Search for: All records

Introduction The effect of testosterone (T) therapy on the vaginal microbiota of transgender men (TGM) is not well characterised, although one cross-sectional study comparing the vaginal microbiota of cisgender women to TGM on T≥1 year found that, in 71% of the TGM, the vaginal microbiota was less likely to be Lactobacillus -dominated and more likely to be enriched with >30 other bacterial species, many associated with bacterial vaginosis (BV). This prospective study aims to investigate changes in the composition of the vaginal microbiota over time in TGM who retain their natal genitalia (ie, vagina) and initiate T. In addition, we will identify changes in the vaginal microbiota preceding incident BV (iBV) in this cohort while investigating behavioural factors, along with hormonal shifts, which may be associated with iBV. Methods and analysis T-naïve TGM who have not undergone gender-affirming genital surgery with normal baseline vaginal microbiota (ie, no Amsel criteria, normal Nugent Score with no Gardnerella vaginalis morphotypes) will self-collect daily vaginal specimens for 7 days prior to initiating T and for 90 days thereafter. These specimens will be used for vaginal Gram stain, 16S rRNA gene sequencing and shotgun metagenomic sequencing to characterise shifts in the vaginal microbiota over time, including development of iBV. Participants will complete daily diaries on douching, menses and behavioural factors including sexual activity during the study. Ethics and dissemination This protocol is approved through the single Institutional Review Board mechanism by the University of Alabama at Birmingham. External relying sites are the Louisiana State University Health Sciences Center, New Orleans Human Research Protection Program and the Indiana University Human Research Protection Program. Study findings will be presented at scientific conferences and peer-reviewed journals as well as shared with community advisory boards at participating gender health clinics and community-based organisations servicing transgender people. Registration details Protocol # IRB-300008073. 

Background Despite more than 60 years of research, the etiology of bacterial vaginosis (BV) remains controversial. In this pilot study, we used shotgun metagenomic sequencing to characterize vaginal microbial community changes before the development of incident BV (iBV). Methods A cohort of African American women with a baseline healthy vaginal microbiome (no Amsel criteria, Nugent score 0–3 with no Gardnerella vaginalis morphotypes) were followed for 90 days with daily self-collected vaginal specimens for iBV (≥2 consecutive days of a Nugent score of 7–10). Shotgun metagenomic sequencing was performed on select vaginal specimens from 4 women, every other day for 12 days before iBV diagnosis. Sequencing data were analyzed through Kraken2 and bioBakery 3 workflows, and specimens were classified into community state types. Quantitative polymerase chain reaction was performed to compare the correlation of read counts with bacterial abundance. Results Common BV-associated bacteria such as G. vaginalis , Prevotella bivia , and Fannyhessea vaginae were increasingly identified in the participants before iBV. Linear modeling indicated significant increases in G. vaginalis and F . vaginae relative abundance before iBV, whereas the relative abundance of Lactobacillus species declined over time. The Lactobacillus species decline correlated with the presence of Lactobacillus phages. We observed enrichment in bacterial adhesion factor genes on days before iBV. There were also significant correlations between bacterial read counts and abundances measured by quantitative polymerase chain reaction. Conclusions This pilot study characterizes vaginal community dynamics before iBV and identifies key bacterial taxa and mechanisms potentially involved in the pathogenesis of iBV. 

Soil moisture–precipitation (SM–PPT) feedbacks at the mesoscale represent a major challenge for numerical weather prediction, especially for subtropical regions that exhibit large variability in surface SM. How does surface heterogeneity, specifically mesoscale gradients in SM and land surface temperature (LST), affect convective initiation (CI) over South America? Using satellite data, we track nascent, daytime convective clouds and quantify the underlying antecedent (morning) surface heterogeneity. We find that convection initiates preferentially on the dry side of strong SM/LST boundaries with spatial scales of tens of kilometers. The strongest alongwind gradients in LST anomalies at 30-km length scale underlying the CI location occur during weak background low-level wind (<2.5 m s⁻¹), high convective available potential energy (>1500 J kg⁻¹), and low convective inhibition (<250 J kg⁻¹) over sparse vegetation. At 100-km scale, strong gradients occur at the CI location during convectively unfavorable conditions and strong background flow. The location of PPT is strongly sensitive to the strength of the background flow. The wind profile during weak background flow inhibits propagation of convection away from the dry regions leading to negative SM–PPT feedback whereas strong background flow is related to longer life cycle and rainfall hundreds of kilometers away from the CI location. Thus, the sign of the SM–PPT feedback is dependent on the background flow. This work presents the first observational evidence that CI over subtropical South America is associated with dry soil patches on the order of tens of kilometers. Convection-permitting numerical weather prediction models need to be examined for accurately capturing the effect of SM heterogeneity in initiating convection over such semiarid regions.

 

Purification of recombinant proteins is a necessary step for functional or structural studies and other applications. Immobilized metal affinity chromatography is a common recombinant protein purification method. Mass spectrometry (MS) allows for confirmation of identity of expressed proteins and unambiguous detection of enzymatic substrates and reaction products. We demonstrate the detection of enzymes purified on immobilized metal affinity surfaces by direct or ambient ionization MS, and follow their enzymatic reactions by direct electrospray ionization (ESI) or desorption electrospray ionization (DESI).

A protein standard, His‐Ubq, and two recombinant proteins, His‐SHAN and His‐CS, expressed inEscherichia coliwere immobilized on two immobilized metal affinity systems, Cu–nitriloacetic acid (Cu‐NTA) and Ni‐NTA. The proteins were purified on surface, and released in the ESI spray solvent for direct infusion, when using the 96‐well plate form factor, or analyzed directly from immobilized metal affinity‐coated microscope slides by DESI‐MS. Enzyme activity was followed by incubating the substrates in wells or by depositing substrate on immobilized protein on coated slides for analysis.

Small proteins (His‐Ubq) and medium proteins (His‐SAHN) could readily be detected from 96‐well plates by direct infusion ESI, or from microscope slides by DESI‐MS after purification on surface from clarifiedE. colicell lysate. Protein oxidation was observed for immobilized proteins on both Cu‐NTA and Ni‐NTA; however, this did not hamper the enzymatic reactions of these proteins. Both the nucleosidase reaction products for His‐SAHN and the methylation product of His‐CS (theobromine to caffeine) were detected.

The immobilization, purification, release and detection of His‐tagged recombinant proteins using immobilized metal affinity surfaces for direct infusion ESI‐MS or ambient DESI‐MS analyses were successfully demonstrated. Recombinant proteins were purified to allow identification directly out of clarified cell lysate. Biological activities of the recombinant proteins were preserved allowing the investigation of enzymatic activity via MS.

 

Purpose This study aims to present theory, practice and original research findings to support the proposition that broad enquiry and problem-based learning (EPBL) approaches provide an appropriate pedagogical lens for sustainability educators to develop the knowledge and skills needed to work effectively within mission-oriented innovation policy (MIP) environments. Design/methodology/approach The research study comprised four elements, each of which used different research methods. The first element involved a literature review mapping the synergies between MIP and EPBL; the second element piloted the use of EPBL for undergraduate modules related to sustainability challenges; the third element involved external stakeholders in the co-creation of a postgraduate programme that brought together innovation and sustainability, with EPBL fundamental to the design and development; the fourth element curated and comparatively analysed international cases of EPBL in the context of MIP, and sustainability challenges in particular, highlighting the versatility of EPBL and the importance of creativity in EPBL design and implementation. Findings The systematic literature review reveals synergies between the key features of EPBL and defining characteristics of MIP, indicating the relevance of applying EPBL to support MIP. Two in situ pilots generated 13 recommendations on the benefits and operational challenges of applying EPBL. These recommendations informed the design and development of a postgraduate programme, involving a transdisciplinary consultation process with key industrial and societal stakeholders. Comparative analysis of four international case studies describing EPBL applied in practice in different international settings show there is no “one size fits all”. Instead, the application of EPBL to different sustainability challenges and for different learner groups demonstrates the versatility of the pedagogical approach and the creativity of the sustainability educators. Originality/value A discourse around the appropriate pedagogical methods and teaching/learning practice to equip the current and future workforce with the knowledge and skills to respond to MIP and global sustainability challenges is nascent but emerging. This paper makes a scientific and practical contribution to the discourse. The authors show how EPBL can underpin the design of programmes to provide learners with the knowledge and skills to support organisations working effectively within an MIP context, especially addressing sustainability challenges. The authors provide recommendations for educators seeking to embed EPBL within their curriculum and call for external stakeholders to proactively engage with educators to co-create programmes with context-specific outcomes. 

Biodiversity collections are experiencing a renaissance fueled by the intersection of informatics, emerging technologies, and the extended use and interpretation of specimens and archived databases. In this article, we explore the potential for transformative research in ecology integrating biodiversity collections, stable isotope analysis (SIA), and environmental informatics. Like genomic DNA, SIA provides a common currency interpreted in the context of biogeochemical principles. Integration of SIA data across collections allows for evaluation of long-term ecological change at local to continental scales. Challenges including the analysis of sparse samples, a lack of information about baseline isotopic composition, and the effects of preservation remain, but none of these challenges is insurmountable. The proposed research framework interfaces with existing databases and observatories to provide benchmarks for retrospective studies and ecological forecasting. Collections and SIA add historical context to fundamental questions in freshwater ecological research, reference points for ecosystem monitoring, and a means of quantitative assessment for ecosystem restoration.

 

The availability of an expanded genetic code opens exciting new opportunities in enzyme design and engineering. In this regard histidine analogues have proven particularly versatile, serving as ligands to augment metalloenzyme function and as catalytic nucleophiles in designed enzymes. The ability to genetically encode multiple functional residues could greatly expand the range of chemistry accessible within enzyme active sites. Here, we develop mutually orthogonal translation components to selectively encode two structurally similar histidine analogues. Transplanting known mutations from a promiscuousMethanosarcina mazeipyrrolysyl‐tRNA synthetase (MmPylRS^IFGFF) into a single domain PylRS fromMethanomethylophilus alvus(MaPylRS^IFGFF) provided a variant with improved efficiency and specificity for 3‐methyl‐L‐histidine (MeHis) incorporation. TheMaPylRS^IFGFFclone was further characterized using in vitro biochemical assays and x‐ray crystallography. We subsequently engineered the orthogonalMmPylRS for activity and selectivity for 3‐(3‐pyridyl)‐L‐alanine (3‐Pyr), which was used in combination withMaPylRS^IFGFFto produce proteins containing both 3‐Pyr and MeHis. Given the versatile roles played by histidine in enzyme mechanisms, we anticipate that the tools developed within this study will underpin the development of enzymes with new and enhanced functions.