabilize.Tables A1 and A2 in the SupportingInformation) following standard methods.31 Free and total chlorine and PO43– concentrationsat the tap were measured on site using a portable DR900 spectrophotometer . Temperature and pH also were monitoredon site using a portable pH and temperature meter  , and ATP was also measured on site usingan AquaSnap Total ATP meter . Total anddissolved concentrations of iron, manganese, copper, and lead weremeasured by inductively coupled plasma mass spectrometry . Prior to analysis, all dissolvedmetal samples were prepared by passing water through a 0.45 μmnylon syringe filter  primed with 5 mLof sample. All analyses, except pH, temperature, and ATP were performedin triplicate.Fourteen water quality parameters weremeasured ,33P. aeruginosa (Orpl gene),34 and NTM(atpE gene)35 were conductedusing previously published primers (Table A4 in the Supporting Information).DNA was extracted fromthe stored filters using the FastDNA Spin Kit  and stored at −20 °C until use. The density (numberof gene copies per unit volume of sample) of total bacteria and Cyanobacteriawas determined using digital droplet PCR (ddPCR) as previously described.n = 98), alongside negativecontrols  and positivecontrols , both of which were negativeand positive, respectively (Table A3 inthe Supporting Information). Positive samples were only determinedabove the LOD for each assay. 22 μL reactions contained 11 μLof 2× ddPCR Supermix ,0.4 μM concentrations of all primers (Integrated DNA Technologies),0.55 μL of bovine serum albumin , and 2 μL of the DNA template. Droplets were generated toa 20 μL reaction volume using the automated droplet generationoil for Sybr (Bio-Rad Laboratories), and the plate was sealed. PCRwas performed on a C1000 Touch thermal cycler (Bio-Rad Laboratories)within 15 min of droplet generation using the reaction conditionspresented in Table A4 in the SupportingInformation. Plates were run on a droplet reader within 1 h of PCRcompletion. Thresholds were set for each ddPCR assay (Table A3 in the Supporting Information) usingQuantasoft v1.0.596 to determine the absolute density of the targettaxa using the method described by Lievens et al.36ddPCR reactions were performedfor all DNA samples using a 97% cutoff using the closed reference OTU-picking protocolin QIIME2 (version 2020.2) using the Silva (version 132.5) referencedatabase. After OTU clustering, the presence of the OTUs in threeor more of the negative control samples was removed  to ensure analysis only of the OTUs in the samples collectedfrom the DWDS.16S rRNA gene amplicon(V4–V5 hypervariable region) library preparation and sequencingwere performed on all samples (collected DWDS samples and negativecontrols) at Argonne National Laboratory following the Illumina EarthMicrobiome Protocol.43– injection, and the remaining four contained DS water post PO43– injection. All reactors were kept indark conditions, placed on individual stir plates  set to 300 rpm and run in parallel for 12 weeks until the reactorswere out of water. To examine direct impacts on NTM growth, threedifferent species of NTM that were found in the DS and have clinicalor laboratory relevance were injected into the reactors: Mycobacterium abscessus , Mycobacterium avium , and Mycobacterium smegmatis . Each mycobacterial species was grownin liquid R2A media, with growth measured via optical density andplate counts prior to inoculation. Once grown, equal concentrationsof all three liquid cultures were mixed, achieving a final concentrationof 1 × 108 cfu/L (representative of the DWDS NTM average), whichwas injected into each reactor. Biweekly samples were collected fromeach reactor and processed for culturable NTM and total NTM, M. smegmatis, M. abscessus, and M. avium absolute density by ddPCR using previously published primers (Table A4 in the Supporting Information).Eight 1.5 L glassbatch reactors  were set up to contain 1L of drinking water obtained from the DWDS within 1 h of sample collection.Four reactors contained DS water pre PO43– addition on NTM aggregation (biofilmformation), an assay developed by DePas et al.40 was utilized to distinguish and quantify aggregated cellsand planktonic cells over time. Briefly, NTM cultures were grown intryptone-yeast extract magnesium sulfate (TYEM) nutrient broth priorto starting the experiment to ensure the same starting concentration.Once grown, liquid culture replicates  were grown in fresh TYEM nutrient broth for 35 h  with different concentrations of PO43– . Cultures were then harvested by passingthe culture through a 10 μm (M. smegmatis) or 5 μm cell strainer , and the optical density(OD600) of both the planktonic fraction  and the aggregates collected on the strainerwere recorded. The OD600 value of the planktonic fractionwas immediately recorded, while aggregates that collected on the strainerwere resuspended in phosphate-buffered saline (PBS) with 6% Tween20. This suspension was then sonicatedto resuspend remaining aggregates before recording the OD600 value . Both OD600 readings were used to calculate theplanktonic to aggregate ratios. Average planktonic/aggregate ratioswere then compared across phosphate concentrations using nonparametricWilcoxon testing .To determine the impacts ofPO41). The transformed OTU data were then used tocalculate pairwise dissimilarities between samples based on the Bray–Curtisdissimilarity index. Resulting matrices were examined for temporaland spatial patterns in the bacterial community structure by NonmetricMultidimensional Scaling as implemented in the Vegan package (version2.5-7) in R (version 4.0.2).42 Significantdifferences in the microbial community compositions (Hellinger transformedOTUs) before and after PO43– additionwere determined by nonparametric permutational multivariate analysisof variance (PERMANOVA) and differential abundance analysis usingDESeq2.43 Relationships between environmentalparameters and patterns in microbial community composition were examinedby canonical correspondence analysis (CCA) with significance testedby ANOVA after removing collinear variables  and reducing the overall suite of environmentalvariables with a stepwise Akaike information criterion model. Additionally,significant differences in the relative and absolute bacterial densitybefore and after PO43– addition and differencesin NTM species aggregation were determined by nonparametric Wilcoxontesting, while the functional relationships between water qualityparameters and bacterial groups were analyzed by stepwise multivariateforward/reverse regression analysis. All statistical analyses wereperformed in R (version 4.0.2)44 with significanceset at a p-value <0.05.Taxonomic andOTU tables generatedfor the samples were transformed using the Hellinger transformationdue to the data set having many rare OTUs (present in a few samples)or low-abundance OTUs and pre and post PO43– addition variation in the DWDS microbial community structures. The observedseasonal differences in microbial community composition in the DSare consistent with previous studies,45 and the observeddifferences in communities based on PO43– dosing corresponds with results found by Douterlo et al.10 Additionally, significant spatial differenceswere observed , which is to be expected as each DWDS site hasa different residence time and differing hydraulics and plumbing materials;however, these spatial differences were only driven by temporary differencesat one or two sites.For the 98 samples collectedfrom the DWDS over the course of 1 year, NMDS 1a,b and 48 with Proteobacteriaand Actinobacteria dominating across all DWDS sites. Furthermore,the Acinetobacter, Pseudomonas, and Sphingomonas genera were theonly three genera within the top 10 most abundant taxa present bothbefore and after PO43– addition . Apart from Cyanobacteria, differentialabundance analysis revealed no other significant changes in the relativeabundance of typical drinking water phyla. At the genus level, therewere multiple significant changes in the abundance of rare drinkingwater taxa  and other uncultured organismsthat could not be identified further. Cyanobacteria  generally represented 10% orless of the DS microbial community and appeared to significantly decrease after PO43– addition into the DWDS.However, while a decrease in Cyanobacteria relative abundance wasobserved, there was also an unexpected significant increase in Cyanobacteriaabsolute density  and related species in complex environmentalmatrices.The average relativeabundance of the most abundant taxa in theDWDS was consistent with other studies,ition SI A4. Apart52 The response in only a few typical taxa andmany more rare could also be indicative of shifts in microbial niches and functionin response to elevated phosphate concentrations  rather than shifts intaxonomic composition. CCA revealed that 16.4% of the variance inthe microbial community composition could be explained by a combinationof factors, including the geographic location of the DS sites, theseason samples were collected in, pH, total copper concentration,total iron concentration, and total phosphorus . Thischange was likely driven by a 2 log 10 increase in the NTM densityat all DWDS sites . Previous work has discussedthe negative correlation between Legionella spp. and Mycobacterium spp.54 that may be due to both DWPIs competing forthe same nutrients in oligotrophic environments. Further regressionanalysis on L. pneumophila densityrevealed 76% of the variance in L. pneumophila was explained by a combination of factors including NTM density and Mycobacterium  do not reflect this significant increase,suggesting that community changes were driven at the subgenus level,as reflected by ddPCR results. It is important to also note that sinceour sequencing assays targeted the 16S rRNA gene which can vary betweenorganisms, another possibility is that Actinobacteria and Mycobacterium differences may be masked bybiasing toward changes in organisms with higher 16S rRNA copies . Possibly with metagenomics,we may have observed changes in the Mycobacterium genus, and future studies should take this into consideration whendesigning and planning to analyze these organisms. It is also importantto note that the differences in assay target can make it challengingto analyze and compare these organisms. When doing amplicon sequencing, the data are generally reported as the relativeabundance of the 16S rRNA gene copies present, while in ddPCR assaysyour gene target can be more specific . This could also accountfor differences in assay results and should be considered when evaluatingchanges in taxa at lower order taxonomic levels .Interestingly, sequencing results (relative abundance) ofboth Actinobacteria species to solubilize and uptake phosphorus in soil, freshwater,and marine environments,57 and as such the observed significantincrease in NTM density could result from the freshwater origins ofthe drinking water. Additionally, other work has shown a positivecorrelation between Actinobacteria abundance in drinkingwater systems and total phosphorus concentration58 while previous metagenomic work has suggested that Actinobacteria may have a key role in phosphorus sequestration59 in the environment. Regression analysis on theNTM density data revealed that 75% of the variance in NTM densitywas explained by a combination of seasonality, total phosphorus concentration, L. pneumophila density, pH, total iron concentration,and turbidity  alsocoincided with a decrease in the water temperature . This observationwas counterintuitive, as previous NTM work has reported seasonal increasesin NTM during warmer times of the year60 and positive correlations with warmer water temperatures.65 As such, elucidating the reason for the observedincrease in the NTM was important.Previous work has detailed the ability of urbidity 1. PrevioFigures A6 and A7 in theSupporting Information), further bench-scale experimentation on NTMgrowth and aggregation (biofilm formation) potential in the presenceand absence of PO43– was conducted toelicit the impacts of PO43– on NTM. Bothviability (plate counts) and ddPCR analyses revealed no significantdifferences in total NTM or NTM species  density between samples treated with orwithout PO43– . Interestingly, M. smegmatis was detected in only 9% of the samplestaken, possibly because the laboratory mc2155 strain wasoutcompeted by the environmental M. abcessus and M. avium strains and other DWmicrobiota. Given the sudden increase in the total NTM density inthe DWDS, the lack of significant differences in batch reactor NTMgrowth and decrease in NTM density 3 years later , it was unlikelythat the PO43– addition caused a significantimpact on NTM growth. Instead, it is possible that the PO43– addition impacted the NTM biofilm processes(formation and sloughing).Since the 2 log 10 increase inNTM density was observed suddenly across all DWDS sites starting inNovember 2019  could likely be an explanation for the observed increase.Biofilm formation is a complex, dynamic,and ongoing process that has several stages, some of which are dependenton nutrient concentrations.600 values of the planktonic NTM(NTM that passed through the strainer) and aggregated NTM (NTM trappedon the strainer) fractions were measured and compared by assessingthe ratio of the NTM in the planktonic phase to the NTM aggregatedphase (hereafter referred to as “NTM aggregation assays”).In nutrient-rich media (primarily C:N dominated), previous work withdomesticated M. smegmatis has observedthat NTM initially aggregates but will then disperse at later culturematurity and grow planktonically.40 Inthe presence of phosphate-augmented media, the domesticated M. smegmatis behaved similarly to what was observedby DePas et al.; however, M. abcessus and M. avium behaved differently loweraverage ratios (more aggregate fractions) in the 0 and 1 μMphosphate conditions compared to the 20 and 100 μM conditions. The increased ratio in the presence of elevated phosphatesuggests that increased phosphate concentrations may cause disaggregation for M. abcessus. In the M. avium assays, elevatedratios were present regardless of the phosphate concentration  may not benefit from elevated phosphorusconcentrations when compared with rapidly growing mycobacteria.68 The results of the aggregation assay data presentedhere are interesting, as previous work has mentioned the impact ofphosphate concentration on the production of extracellular polymericsubstance (EPS)70 and biofilm structural mechanics.72 Specifically, it has been reported that increasing phosphorus concentrationscan result in a reduction in EPS or an increase in the amount of porespresent in the biofilm, thus weakening biofilms and making them moresusceptible to detachment under drinking water pipe flows.71 Additionally, previous spatial work has demonstratedthat NTM are late colonizers of biofilms in drinking water.73 Therefore, it is possible that NTM could beon the outer surface of drinking water biofilms, making detachmenteasier; however, more work is needed on the localization of NTM indrinking water biofilms. Furthermore, it is also possible that phosphateinteracts with the constituents of the outer membrane of mycobacterialcells and causes a change in the cell hydrophobicity; however, tothe authors’ knowledge this has not been explored.The differences observedin NTM species could be attributed todifferences in nutrient requirements between rapid- and slow-growingNTM, as one study posits that slow-growing mycobacteria , it is also important to considerthe time scales of impact. Previous work has examined the impact ofnutrient starvation on biofilm formation and suggested that prolongednutrient starvation can lead to increased biofilm detachment.74 In the presented system, the shift from excessPO43– dosed at 3.0 mg/L down to 1.8mg/L could have been enough of a shift to trigger a starvation responsefrom the biofilm. It could also be possible that the length of theelevated NTM concentrations was a delayed response to the change inDWDS PO43– concentration .Future studies should identify the drivers of NTM disaggregation inthe presence of elevated phosphate concentrations, create a more normalizedprocess for examining disaggregation that captures robust speciesdynamics, and elucidate the species differences in biofilm formationpotential, as well as determine the time scale of sloughing eventsin the DWDS.Giventhe data presented, it is possible that the sudden increasein NTM in the DWDS was due to a large biofilm sloughing event, aninteraction between phosphate and the NTM cell walls, or a combinationof both. Moreover, given what appears to be a gradual decrease toa new increased baseline NTM density to (1) conduct more targeted analysis , (2) determine what specific types of NTM or otherDWPIs are present in a full-scale DWDS, and (3) continue to developour understanding of the impacts of nutrients on these organisms andbiofilm formation. Likewise, although no changes in NTM pulmonarydisease incidence have been observed to date, additional longitudinalstudies are required to ensure that no adverse health impacts arise.Overall, the results presented here suggest thatPO


SARS-CoV-2 variants have continuously emerged in the face of effective vaccines. Reduced neutralization against variants raises questions as to whether other antibody functions are similarly compromised, or if they might compensate for lost neutralization activity. Here, the breadth and potency of antibody recognition and effector function is surveyed following either infection or vaccination. Considering pregnant women as a model cohort with higher risk of severe illness and death, we observe similar binding and functional breadth for healthy and immunologically vulnerable populations, but considerably greater functional antibody breadth and potency across variants associated with vaccination. In contrast, greater antibody functional activity targeting the endemic coronavirus OC43 is noted among convalescent individuals, illustrating a dichotomy in recognition between close and distant human coronavirus strains associated with exposure history. This analysis of antibody functions suggests the differential potential for antibody effector functions to contribute to protecting vaccinated and convalescent subjects as novel variants continue to evolve. As the SARS-SoV-2 pandemic progressed and the vaccinated population increased, a number of variants of concerns that circumvented the vaccine induced immunity emerged. Here the authors compare SARS-SoV-2 mRNA vaccine responses in pregnant women and other people, characterising the antibody response and Fc functions, and potency against a range of variants of concern. Notable SARS-CoV-2 variants have arisen and expanded across the globe, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and most recently, Omicron (B.1.1.529). While vaccination is a critical intervention in combatting the pandemic, many studies of both infection and vaccination have shown reduced neutralizing potency toward variants of concern (VOC)14.Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the virus causing the COVID-19 pandemic, has continued to evolve despite the widespread use of multiple highly effective vaccines23. Because binding antibodies can elicit potent antibody Fc domain-dependent effector functions that contribute to protection even in the absence of neutralization27, they may help to compensate for compromised neutralization potency. These activities, including antibody-dependent cellular phagocytosis (ADCP), cellular cytotoxicity (ADCC), and complement deposition (ADCD), rely on interactions with soluble and cell-expressed antibody Fc receptors on diverse innate immune cells that can drive direct lysis of virions or infected cells, as well as trigger inflammatory cascades to amplify host defense30. Fc effector functions have correlated with protection in animal models of SARS-CoV-2 and in humans in other disease settings34. The potential clinical relevance of antibody-mediated Fc effector functions is suggested by observations that vaccines appear to remain highly effective in preventing serious disease despite reduced levels of neutralizing antibodies.Fortunately, studies have also shown that both infection and vaccination lead to measurable levels of antibodies and T-cell responses against VOC35, and antibodies with the capacity to elicit multiple effector functions appear to recognize diverse epitopes on the spike (S) protein36. SARS-CoV-2 variants generally accrue most mutations in the receptor binding domain (RBD) and the N terminal domain (NTD), where the most common epitopes for neutralizing Abs are found39. The emergence of the Omicron variant, with far more mutations than other VOC, including in other regions of the spike protein, has led to reductions in neutralization potency against this variant41. These observations have raised interest in determining whether and which other antibody activities may help confer protection from severe disease from sequence distant strains and how well these responses are induced by infection or vaccination.Importantly, studies have shown that Fc effector activities are elicited following vaccination and infection43. Within groups, individuals also show considerable variability in response magnitudes and characteristics46. Lastly, populations at increased risk for severe disease, such as pregnant women47 may also exhibit further distinctions in humoral response attributes48. How these distinct axes of variability associate with the magnitude and breadth of antibody effector functions across VOC is not known, but it has important implications for continued protection of diverse individuals and in the face of further viral diversification. The breadth of Fc effector functions across VOC in vulnerable and healthy subjects following natural infection or vaccination can provide new insight into the potential contributions of mechanisms beyond neutralization to protection from SARS-CoV-2 disease.To this end, immunity resulting from vaccination and natural infection are known to exhibit a number of distinctions with respect to antibody responsesn = 87), were previously infected when the Wuhan strain was predominant (n = 57) or were SARS-CoV-2 naïve (n = 38)  and convalescent (n = 38) individuals were collected from pregnant women who are at greater risk of hospitalization for covid-1947. To define similarities and differences in antibody profiles among seropositive individuals, dimensionality reduction was performed on biophysical antibody features using Uniform Manifold Approximation and Projection (UMAP)49. Subjects were distributed across the profile landscape into three distinct clusters, which were almost perfectly segregated by whether their responses were elicited by vaccination or infection , suggesting that not only sequence but conformational state are key factors in defining functional antibody cross-reactivity profiles among even distantly related coronaviruses, as has recently been described for neutralization and binding activity14. This fear is further exacerbated in vulnerable populations, among which vaccine effectiveness is already reduced61. Whereas many studies have shown that vaccination results in reduced neutralization of variants as compared to the Wuhan strain53, vaccines remain protective from severe disease62, suggesting that either a threshold effect exists or that contributions from other cellular and humoral mechanisms may be at play. This latter possibility is well-supported by studies providing evidence that Fc effector functions are more resistant to changes from VOC, pointing to a potential component of protection from severe disease54.The continued emergence of SARS-CoV-2 variants has raised concern that vaccines based on the Wuhan strain will exhibit reduced efficacy over time in the face of viral evolutionThe continually evolving viral landscape of SARS-CoV-2 strains shows the importance of needing to further understand how effector functions may provide protection from severe disease when vaccine neutralization is reduced. To this end, and relative to the more considerably reduced neutralization potencies expected, we report well-maintained antibody effector functions across diverse VOC. Broad effector function was observed for both vaccinated and convalescent subjects and for both whole spike and RBD, although the former was somewhat broader than the latter. This data suggests that for VOC against which neutralization activity may be insufficient, Fc-mediated effector functions have the potential to compensate for decreased neutralization and contribute to protection.63. Additionally, binding antibody breadth does not appear to simply scale with greater IgG response magnitude, as for each antigen tested, at least some convalescent subjects exhibited responses of similar magnitude to vaccinated subjects, yet no convalescent subject exhibited a similar breadth score. Instead of response magnitude, these differences in breadth across VOC may relate to differences at the level of B cell responses between vaccinees and convalescent individuals64. Considerable antibody breadth and potency across VOC were similarly observed for diverse antibody effector functions, including phagocytosis mediated by monocytes, a surrogate measure of ADCC, and complement deposition. Among these functions, complement deposition was generally more sensitive to both antigenic variation and to serum concentration than the other activities tested; and among VOC, beta and gamma RBD variants showed a greater decrease in ADCC and complement deposition activity than more distant VOC, a pattern that was not easily explained by mutational loads or identities.This study shows that the breadth of antibody recognition across SARS-CoV-2 VOC varies by isotype and by antigen exposure history. Whereas vaccinated subjects showed considerably greater FcγR-binding IgG antibodies of diverse subclasses, convalescent subjects exhibited greater IgM breadth. IgA breadth was comparable between populations. The distinctions among isotypes may reflect differences in exposure route, duration, and costimulatory factors. As mucosal vaccines continue to advance toward the clinic, these possibilities can be tested. Our data also suggests that the differences observed in the breadth of recognition of VOC and emergent CoV as compared to endemic CoV between vaccination and infection likely at least partially relate to distinctions in antigenic conformations between native and proline-stabilized forms of spikeWhile the observation of greater breadth and potency induced by vaccination than natural infection could be extended to SARS-CoV-1, neither exposure induced functional antibodies to MERS, and depending on spike conformation, the opposite pattern was observed for the endemic CoV OC43. These observations establish an intriguing dichotomy: breadth across CoV-2 strains was greater among vaccine recipients, but breadth across more distant beta and alpha coronaviruses was greater among convalescent subjects. While the origin of this dichotomy cannot be defined from this study, the observation that functional breadth toward stabilized and unstabilized spikes differed between populations suggests that it may be related to the conformational state of the spike antigen to which the immune system was exposed. This data suggests differential antigenicity and immunogenicity of the stabilized spike protein used in mRNA vaccines and has important implications for efforts to develop pan-CoV vaccines.69, only three activities were evaluated. Similarly, only five major VOCs were evaluated, and subjects were infected or vaccinated with the Wuhan strain. The breadth of effector functions induced by strains other than Wuhan or against future viral variants may exhibit different or similar degrees of conservation, as observed here. While some comparability testing was performed herein, other limitations include the use of surrogate measures of effector function reliant on recombinant antigens and cell lines as opposed to infected cells or virions and primary effectors. For example, the expression of regulatory factors expected to potentially impact complement deposition activity was not captured in the assay employed in this study.We initially set out to characterize functional breadth in a vulnerable population but found only limited differences in the binding and functional profiles of antibodies in pregnant women as compared to healthy controls. While this observation has important implications for vulnerable populations, it is important to note that even in the context of similarly functional antibodies, the effector capacity of susceptible individuals may be compromised due to alterations in the cellular effectors and the availability or regulation of complement cascade factors. While the assays used herein have correlated with improved protection in vivo in various settings70 and monoclonal antibody therapy72, our observations support the potential value of these interventions even in the face of continually diversifying viruses. Our observations of the enhanced breadth of IgG-dependent Fc effector functions provide additional evidence of beneficial aspects of vaccine-induced immunity as compared to natural infection. Future studies could define the impact hybrid immunity resulting from combinations of infection and vaccination or after a combination of distinct vaccine regimens and variant-specific boosters may have on functional breadth in healthy and immune-vulnerable populations. Overall, this work provides insights into how vaccines and prior natural infection may provide protection by antibody functional mechanisms. These observations promote vaccination as able to drive superior functional antibody breadth, and set expectations for cross-variant antibody effector function and may serve as a useful comparator in studies of candidate vaccines aimed at improving the breadth of protection and in evaluating population susceptibility as exposure histories and infecting virus continue to evolve.Nonetheless, these observations of well-maintained functional antibody breadth following vaccination and infection, even among vulnerable individuals, have important implications. They suggest that antibodies elicited by either prior infection or vaccination with a given strain have the potential to restrict the replication of disease caused by future variants. Similarly, given evidence that these activities contribute to the efficacy of convalescent plasman = 87) received two doses of either mRNA-1273 (n = 2) or BNT162b2 (n = 85) vaccines. Vaccinated subjects were pregnant women in Israel (n = 50) who were screened for lack of anti-N SARS-CoV-2 antibody responses or non-pregnant subjects from the United States (n = 37), among which four subjects had a prior history of SARS-CoV-2 infection. These subjects were included in the analysis as they were not clearly distinct from other vaccinated subjects in multivariate (UMAP) analysis. Convalescent subjects were pregnant women from Belgium (n = 38) or non-pregnant subjects from Dartmouth Hitchcock Medical Center in the United States (n = 19) with infection status defined by RT-PCR. Collection of these samples occurred when Wuhan was the dominant strain in circulation, but viruses were not typed. Naïve serum was obtained from a commercial source prior to the approval of vaccines and was screened for anti-N SARS-CoV-2 antibody responses to exclude donors with previous infection. Characteristics for each cohort are described in Supplemental Table This study complies with relevant human subject research regulations and was reviewed and approved by the Dartmouth College Committee for Protection of Human Subjects. Vaccinated subjects  using two-step carbodiimide chemistry. Experimental controls included pooled human polyclonal serum IgG (IVIG), S309, an antibody from a SARS-CoV patient that cross-reacts SARS-CoV and SARS-CoV-2, and VRC01, an HIV-specific antibody76. Serum dilutions used in experiments were based on experience from previous work and a small pilot experiment of test concentrations. Final dilutions used in assays varied from 1:250 to 1:5000 depending on the detection reagent (see reporting summary). Antigen-specific antibodies were detected by R-phycoerythrin-conjugated secondary reagents specific to human immunoglobulin isotypes and subclasses and by Fc receptor tetramers as described previously78. Median fluorescent intensity data was acquired on a FlexMap 3D array reader (Luminex Corporation). Samples were run in technical duplicates.Antigen-specific antibodies were characterized using the Fc array assay79 for a subset of sera samples . Briefly, serial dilutions of heat-inactivated serum  were incubated for 1 hr at 37 °C and 7% CO2 with 3×TCID100 of Wuhan strain  and Omicron strain BA.1 . A volume of 100 μL of sample-virus mixture was added to 100 μL of Vero cells  in a 96-well plate and incubated for 5 days at 37 °C and 7% CO2. The cytopathic effect caused by viral growth was scored microscopically. The Reed-Muench method was used to calculate the nAb titer that reduced the number of infected cells by 50% (NT50), which was used as a proxy for the nAb concentration in the sample. In accordance with WHO guidance, an internal reference standard composed of a pool of serum from naturally infected and vaccinated adults was included in each nAb assay run. This internal standard was calibrated against the Internationals Standard 21/234 (NIBSC), and NT50 values were recalculated to IU/mL.SARS-CoV-2 neutralizing antibodies (nAb) were quantified80. Briefly, 1 μM yellow-green fluorescent beads  were covalently conjugated to spike or RBD antigens. Beads were then incubated with serum samples for 4 h with THP-1 cells (ATCC TIB-202) at 37 °C in 5% CO2. Cells were fixed and analyzed by flow cytometry  cells were cultured according to manufacturer’s instructions. Cultured cells express CD16 (FcγRIIIa), which, when engaged on the cell surface, leads to luciferase secretion from the cell. First, high-binding 96-well plates were coated overnight at 4 °C with 1 μg/mL of spike or RBD antigen. Following incubation, plates were washed (PBS + 0.1% Tween20) and blocked (PBS + 2.5% BSA) at room temperature (RT) for 1 h. Following plate washing, 100,000 cells per well and dilute serum samples were added to each well in cell culture media lacking antibiotics in a 200 μL volume. Following 24 h incubation, 25 μL of supernatant from each well was transferred into a white 96-well plate in which 75 μL of quantiluc substrate was immediately added. Following 10 min incubation, plates were read on a SpectraMax plate reader (Molecular Devices). VRC01 was used as a negative control; cell stimulation cocktail  and ionomycin and S309 served as positive controls. Concentrated pooled polyclonal serum IgG (Sigma Aldrich I4506) was used as a positive control for endemic CoV , which were tested in order to evaluate the concordance between FcγRIIIa signaling determined in the reporter assay and cell killing  was diluted 1:100 in gel veronal buffer  and mixed with samples at RT with shaking for 1 h. After washing, samples were incubated with murine anti-C3b (Cedarlane #CL7636AP) at a 1:500 dilution at RT for 1 h, followed by staining with anti-mouse IgG1-PE secondary Ab (Southern Biotech #1070-09) at a 1:1000 dilution at RT for 30 min. A final wash was performed, and samples were resuspended into Luminex sheath fluid, and MFI was acquired on a FlexMap 3D reader. Assay controls with no antibody, an irrelevant VRC01 antibody, and a heat-inactivated complement were used as negative controls. S309 was used as a positive control. Concentrated pooled polyclonal serum IgG (Sigma Aldrich I4506) was used as a positive control for endemic CoV  experiments were performed essentially as previously describedSigma, S164 was diα = 0.05) of differences between groups.Statistical analysis was performed in GraphPad Prism (version 9.7). Breadth–potency curves were defined as the proportion of antigen-specificities exhibiting a signal above a given intensity. Curves were generated using the LOWESS curve fit method in Prism for each respective subject group. Breadth scores were calculated by taking the geometric mean across antigen specificities for each subject. The sample size for each figure includes all subjects from their respective groups unless otherwise noted. Data graphed in figures is provided in Source Data File Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


Osteoporosis is a metabolic bone disorder that threatens holistic well-being and economic stability worldwide due to its high prevalence in the elderly. Osteoporosis is associated with systemic reduction in bone mass and microarchitectural deterioration of bone tissue causing an increased risk of bone fracture. Osteoporosis together with other age-related conditions disturbs the pace and process by which osteoclasts (OCs) and osteoblasts (OBs) activities are coupled and balanced, and therefore normal bone remodeling and calcium homeostasis are deranged. This collection of seven papers constitutes the Research Topic and brings together vital studies investigating osteoporosis’s molecular mechanisms and related complications, and the novel advances in drug development targeting functional genes/proteins involved in the pathology of osteoporosis.Hu and Wang reviewed the multifaceted role of SIRT3 in osteoporosis, and they elaborated on the cell types involved in bone remodeling and thereby built a theoretical basis for SIRT3 being a therapeutic target for osteoporosis. In another article, Yang et al. studied the role of p.Ser267Phe mutation in NTCP deficiency and its subsequent long-term impacts. Their study offered insightful revelations and underscored the value of genetic research in unraveling novel and potential therapeutic approaches. Yang et al. revealed that NTCP deficiency, caused by the p.Ser267Phe mutation in the SLC10A1 gene, affected the circulating level of bile acid leading to vitamin D deficiency, bone loss, and gallbladder abnormalities. Therefore, regular monitoring on level of bile acids, amount of vitamin D storage, and bone density in those with NTCP deficiency was recommended. Research advances on WNT1 and PLS3 mutations by Loid et al. demonstrated the association between plasma lipocalin-2 level and fibroblast growth factor-23 (FGF23). This association could be linked to abnormal WNT1 and PLS3 signaling which indicated its significant role in bone metabolism. This study revealed that lipocalin-2 was not altered in WNT1 or PLS3 mutation-positive subjects, while it was associated with C-terminal and intact FGF23 in WNT1 and PLS3 patients respectively. This finding indicates a possible role of lipocalin-2 in the FGF23-mediated signaling pathway in the osteoporosis caused by these mutations. This study provides novel insights into targeted therapeutics in osteoporosis. The correlation of lipocalin-2 with serum transferrin in WNT1 potentially outlines future areas of exploration regarding the role of iron status in abnormal WNT1 signaling. Zhao et al. used robust methods to identify hub genes that interact strongly during osteoporosis progression, including gene co-expression network analysis (WGCNA) and differential analysis on gene expression profiles from the GEO database. Their meticulously conducted study uncovered significant insights into the role oxidative stress plays in osteoporosis, alongside a potentially promising treatment development strategy targeting the MAPKAPK2 proteins, such as PPP1R15A, CYB5R3, BCL2L1, and MAPKAPK2. These markers provide further insights into the role of oxidative stress in osteoporosis development and potential treatment targets.A total of four articles are included in this part to discuss the recent advancements in functional genes in osteoporosis. These studies aimed to establish a foundation for translating insights from human genetic research into innovative treatment solutions for osteoporosis. Recent studies suggest that SIRT3 contributes to the physiological processes associated with the senescence of bone marrow mesenchymal stem cells (BMSCs) and the differentiation of BMSCs and osteoclasts. Nevertheless, the exact effects and mechanisms underlying SIRT3’s role in osteoporosis remain unclear. Zhang et al. highlighted the potential therapeutic use of berbamine in treating postmenopausal osteoporosis (PMOP). Their results showed that berbamine inhibited bone resorption in PMOP mice and offered a fascinating spotlight on this traditional Chinese medicinal plant with its potential in treating diseases beyond its current clinical utilization for leukopenia. The detailed depiction of berbamine’s inhibitory role against RANKL-induced osteoclastogenesis denoted a promising shift in osteoporosis treatment. Another novel study by Che et al. unraveled a new dimension in the treatment of postmenopausal osteoporosis with FDA-approved PTH 1-34. The research delved into the less-explored area of iron metabolism and studied its role in the regulation of unloading-induced osteoporosis. Their study documented implications of iron overload as a significant risk factor for bone loss which enhanced the current understanding of osteoporosis and paved the way for future novel therapeutic strategies. By illuminating how PTH 1-34 moderates the imbalance of iron metabolism in unloading mice via Nrf2 activation and subsequent increase in hepatic hepcidin expression, Che et al. provided compelling evidence supporting the osteoprotective effect of PTH 1-34 beyond its primary mechanism of action. On top of that, their results also indicated that Nrf2 inhibitors might have an unanticipated adverse effect on the benefits of PTH 1-34 treatment. Kulkarni et al. offered a new perspective on the bone-conserving properties of the plant Coleus forskohlii root extract (CFE) which is typically known for its valuable role in preventing weight gain amongst obese individuals. Their study suggested that forskolin consequently fostered an osteogenic effect. Forskolin is the predominant compound present in CFE and is notable as an adenylate cyclase (AC) activator that raises intracellular cyclic adenosine monophosphate (cAMP) levels in osteoblasts. Kulkarni et al. utilized an examination in adult ovariectomized (OVX) rats and demonstrated how CFE sustains bone mass and microarchitecture to levels comparable to sham-operated rats. Intriguingly, the surface-referent bone formation is significantly increased in CFE-treated rats, amidst several other vital findings pertaining to the preservation of bone mass, microarchitecture, and strength.A total of three articles about advancements in disease treatment for osteoporosis are included in this part. In conclusion, potential therapeutics, from plant extracts to FDA-approved drugs and novel genetic targets, are revealing molecules and pathways for preventing and treating osteoporosis. Ongoing research continues to elucidate molecular and cellular mechanisms, paving the way for the discovery and optimization of future treatments for this prevalent disease.ZL: Writing – original draft, Writing – review & editing. KY: Writing – original draft, Writing – review & editing.


Hypoxia‐inducible Factor‐3α (HIF3α) and Peroxisome Proliferator-activated Receptor Gamma-coactivator-Alpha (PGC1-α), and the decreased methylation of one gene, Peroxisome Proliferator Activated Receptor Alpha (PPARα), in women with GDM compared to pregnant women with normoglycemia that were consistently methylated across diverse populations with varying pregnancy durations, and using different diagnostic criteria, methodologies and biological sources. These findings support the candidacy of these three differentially methylated genes as biomarkers for GDM. Furthermore, these genes may provide insight into the pathways that are epigenetically influenced during maternal diabetes and which should be prioritized and replicated in longitudinal studies and in larger populations to ensure their clinical applicability. Finally, we discuss the challenges and limitations of DNA methylation analysis, and the need for DNA methylation profiling to be conducted in different types of maternal diabetes in pregnancy.Diabetes in pregnancy is associated with adverse pregnancy outcomes and poses a serious threat to the health of mother and child. Although the pathophysiological mechanisms that underlie the association between maternal diabetes and pregnancy complications have not yet been elucidated, it has been suggested that the frequency and severity of pregnancy complications are linked to the degree of hyperglycemia. Epigenetic mechanisms reflect gene-environment interactions and have emerged as key players in metabolic adaptation to pregnancy and the development of complications. DNA methylation, the best characterized epigenetic mechanism, has been reported to be dysregulated during various pregnancy complications, including pre-eclampsia, hypertension, diabetes, early pregnancy loss and preterm birth. The identification of altered DNA methylation patterns may serve to elucidate the pathophysiological mechanisms that underlie the different types of maternal diabetes during pregnancy. This review aims to provide a summary of existing knowledge on DNA methylation patterns in pregnancies complicated by pregestational type 1 (T1DM) and type 2 diabetes mellitus (T2DM), and gestational diabetes mellitus (GDM). Four databases, CINAHL, Scopus, PubMed and Google Scholar, were searched for studies on DNA methylation profiling in pregnancies complicated with diabetes. A total of 1985 articles were identified, of which 32 met the inclusion criteria and are included in this review. All studies profiled DNA methylation during GDM or impaired glucose tolerance (IGT), while no studies investigated T1DM or T2DM. We highlight the increased methylation of two genes,  ThuPGC-1α reported increased placental methylation in Chinese and French-Canadian women with GDM  and may lead to the identification of distinct methylation profiles , 96. In In silico analysis revealed that these differentially methylated genes are predominantly involved in metabolic disease pathways, including glucose metabolism-related disorders which were amongst the top ranked pathways in both placenta and cord blood. Finer et al. identified 1418 methylated variable positions in cord blood and 1373 methylated variable positions in the placenta of South Asian women with GDM compared to women without GDM (constitutive photomorphogenic homolog subunit 8 (COPS8), phosphoinositide 3-kinase regulatory subunit 5 (PIK3R5), 3-hydroxyanthranilate 3,4-dioxygenase (HAAO), coiled-coil domain containing 124 (CCDC124), and chromosome 5 open reading frame 34 (C5orf34), were identified and validated using bisulfite pyrosequencing. Methylation differences of these CpG sites were detected early during pregnancy and may prove useful as predictive biomarkers of GDM. However, this study included 11 women with and without GDM only, and thus, requires further validation in a larger sample size (Rho Guanine Nucleotide Exchange Factor 11 (ARHGEF11), a key gene associated with metabolic pathways, which was negatively correlated with glucose levels and neonatal birth weight (aryl hydrocarbon receptor repressor (AHRR) and protein tyrosine phosphatase receptor type N2 (PTPRN2), were differentially methylated in both cord blood and placental samples. Functional analysis of the differentially methylated genes revealed that the top biological pathways were enriched for antigen processing and presentation via MHC class 1, which plays a vital role in regulating immune responses. Together, these findings indicate that differentially methylated genes, identified in the placenta, cord blood, adipose tissue, and peripheral blood of women with GDM are involved in several metabolic processes that may play an important role in the development of GDM. However, future studies should consider validation or verification of these differentially methylated genes in different populations to confirm their feasibility.Eight of the 12 studies investigated in this review used the Illumina HumanMethylation450 BeadChip Array, which interrogates more than 480,000 methylation sites and covers 96% of CpG islands, as well as additional island shores . Ruchat hout GDM . Of theshout GDM . Haertlehout GDM . Using mhout GDM . Functiohout GDM . Using aple size . Yan et ple size . The auth weight . Using ah weight . More reh weight . Two of (JAK2/STAT-3) and mitogen activated protein kinase (MAPK) in women with GDM compared to women without GDM. Conversely, in a more recent study, Kasuga et al. quantified DNA methylation at 754 255 CpG sites in the cord blood of 230 Japanese women and showed no significant differences between women with GDM compared to women without GDM  and pre-existing T2DM (n=3). However, this study correlated DNA methylation with offspring sex and did not compare DNA methylation across diabetes groups, therefore was not included in this review .None of the four studies that measured global DNA methylation reported consistent associations between global DNA methylation and GDM , 43, 53.HIF3α and PGC1-α and the decreased methylation of PPARα in women with GDM compared to women without GDM were consistent across studies in diverse populations, using different measurement platforms and methodologies, biological material and diagnostic criteria, supporting their involvement in GDM. The results reported in this review showed that CpG islands in the promoter region of HIF3α were more methylated in women with GDM compared to women without GDM in European and Chinese populations   . Similaripidemia  and obesipidemia  in offspoduction , 112. Thp-values <0.05 for their analysis , Thuthuka Grant No: 129844 to SD and Grant No: 129897 to TW, and the NRF Competitive Programme for Rated Researchers Grant No: 120832 to CP. Baseline funding from the South African Medical Research Council (SAMRC) is also acknowledged.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.The content hereof is the sole responsibility of the authors and do not represent the official views of the NRF or SAMRC.


Inadequate differentiated diagnostic features of predominantly colonic inflammatory bowel diseases i.e., ulcerative colitis and Crohn’s colitis, may lead to inexact diagnosis of “indeterminate colitis”. About 15% of indeterminate colitis patients are diagnosed at colonoscopy, in colonic biopsies, and/or at colectomy. Managing outcomes of indeterminate colitis, given its unpredictable clinical presentation, depends on future diagnosis of colitis, Crohn’s colitis or ulcerative colitis.Overview the diagnostic efficacy of ectopic colonic ileal metaplasia and human α-defens 5  for accurate delineation of indeterminate colitis into authentic Crohn’s colitis and/ or ulcerative colitis.We describe a targeted protein for potentially differentiating indeterminate colitis into an accurate clinical subtype diagnosis of inflammatory bowel diseases i.e., ulcerative colitis and Crohn’s colitis.Twenty-one patients with the clinically inexact diagnosis of indeterminate colitis were followed, reassessed and data analyzed.We observed that (i) some patients had their original diagnosis changed from indeterminate colitis to either ulcerative colitis or Crohn’s colitis; and (ii) human α-defensin 5 is aberrantly overexpressed in Crohn’s colitis.Fifteen of the twenty-one (71.4%) patients with indeterminate colitis had their inconclusive diagnosis changed; nine patients changed to ulcerative colitis and six to Crohn’s colitis. In human colon surgical samples, Human α-defensin-5 was significantly upregulated in Crohn’s colitis. In addition, Human α-defensin 5 processing enzyme, matrix metalloptotease-7 was inversely expressed compared to Human α- Defensin 5.Due to the sequence homology of the α-defensin class of proteins, preceding efforts to raise antibodies (Abs) against DEFA5 have limitations to produce adequate specificity. The Abs used in previous assays recognizes the α-defensins, active α-defensins 5 and inactive pro- α-defensins 5. Monoclonal antibodies (mAbs) to determine specificity and sensitivity of α-defensins 5, which is diagnostic of CC disease, and NOT other α-defensins is the limitation to overcome.It is feasible to differentiate ulcerative colitis from Crohn’s colitis among patients with inexact diagnosis of indeterminate colitis using Human α-defensin 5 as a molecular biosignature delineator. Inadequate differentiated diagnostic features of the two predominantly colonic inflammatory bowel diseases (IBD) i.e., ulcerative colitis (UC) and Crohn’s colitis (CC), may lead to an inexact/inconclusive diagnosis of indeterminate colitis (IC). 5novo Crohn’s ileitis after surgical intervention. In colorectal surgery, the distinction between UC and CC is of utmost importance when determining a patient’s candidacy for pouch surgery, the restorative proctocolectomy with ileal pouch-anal anastomosis (RPC-IPAA). DEFA5 gene and is highly expressed in secretory granules of Paneth cells of the small intestine (ileum). 16DEFA5 is a human protein that is encoded by the In determining levels of DEFA5 in the IBD, we overviewed its possible mechanism. It is known that MMP-7 is responsible for cleaving and activating DEFA5. DEFA5, also abbreviated as HD5, as a biomarker to molecularly differentiate CC from UC in otherwise inexact IC diagnosis is a conceptual innovative strategy for identifying appropriate patients with colonic IBD for recommended surgical treatment regimens.To date, there is no available single molecular biomarker that has proven to have the desired diagnostic qualities in IBD. In this overview, we provide DEFA5 as a possible signature biomarker to solve the diagnostic ambiguity and delay in IBD. Several lines of evidence herein presented suggest that the use of Studies were approved by the Meharry Medical College (IRB file number: 100916AM206) and Vanderbilt University (IRB file numbers: 080898 and 100581) Institutional Ethical Review Board Committees and conducted in accordance with the Second International Helsinki Declaration. 4Pathology teams at Meharry Medical College  and Vanderbilt University Schools of Medicine (MKW) used the following protocol criteria for the final surgical pathology reporting.Characteristic pattern of involvement of colon, worse distally in untreated patients; lack of perianal or fistulizing disease; no granulomas, except in association with ruptured/injured crypts; no transmural lymphoid aggregates or other transmural inflammation; no involvement of terminal ileum, except mild “backwash ileitis” in cases with severe cecal involvement and no pyloric metaplasia in terminal ileum.Involvement of other sites in GI tract ; perianal or fistulizing disease; granulomas, not in association with ruptured/injured crypts and terminal ileum involvement.Distribution favors UC, but focal transmural inflammation, or inflammation in ileum more than expected in backwash ileitis and no fistulizing disease.The availability of a detailed IBD patient database registry at VUMC makes chart review and follow-up surveillance possible. Medical records data on patient demographics, preoperative variables prior to and after the time of RPC-IPAA surgery, surveillance endoscopic and clinical findings, and medical and surgical treatment history are able to be retrieved retrospectively. Further, VUMC has many health centers of practices located throughout greater Nashville metropolitan area and parts of Kentucky that provide referrals to the IBD center.A retrospective investigation was conducted to identify a cohort of patients diagnosed with IC and registered in the IBD Center at VUMC. Twenty-one patients, initially classified as IC at the time of diagnosis between years 2000 - 2007, were identified and reevaluated for disease course in 2014 after a mean surveillance follow-up of 8.7±3.7  years in order to identify the rates of diagnosis resolution to UC or CC. Diagnosis for each patient was determined based on standard clinical and pathologic features as described in the diagnostic criteria in the Whole-transcriptome microarray with RNA extracted and pooled from human full thickness colon samples from UC and CC patients (n = 5/group)  as previously described. 23_RNA from experimental and control cell was processed following NanoString  recommended procedure to determine gene expression level according to the manufacturer protocol. 24qPCR was used to measure transcript levels of DEFA5 and MMP-7 for MicroRNA signatures to differentiate CC from UC as previously described. Western blot was utilized to assess any differences of DEFA5 and MMP-7 at the protein level as previously described  29). Pr. Pr29). Five colon tissue protein extracts and staining of DEFA5 per disease by IHC was done as previously described. Quantification of DEFA5 staining was analyzed manually by microscopy / automatically quantified using Nikon's Eclipse Ti microscope with built-in NIS-Elements Advanced Research software (NEARAS). 28Nikon Elements Advanced Research Software (NEARAS)  was used to manually calculate the number of cells with DEFA5 staining in IHC tissue slides. Statistical analyses were performed using GraphPad Prism v6 software  qRT-PCR,Twenty-one (#21) patients, Diagnostic ambiguity and delay among IBD patients with IC has remained a major challenge in endoscopic medicine 27A standard surgical procedure for treating UC, RPC-IPAA typically does not benefit and can be harmful to most CC patients because of higher rates of pouch failure. Therefore, it is important for surgeons to choose the appropriate treatment for their IBD patients To circumvent the diagnosis ambiguity and delay in IBD clinical setting we analyzed colonic surgical pathology samples of patients with unambiguous CC and UC undergoing colectomy in connection with RPC. To date, there is no diagnostic “Gold standard” tool for IBD. Differentiating UC and CC among patients with IC has remained a major challenge in endoscopic medicine. 66Curative treatment for UC is often surgical. 70UC and CC share many demographic and clinical features yet present significant differences in tissue inflammation and damage, suggesting a distinct etiopathogenic trigger. p < 0.05). The definitive CC and CI diseases do not share demographic and clinical features and/or molecular biometrics of tissue inflammation and damage suggesting distinct etiology and mechanisms. This is intriguing and needs further elucidation. These studies have reconciled all IC patient samples into authentic either UC or CC using DEFA5 and patient outcomes, Contrary, in line with the discussed data herewith presented, it is evidently observed in a newly assembled cohort that patients with “Crohn’s ileitis (CI)” are characterized with a deficiency of DEFA5,  as shownEarly timely diagnosis accuracy leads to appropriate therapeutic options and will aid in determining candidates for RPC-IPAA and avoid unnecessary surgeries. 5DEFA5 expressed differentially in IBD and that aberrant expression, localization, and/or activation of DEFA5 underlies the tissue inflammation and damage associated with authentic CC form of colitis. DEFA5 is a reliable signature to distinguishCC from UC among IC patient cohort. It is promosing to use DEFA5 to determine patient candidacy for RPC-IPAA surgery.The high degree of similarity of DEFAs implies that antibodies against DEFA5 may not be specific enough to distinguish DEFA5 from other DEFAs. The rate limiting step is the generation of monoclonal antibodies to the DEFA5 peptides. We are confident that antibodies against the segments of DEFA5, dones 1A8 and 4FD will be successful.


Wang et al. Nature 10.1038/s41586-023-06314-y (2023)1 argue that our report2 of lower mutation rates in gene bodies, essential genes and regions marked by H3K4me1 must result from DNA sequencing errors. We appreciate the issues raised by them and by other colleagues3. Although we overlooked some sources of errors, these are insufficient to invalidate our conclusions, which are confirmed by more stringent reanalyses of our original data, new analyses restricted to high-confidence germline mutations4, and direct demonstration of plant DNA repair proteins being recruited to gene bodies, essential genes and H3K4me1, where they reduce local mutation rates6.Wang and colleagues1 identify issues with somatic mutation calling, suggesting that homopolymer bleed-through errors in Illumina sequencing are responsible for patterns observed in somatic mutations, and that elevated cytosine deamination in transposable elements is responsible for the patterns in germline mutations. Here we address these concerns.Wang and colleagues7. At the same time, homopolymer regions have higher true mutation rates even at local but non-adjacent sites . Wang and colleagues1 found that the distribution of simulated homopolymer errors mirrors the overall distribution of mutations we reported around genes  could be potential homopolymer-adjacent bleed-through errors, and thus on their own cannot explain the approximately 50% mutation rate reduction we observed in gene bodies relative to intergenic regions2.Wang and colleagues1 reports that the proportion of potential homopolymer bleed-through errors in our data is actually higher in gene bodies (exons plus introns), which should lead to gene body mutation rates being overestimated, not underestimated. We confirm across our datasets that the proportion of potential homopolymer bleed-through errors is not lower in gene bodies  are enriched outside gene bodies, as reported by Wang and colleagues1 further suggest that the patterns we observed in germline mutations might result largely from elevated deamination of methylated cytosines (GC-to-AT mutations) in transposable elements. Several findings are inconsistent with this hypothesis: cytosine methylation was included as a covariate in our original models, mutation accumulation experiments consistently indicate that mutation rates are lower in gene bodies relative to non-transposable element intergenic regions in A. thaliana . The same has been demonstrated for mutation rate variation in rice, in which mutation rates are lower in gene bodies relative to both intergenic regions and transposable elements6.Wang and colleagues10 by mapping reads to an improved reference genome11 and applying more stringent filtering . The notable exception comes from plants lacking the mismatch repair protein MSH2 . A similar pattern is seen when somatic mutations were called with very stringent criteria in plants deficient for the MSH2 partner MSH6, using a tool specifically designed for rare somatic mutations14  and References.Reporting Summary


In vitro evolutionstrategies have been used for >30 years to generatenucleic acid aptamers against therapeutic targets of interest, includingdisease-associated proteins. However, this process requires many iterativecycles of selection and amplification, which severely restricts thenumber of target and library design combinations that can be exploredin parallel. Here, we describe a single-round screening approach toaptamer discovery that relies on function-enhancing chemotypes toincrease the distribution of high-affinity sequences in a random-sequencelibrary. We demonstrate the success of de novo discovery by affinityselection of threomers against the receptor binding domain of theS1 protein from SARS-CoV-2. Detailed biochemical characterizationof the enriched population identified threomers with binding affinityvalues that are comparable to aptamers produced by conventional SELEX.This work establishes a highly parallelizable path for querying diversechemical repertoires and may offer a viable route for acceleratingthe discovery of therapeutic aptamers. Although machine learning algorithmscould 1 day solve this problem,14 reinforcementlearning requires large amounts of data that are difficult to generateusing the traditional model of one target, one library, and many roundsof selection.Darwinianevolution has long been viewed as the cornerstone ofbiology and the inspiration for laboratory methods capable of discoveringand refining biopolymer function through iterative rounds of selection.24 uniquesequences, which is considerably larger than the number of sequencessampled in a typical aptamer selection (∼1014).15 Estimates on the frequency of folded and functionalaptamers vary according to target but can be as low as 10–10, emphasizing the magnitude of the problem facing those attemptingto bypass the evolutionary process.16 Toinvestigate the feasibility of converting in vitro selection intoa high-throughput screen, we considered the potential for function-enhancingside chains to increase the abundance of high-activity sequences ina random-sequence library  of <1 nM.23 This finding is consistent with the ability of base-modified aptamersto effectively mimic the paratope surface of an antibody.24The requirement for recursive rounds of selectionreflects thedifficulty in surveying large pools of random sequences for membersthat can fold into structures that function with a predefined activity.A nucleic acid library consisting of 40 randomized positions, forinstance, has a theoretical diversity of ∼10 library 1a. We we25 Our results indicate that a single round of high-throughput screeningis sufficient to generate threomers that can bind their target proteinwith low to subnanomolar affinity and discriminate against off-targetproteins known to bind nonspecifically to nucleic acid sequences.Here, we wished to testthe hypothesis that side chains could increasethe abundance of functionally enhanced TNA aptamers, termed threomers,in a pool of random sequences 1a. To teT/L) ratios, and various buffer conditions for proteinbinding and bead washing. Accordingly, each library aliquot (1012 molecules) was passed through a negative selection stepto remove sequences with affinity to Ni-NTA beads and a positive selectionstep for binding to a representative therapeutic protein  is∼6.9 × 1010, each library aliquot should containa similar distribution of sequences. Molecules that bound to the S1protein in solution were partitioned away from the unbound materialby passing the mixture over Ni-NTA beads to capture the bound aptamer–proteincomplexes on a solid-support matrix. Following several wash steps,the dsDNA-encoding tags were photocleaved and quantified relativeto their starting molar amount using a quantitative polymerase chainreaction (qPCR) . Selectionconditions with higher library to elution values (L/E) were viewed as more optimal for a protein bindingassay as the elution fractions were expected to be more enriched infunctional aptamers than conditions with lower L/E values.Aptamer discovery based on a single round ofprotein target bindingdemands stringent binding conditions to partition functional sequencesaway from the nonfunctional pool. To probe the partitioning of activeand inactive library members, we subjected a Phe chemotype libraryto a competitive binding assay that explored a broad range of conditions,which included aptamer refolding, target-to-library  revealed that a 1:10 ratio was optimal for a naïve library.Surprisingly, the inclusion of bovine serum albumin (BSA) and salmonsperm DNA (ssDNA) as competitive proteins and nucleic acid agents,respectively, in the binding buffer (buffer 2) exhibited only a modest2-fold increase in partitioning efficiency. However, the most strikingdifference (∼2500-fold partitioning) was observed with conditions13–15, which combined the cumulative effects of these smallerchanges to the standard binding and washing protocol with an aptamerrefolding step. Control experiments show that the aptamer refoldingstep and urea wash did not disrupt the dsDNA region of the DNA displaylibrary . As such, condition15, which included an aptamer refolding step, BSA, and ssDNA in thebinding buffer, 10-fold less protein to the library, and urea in thewash buffer, was chosen as the optimal condition for our single-roundaptamer screening approach.The results of our binding assays show the profoundeffect thatthe binding and wash conditions can have on the partitioning efficiencyof functional aptamers from weakly active and inactive library members2b. The iFigure S3) that simplifies the selection by avoidingthe need for a separate reverse transcription step,27 which is inefficient at low concentrations of TNA.28 Relative to prior work,29 aphotocleavable linker was included in the region connecting the dsDNAto the TNA sequence to promote efficient DNA recovery and amplificationby droplet PCR (S3). The libraries were designedat the DNA level to contain a 30 nucleotide (nt) random region  that was flankedon both sides with fixed-sequence primer binding sites (PBS) and aunique bar code positioned between the 5′ PBS and random region(Tables S1 and S2). Each linear single-strandedDNA library was ligated at the 3′ end to a DNA stem-loop structurethat served as the primer for TNA synthesis on the DNA template 25  in a parallel binding assay.The screens were performed using a DNA-display library format (plet PCR 1d and S312 molecules) was passed through a negative selection step to removesequences with an affinity for Ni-NTA beads and a positive selectionstep for binding to the S1-RBD protein. Sequences that bound to theprotein were partitioned away from the unbound material by passingthe mixture over Ni-NTA beads to capture the aptamer–proteincomplexes on a solid-support matrix. The beads were washed 3 timeswith selection binding buffer, once with 2 M urea, and twice withPCR buffer . The double-strandedDNA region encoding the bound TNA molecules was photocleaved and amplifiedby droplet PCR to ensure even amplification of the eluted material.The resulting amplicons were further amplified in solution by bulkPCR and submitted for HTS.An activity screen was performed in parallel to evaluatethe propensityfor the various function-enhancing side chains  to increase the abundance of TNA aptamers present inunbiased pools of random sequences. Guided by the results of the bindingassay, the screen was performed with condition 15 , which included 0.5mg/mL BSA and 0.05 mg/mL ssDNA as general protein and DNA competitors,respectively. Similar to the binding assay 1d, each 32 In contrast to traditionalparticle display,33 whereby DNA aptamersare attached directly to the surface of a magnetic particle usingan emulsion PCR and template stripping protocol, hydrogel aptamerparticles are prepared by extension of a DNA primer that has beencross-linked into the gel matrix of a polyacrylamide hydrogel shellencapsulating a magnetic particle.32 TheTNA aptamer is generated by primer extension using a DNA templateencoding the desired TNA sequence. This microfluidics-free approach,which allows reagents and enzymes to pass freely in and out of thematrix, is necessary because a polymerase has not yet been discoveredthat can amplify XNAs by PCR. Following TNA synthesis, the templateis removed and the TNA aptamers are folded by heating and coolingthe hydrogel particles in binding buffer . The resulting monoclonal TNAaptamer hydrogel particles are evaluated for binding to a biotin-modifiedversion of S1-RBD in 96-well format using an ELISA-type assay thatis compatible with flow cytometry , viewed here as a background control, are predictedto function with a higher target binding affinity due to their abilityto remain bound to the target protein following iterative wash steps.This approach dramatically accelerates the pace of XNA aptamer characterizationby reducing the scale of aptamer synthesis and eliminating the needto individually purify each TNA sequence by denaturing polyacrylamidegel electrophoresis.To illuminate the relationship betweenlibrary chemotype and function,96 representative members from each chemotype family were evaluatedfor binding to S1-RBD using a highly parallel hydrogel aptamer particlestrategy.ytometry 3a. In th34 Coincidentally, the Trp side chain is also commonlyfound on the paratope of protein–antibody cocrystal structures,35 implying that our design converged on a solutionthat mimics nature’s approach to protein binding affinity reagents.Flow cytometry analysis of the six chemotype families(576 sequences)offered striking insights into the potential for function-enhancingside chains to increase the abundance of TNA aptamers in a random-sequencelibrary. In an effort to minimize bias, we standardized the sequenceselection criteria across aptamer binding experiments by randomlychoosing sequences that contained 30% chemical modifications (or Tfor the std chemotype) and no more than three identical nucleotidesin a row from the NGS data collected from the elution fractions ofeach chemotype library screen. Other levels of chemical modificationswere not evaluated in this study. The resulting flow cytometry dataindicate 3bthat t15 However, it was interesting to note that twoof the threomers isolated from the 25 nt library exhibited bindingresponse levels that are similar in magnitude (18-fold) to those ofthe best 30 and 40 nt threomers, indicating that shorter Trp-modifiedaptamers, though more rare, are still capable of achieving high-affinitybinding to S1-RBD.Inspired by the enhanced functional activity exhibited by the Trp-modifiedTNA library, we wished to gain a deeper understanding of the lengthdependency of the random region on the discovery rate of functionalthreomers. We were particularly interested in the question of whethercertain highly active chemotypes could promote the discovery of shorter-lengththreomers. If so, such an approach would offer a viable route to threomersthat could eventually be produced, resulting in higher yields andlower costs than equivalent reagents of longer lengths. To explorethis question, we screened three new Trp chemotype libraries withrandom regions of 20, 25, and 40 unbiased nucleotides for bindingto S1-RBD. Flow cytometry analysis of 96 representative members fromeach Trp chemotype library (384 sequences) revealed a positive correlationbetween library length and activity 3c, consiTable S2). The resulting aptamers were PAGE purified, electroeluted, andUV quantified. Aptamers were immobilized on SA-coated biosensor tipsand evaluated for binding to the S1 protein. Curve fitting revealsthat the data conform to a 1:1 binding model with calculated dissociationconstants (KD) spanning a narrow rangeof 0.8–3.7 nM (Table S3). Closeinspection of the binding curves  demonstrates that the 17 threomers function with off-rates (koff 1.3–8.8 × 10–4 s–1) that are comparable to high-quality antibodies.37 Interestingly, the two top performing 25 nt threomers ranked secondand third out of the 17 threomers evaluated by BLI  and were only ∼2-fold weaker in activity thanthe top threomer (KD = 0.8 nM) identifiedin our screen  for the top 17 threomers ranked according to their observedfold change in our ELISA-based flow cytometry assay. Each aptamerwas prepared by primer extension using a 5′ biotinylated DNAprimer and complementary DNA template (g curves S1 demonsp < 0.001) on the Trp chemotype for S1-RBD binding activity . The data show that high-activitybinding is abrogated when the Trp side chain is replaced with a methylgroup from the analogous thymine base found in the std library ora uracil residue carrying Phe at the C-5 position.Recognizing the Trp side chain as a critical factorresponsiblefor achieving high-affinity binding, we decided to perform a structure–activityrelationship (SAR) study to confirm that the S1-RBD binding activitywas dependent on the presence of the Trp moiety. Flow cytometry analysisperformed on the top 17 S1-RBD binding threomers identified by hydrogelparticle screening 3c, each Concernedthat threomers obtained from a high-throughput screenmight function with general protein-binding activity, we chose toexamine the target binding specificity of the top 17 S1-RBD bindingthreomers in the particle display format. In separate flow cytometryassays, the aptamer hydrogel particles were challenged to distinguishtheir cognate S1-RBD target as well as the full-length S1 proteincontaining the RBD domain from two off-target proteins, which includedhuman serum albumin (HSA) and SA 5a. HSA a15 Top-performing sequences are then refined by directed evolutionor via a post-SELEX chemical optimization process in which additionalchemistry is used to enhance the physicochemical properties of themolecule.41 This was the approach taken to discoverPegaptanib (Macugen), the first therapeutic aptamer to receive approvalby the US Food and Drug Administration (FDA) for the treatment ofage-related macular degeneration.42 However,the inherently slow nature of this methodology, which can take severalmonths to complete, severely restricts the pace of aptamer discovery.While automated protocols have been established to streamline thediscovery of DNA and RNA aptamers,43 suchapproaches require specialized equipment and follow procedures thatare not readily transferrable to XNAs due to a lack of polymerasesthat can amplify XNA by PCR. Although machine learning algorithmsprovide an opportunity to address this problem,14 reinforcement learning necessitates large amounts of datathat are not presently available for alternative genetic systems,like TNA, that are only beginning to emerge as putative genetic systemsfor therapeutic aptamers.44Aptamers are typicallydiscovered through an iterative processof Darwinian evolution in which many recursive cycles of in vitroselection and amplification are performed to identify individual sequencesthat can bind to a desired target with high affinity.37 and aptamers evolved from conventional in vitro selection protocols.39 SAR studies confirmed the importance of the function-enhancing sidechain, a Trp chemotype, as a critical element for target binding affinityand specificity. Functional analysis of hundreds of TNA sequencesrevealed that the Trp side chain is able to augment the TNA scaffoldwith physicochemical properties that were not observed in screensperformed against other chemotype categories. This example is qualitativelysimilar to the complementary determining region of antibodies, whichuse large aromatic residues to shape the paratope surface with hydrophobicinteractions that help drive epitope binding. However, unlike antibodies,threomers are conceptually much easier to discover as targets canbe queried entirely by in vitro means that avoid the use of animalmodels and complex cellular expression systems that are prone to contaminationand difficult to scale.The currentstudy describes a new approach to aptamer discoverythat bypasses the Darwinian evolution process by using a library screeningstrategy that relies on the presence of function-enhancing side chainsto increase the abundance of functional aptamers in unbiased poolsof random sequences. We demonstrated the effectiveness of this approachby identifying threomers from a single round of high-throughput screeningthat bind the S1-RBD protein with binding affinities values (low tosubnanomolar) comparable to monoclonal antibodies45 As these studies continue, it will be interestingto see how their results shape our understanding of such fundamentalquestions as how reproducible evolutionary trajectories are in sequencespace and what underlying chemical forces enable chemotypes to enhancebiopolymer function. In the area of biotechnology, highly parallelizablescreening platforms, such as the one described herein, could fast-trackthe rise of therapeutic aptamers by drastically lowering the barrierrequired for reagent discovery. The data generated from these experimentscould accelerate the development of computational tools that can identifyaptamers de novo or predict sequences that function with even greateractivity than what might be observed in an experimental dataset. SinceTNA is invisible to biological nucleases, the resulting reagents canbe used directly without the need for the medicinal chemistry stepsrequired to stabilize the backbone structure of DNA and RNA aptamers.Collectively, these findings representa possible paradigm shiftin the way that aptamers are discovered. Rather than following thetraditional model of one target, one library, and many rounds of selection,researchers now have an opportunity to investigate a range of targetsand library designs in parallel and under experimental conditionsthat minimize the problem of amplification bias that arises when multiplerounds of selection are performed against the same target.We demonstrate that high-throughput screeningof functionally enhancedTNA libraries offers a viable route for discovering threomers thatcan bind their targets with high affinity and high specificity. Wesuggest that the ability to bypass conventional in vitro selectionprotocols could help narrow the gap between aptamers and antibodiesby providing a highly parallelizable path for querying diverse chemicalrepertoires and may offer a viable route for accelerating the discoveryof therapeutic aptamers.