Gender Is a Spectrum: Evaluating Current and Novel Ways to Inquire About Gender Identity in the Health Care Setting.

PURPOSE: The use of visuals to inquire about gender in the clinical setting has been rare. We developed a survey that included a visual spectrum to assess perceptions about the most and least inclusive ways of inquiring about gender in patients with gender dysphoria. METHODS: The survey included a multiple-choice question (MCQ), free-response question, and a visual spectrum on which respondents were asked to select one box that best depicts their gender. The survey was administered to all patients diagnosed with gender dysphoria at our institution between April and June 2022. RESULTS: A total of 223 of 856 patients responded. Those with more masculine gender identities selected boxes near the visual spectrum corner of "man," whereas responses were more variable for more feminine genders. The free-response question was identified by 59% of respondents as the most inclusive. The MCQ was identified as least inclusive by 70.4%. The visual spectrum was considered the most inclusive method by the majority of patients who self-identified as woman and demiwoman/demifemale. Being asked about pronouns was extremely or very important in the health care setting for 52% of respondents, but 68.6% indicated that they are rarely or sometimes asked about their pronouns in this setting. CONCLUSIONS: The traditional MCQ format for self-identifying gender may be lacking in inclusivity and fails to represent the nuances of gender identity. Free response was considered the most inclusive way to inquire about gender among our respondents. These findings highlight the importance of formatting gender identity questionnaires to foster inclusivity for transgender patients.

Soluble Nogo-Receptor-Fc decoy (AXER-204) in patients with chronic cervical spinal cord injury in the USA: a first-in-human and randomised clinical trial.

BACKGROUND: Spinal cord injury (SCI) causes neural disconnection and persistent neurological deficits, so axon sprouting and plasticity might promote recovery. Soluble Nogo-Receptor-Fc decoy (AXER-204) blocks inhibitors of axon growth and promotes recovery of motor function after SCI in animals. This first-in-human and randomised trial sought to determine primarily the safety and pharmacokinetics of AXER-204 in individuals with chronic SCI, and secondarily its effect on recovery. METHODS: We conducted a two-part study in adults (aged 18-65 years) with chronic (>1 year) cervical traumatic SCI at six rehabilitation centres in the USA. In part 1, AXER-204 was delivered open label as single intrathecal doses of 3 mg, 30 mg, 90 mg, or 200 mg, with primary outcomes of safety and pharmacokinetics. Part 2 was a randomised, parallel, double-blind comparison of six intrathecal doses of 200 mg AXER-204 over 104 days versus placebo. Participants were randomly allocated (1:1) by investigators using a central electronic system, stratified in blocks of four by American Spinal Injury Association Impairment Scale grade and receipt of AXER-204 in part 1. All investigators and patients were masked to treatment allocation until at least day 169. The part 2 primary objectives were safety and pharmacokinetics, with a key secondary objective to assess change in International Standards for Neurological Classification of SCI (ISNCSCI) Upper Extremity Motor Score (UEMS) at day 169 for all enrolled participants. This trial is registered with ClinicalTrials.gov, NCT03989440, and is completed. FINDINGS: We treated 24 participants in part 1 (six per dose; 18 men, six women), and 27 participants in part 2 (13 placebo, 14 AXER-204; 23 men, four women), between June 20, 2019, and June 21, 2022. There were no deaths and no discontinuations from the study due to an adverse event in part 1 and 2. In part 2, treatment-related adverse events were of similar incidence in AXER-204 and placebo groups (ten [71%] vs nine [69%]). Headache was the most common treatment-related adverse event (five [21%] in part 1, 11 [41%] in part 2). In part 1, AXER-204 reached mean maximal CSF concentration 1 day after dosing with 200 mg of 412 000 ng/mL (SD 129 000), exceeding those concentrations that were efficacious in animal studies. In part 2, mean changes from baseline to day 169 in ISNCSCI UEMS were 1·5 (SD 3·3) for AXER-204 and 0·9 (2·3) for placebo (mean difference 0·54, 95% CI -1·48 to 2·55; p=0·59). INTERPRETATION: This study delivers the first, to our knowledge, clinical trial of a rationally designed pharmacological treatment intended to promote neural repair in chronic SCI. AXER-204 appeared safe and reached target CSF concentrations; exploratory biomarker results were consistent with target engagement and synaptic stabilisation. Post-hoc subgroup analyses suggest that future trials could investigate efficacy in patients with moderately severe SCI without prior AXER-204 exposure. FUNDING: Wings for Life Foundation, National Institute of Neurological Disorders and Stroke, National Center for Advancing Translational Sciences, National Institute on Drug Abuse, and ReNetX Bio.

Microfluidics-free single-cell genomics with templated emulsification.

Current single-cell RNA-sequencing approaches have limitations that stem from the microfluidic devices or fluid handling steps required for sample processing. We develop a method that does not require specialized microfluidic devices, expertise or hardware. Our approach is based on particle-templated emulsification, which allows single-cell encapsulation and barcoding of cDNA in uniform droplet emulsions with only a vortexer. Particle-templated instant partition sequencing (PIP-seq) accommodates a wide range of emulsification formats, including microwell plates and large-volume conical tubes, enabling thousands of samples or millions of cells to be processed in minutes. We demonstrate that PIP-seq produces high-purity transcriptomes in mouse-human mixing studies, is compatible with multiomics measurements and can accurately characterize cell types in human breast tissue compared to a commercial microfluidic platform. Single-cell transcriptional profiling of mixed phenotype acute leukemia using PIP-seq reveals the emergence of heterogeneity within chemotherapy-resistant cell subsets that were hidden by standard immunophenotyping. PIP-seq is a simple, flexible and scalable next-generation workflow that extends single-cell sequencing to new applications.

Alzheimer risk gene product Pyk2 suppresses tau phosphorylation and phenotypic effects of tauopathy.

BACKGROUND: Genetic variation at the PTK2B locus encoding the protein Pyk2 influences Alzheimer's disease risk. Neurons express Pyk2 and the protein is required for Amyloid-ß (Aß) peptide driven deficits of synaptic function and memory in mouse models, but Pyk2 deletion has minimal effect on neuro-inflammation. Previous in vitro data suggested that Pyk2 activity might enhance GSK3ß-dependent Tau phosphorylation and be required for tauopathy. Here, we examine the influence of Pyk2 on Tau phosphorylation and associated pathology. METHODS: The effect of Pyk2 on Tau phosphorylation was examined in cultured Hek cells through protein over-expression and in iPSC-derived human neurons through pharmacological Pyk2 inhibition. PS19 mice overexpressing the P301S mutant of human Tau were employed as an in vivo model of tauopathy. Phenotypes of PS19 mice with a targeted deletion of Pyk2 expression were compared with PS19 mice with intact Pyk2 expression. Phenotypes examined included Tau phosphorylation, Tau accumulation, synapse loss, gliosis, proteomic profiling and behavior. RESULTS: Over-expression experiments from Hek293T cells indicated that Pyk2 contributed to Tau phosphorylation, while iPSC-derived human neuronal cultures with endogenous protein levels supported the opposite conclusion. In vivo, multiple phenotypes of PS19 were exacerbated by Pyk2 deletion. In Pyk2-null PS19 mice, Tau phosphorylation and accumulation increased, mouse survival decreased, spatial memory was impaired and hippocampal C1q deposition increased relative to PS19 littermate controls. Proteomic profiles of Pyk2-null mouse brain revealed that several protein kinases known to interact with Tau are regulated by Pyk2. Endogenous Pyk2 suppresses LKB1 and p38 MAPK activity, validating one potential pathway contributing to increased Tau pathology. CONCLUSIONS: The absence of Pyk2 results in greater mutant Tau-dependent phenotypes in PS19 mice, in part via increased LKB1 and MAPK activity. These data suggest that in AD, while Pyk2 activity mediates Aß-driven deficits, Pyk2 suppresses Tau-related phenotypes.

Development of a paddy-based biorefinery approach toward improvement of biomass utilization for more bioproducts.

Improvement of biomass utilization productivity following cascading strategy is a priority for the biorefinery-based circular bioeconomy. In recent years, the field of energy research has seen an increasing interest in bio-products from paddy-based biorefinery, but the utilization of the entire value of paddy biomass to guide the commercial viability of its products has not been got feasible outcomes. Here we propose a potential pathway for a conceptual paddy biorefinery framework by addressing wastes for producing more products. The feasibility of the integrated biorefinery was demonstrated by the conversion of wastes into value-added products such as nano-silica and lignin. In particular, this is the first time that silica recovered from bioethanol system was continued to be reused to produce ZSM-5 and Ni/ZSM-5 as catalysts of rice straw lignin depolymerization achieving high conversion of lignin up to 95% and fair yield of phenolic products up to 41%. Material flow of an integrated biorefinery model was reported to give a future outlook for making most of the processing routes of rice residues. We also established a life cycle that follows the circular bioeconomy concept and discussed the relationship between each of potential bioproducts and their market opportunities.

Organoid models for mammary gland dynamics and breast cancer.

The mammary gland is a highly dynamic tissue that undergoes repeated cycles of growth and involution during pregnancy and menstruation. It is also the site from which breast cancers emerge. Organoids provide an in vitro model that preserves several of the cellular, structural, and microenvironmental features that dictate mammary gland function in vivo and have greatly advanced our understanding of glandular biology. Their tractability for genetic manipulation, live imaging, and high throughput screening have facilitated investigation into the mechanisms of glandular morphogenesis, structural maintenance, tumor progression, and invasion. Opportunities remain to enhance cellular and structural complexity of mammary organoid models, including incorporating additional cell types and hormone signaling.

Evolution of Interdisciplinary Landscapes of HIV/AIDS Studies from 1983 to 2017: Results from the Global Analysis for Policy in Research (GAPRESEARCH).

In recent years, there have been numerous calls by researchers to adopt multi-disciplinary and international perspectives to address the HIV pandemic. Meaningful and prudent public health policy should be based on sound empirical data and research. Henceforth, our study aims to contribute to the current literature by conducting a comprehensive global mapping and determine the landscapes of HIV/AIDS research covering the years between 1983 and 2017. Bibliometric and content analysis was used to describe trends in research productivity, usages, research collaborations, and clusters of research topics. Exploratory factor analysis, Jaccard's similarity index, and Ward dendrogram were applied to abstracts' contents to determine the development of interdisciplinary research landscapes. The United States of America continues to lead in research production and be main hub for author- and country-level collaborations. Research employing an epidemiological, social, and/or behavioral perspective for studying HIV/AIDS was found to dwarf in the presence of basic and biomedical HIV research. Interdisciplinary approaches to HIV research have been increasing with the creation of various research landscapes: strong constructs of studies examining health status, clinical responses, and HIV treatment, risk behaviors have been formed, while research topics relating to psycho-behavioral and cultural aspects as well as services have emerged along. To effectively prevent and control the disease, more researches are needed to provide culturally relevant and/or contextualized evidence of effective interventions. It is also necessary to enhance the ability and partnership of local researchers as well as invest in research infrastructure at national and regional levels to implement high-quality studies since they are the "gate-keepers" who could respond to local changes in a timely manner. These types of research could be a helpful guide for international donors, governments, and academicians to set up research priorities in target groups and settings, and to develop future research agendas globally.

Reconstructing the Human Renal Vascular-Tubular Unit In Vitro.

Engineered human kidney-on-a-chip platforms show tremendous promise for disease modeling and drug screening. Outstanding challenges exist, however, in reconstructing the complex architecture, cellular make-up, and matrix composition necessary for the proper modeling of kidney function. Herein, the first fully tunable human kidney-on-a-chip platform is reported that allows the reconstruction of the native architecture of the renal endothelial-epithelial exchange interface using entirely cell-remodelable matrix and patient-derived kidney cells. This platform consists of a double-layer human renal vascular-tubular unit (hRVTU) enabled by a thin collagen membrane that replicates the kidney exchange interface. It is shown that endothelial and epithelial cells lining their respective lumens remodel the membrane in culture into a ≈1 µm thick exchange interface composed of native basement membrane proteins. This interface displays sufficient mechanical integrity for media flow and blood perfusion. As a proof of principle, it is demonstrated that the hRVTU performs kidney-specific functions including reabsorption of albumin and glucose from the epithelial channel. By incorporating multiple cell populations from single donors, it is demonstrated that the hRVTU may have utility for future precision medicine applications. The success of the system provides new opportunities for the next generation of organ-on-a-chip models.

Microvasculature-directed thrombopoiesis in a 3D in vitro marrow microenvironment.

Vasculature is an interface between the circulation and the hematopoietic tissue providing the means for hundreds of billions of blood cells to enter the circulation every day in a regulated fashion. The precise mechanisms that control the interactions of hematopoietic cells with the vessel wall are largely undefined. Here, we report on the development of an in vitro 3D human marrow vascular microenvironment (VME) to study hematopoietic trafficking and the release of blood cells, specifically platelets. We show that mature megakaryocytes from aspirated marrow as well as megakaryocytes differentiated in culture from CD34+ cells can be embedded in a collagen matrix containing engineered microvessels to create a thrombopoietic VME. These megakaryocytes continue to mature, penetrate the vessel wall, and release platelets into the vessel lumen. This process can be blocked with the addition of antibodies specific for CXCR4, indicating that CXCR4 is required for megakaryocyte migration, though whether it is sufficient is unclear. The 3D marrow VME system shows considerable potential for mechanistic studies defining the role of marrow vasculature in thrombopoiesis. Through a stepwise addition or removal of individual marrow components, this model provides potential to define key pathways responsible for the release of platelets and other blood cells.

Engineering a multicellular vascular niche to model hematopoietic cell trafficking.

BACKGROUND: The marrow microenvironment and vasculature plays a critical role in regulating hematopoietic cell recruitment, residence, and maturation. Extensive in vitro and in vivo studies have aimed to understand the marrow cell types that contribute to hematopoiesis and the stem cell environment. Nonetheless, in vitro models are limited by a lack of complex multicellular interactions, and cellular interactions are not easily manipulated in vivo. Here, we develop an engineered human vascular marrow niche to examine the three-dimensional cell interactions that direct hematopoietic cell trafficking. METHODS: Using soft lithography and injection molding techniques, fully endothelialized vascular networks were fabricated in type I collagen matrix, and co-cultured under flow with embedded marrow fibroblast cells in the matrix. Marrow fibroblast (mesenchymal stem cells (MSCs), HS27a, or HS5) interactions with the endothelium were imaged via confocal microscopy and altered endothelial gene expression was analyzed with RT-PCR. Monocytes, hematopoietic progenitor cells, and leukemic cells were perfused through the network and their adhesion and migration was evaluated. RESULTS: HS27a cells and MSCs interact directly with the vessel wall more than HS5 cells, which are not seen to make contact with the endothelial cells. In both HS27a and HS5 co-cultures, endothelial expression of junctional markers was reduced. HS27a co-cultures promote perfused monocytes to adhere and migrate within the vessel network. Hematopoietic progenitors rely on monocyte-fibroblast crosstalk to facilitate preferential recruitment within HS27a co-cultured vessels. In contrast, leukemic cells sense fibroblast differences and are recruited preferentially to HS5 and HS27a co-cultures, but monocytes are able to block this sensitivity. CONCLUSIONS: We demonstrate the use of a microvascular platform that incorporates a tunable, multicellular composition to examine differences in hematopoietic cell trafficking. Differential recruitment of hematopoietic cell types to distinct fibroblast microenvironments highlights the complexity of cell-cell interactions within the marrow. This system allows for step-wise incorporation of cellular components to reveal the dynamic spatial and temporal interactions between endothelial cells, marrow-derived fibroblasts, and hematopoietic cells that comprise the marrow vascular niche. Furthermore, this platform has potential for use in testing therapeutics and personalized medicine in both normal and disease contexts.

Micropatterning and Assembly of 3D Microvessels.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 µm. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

A prognostic nomogram to predict overall survival in women with recurrent ovarian cancer treated with bevacizumab and chemotherapy.

OBJECTIVE: To develop a nomogram to predict overall survival (OS) in women with recurrent ovarian cancer treated with bevacizumab and chemotherapy. METHODS: A multicenter retrospective study was conducted. Potential prognostic variables included age; stage; grade; histology; performance status; residual disease; presence of ascites and/or pleural effusions; number of chemotherapy regimens, treatment-free interval (TFI) prior to bevacizumab administration, and platinum sensitivity. Multivariate analysis was performed using Cox proportional hazards regression. The predictive model was developed into a nomogram to predict five-year OS. RESULTS: 312 women with recurrent ovarian cancer treated with bevacizumab and chemotherapy were identified; median age was 59 (range: 19-85); 86% women had advanced stage (III-IV) disease. The majority had serous histology (74%), high grade cancers (93.5%), and optimal cytoreductions (69.5%). Fifty-one percent of women received greater than two prior chemotherapeutic regimens. TFI (AHR=0.98, 95% CI 0.97-1.00, p=0.022) was the only statistically significant predictor in a multivariate progression-free survival (PFS) analysis. In a multivariate OS analysis, prior number of chemotherapy regimens, TFI, platinum sensitivity, and presence of ascites were significant. A nomogram to predict five-year OS was constructed and internally validated (bootstrap-corrected concordance index=0.737). CONCLUSION: Our multivariate model identified prior number of chemotherapy regimens, TFI, platinum sensitivity, and the presence of ascites as prognostic variables for OS in women with recurrent ovarian cancer treated with bevacizumab combined with chemotherapy. Our nomogram to predict five-year OS may be used to identify women who may benefit from bevacizumab and chemotherapy, but further validation is needed.

Thermal inactivation reaction rates for ricin are influenced by pH and carbohydrates.

Ricin is a lethal protein toxin produced by the castor bean plant. Ricin is known to possess significant heat resistance. Therefore, we placed it in a variety of foods to study the influence of the food matrix on behavior of a thermally stable protein toxin. First order rate constants for the thermal inactivation of ricin in foods and simple buffers were measured using cytotoxicity assays. We observed greater thermal stability at 75 °C for the cytotoxic activity of ricin when it was placed in a yogurt-containing fruit drink compared to its stability when placed in the other foods tested. We found that galactose and high molecular weight exopolysaccharides present in various dairy products contributed to the thermal stability of ricin. Differential scanning calorimetry also showed enhanced thermal stability for ricin at pH 4.5. Our results demonstrate the importance of considering pH and the presence of stabilizing ligands in the thermal inactivation of protein toxins in foods.

Chemical inactivation of protein toxins on food contact surfaces.

We compared the kinetics and efficacies of sodium hypochlorite, peracetic acid, phosphoric acid-based detergent, chlorinated alkaline detergent, quaternary ammonium-based sanitizer, and peracetic acid-based sanitizer for inactivating the potential bioterrorism agents ricin and abrin in simple buffers, food slurries (infant formula, peanut butter, and pancake mix), and in dried food residues on stainless steel. The intrinsic fluorescence and cytotoxicity of purified ricin and abrin in buffers decreased rapidly in a pH- and temperature-dependent manner when treated with sodium hypochlorite but more slowly when treated with peracetic acid. Cytotoxicity assays showed rapid and complete inactivation of ricin and crude abrin in food slurries and dried food residues treated 0-5 min with sodium hypochlorite. Toxin epitopes recognized by ELISA decayed more gradually under these conditions. Higher concentrations of peracetic acid were required to achieve comparable results. Chlorinated alkaline detergent was the most effective industrial agent tested for inactivating ricin in dried food residues.

Prognostic discrimination of subgrouping node-positive endometrioid uterine cancer: location vs nodal extent.

BACKGROUND: The 2009 International Federation of Gynecologists and Obstetricians elected to substage patients with positive retroperitoneal lymph nodes as IIIC 1 (pelvic lymph node metastasis only) and IIIC 2 (paraaortic node metastasis with or with positive pelvic lymph nodes). We have investigated the discriminatory ability of subgrouping patients with retroperitoneal nodal involvement based on location, number, and ratio of positive nodes. METHODS: For 1075 patients with stage IIIC endometrioid corpus cancer abstracted from the Surveillance, Epidemiology, and End Results databases for 2003-2007, Kaplan-Meier analyses, Cox proportional hazard models, and other quantitative measures were used to compare the prognostic discrimination for disease-specific survival (DSS) of nodal subgroupings. RESULTS: In univariate analysis, the 3-year DSS were significantly different for subgroupings by location (IIIC 1 vs IIIC 2; 80.5% vs 67.0%, respectively, P=0.001), lymph node ratio (≤ 23.2% vs >23.2%; 80.8% vs 67.6%; P<0.001), and number of positive lymph nodes (1, 2-5, >5; 79.5, 75.4, 62.9%, P=0.016). The ratio of positive nodes showed superior discriminatory substaging in Cox models. CONCLUSION: Subgrouping of stage IIIC patients by the ratio of positive nodes, either as a dichotomized or continuous parameter, shows the strongest ability to discriminate the survival, controlling for other confounding factors.

Plasmid-mediated quinolone resistance in pseudomonas putida isolates from imported shrimp.

Fourteen quinolone-resistant Pseudomonas putida isolates were recovered from imported frozen shrimp sold in the United States. Two isolates harbored plasmids with qnrA and qnrB genes. PCR and DNA sequencing of quinolone resistance-determining regions identified novel substitutions in GyrA (His139→Glu and Thr128→Ala) and GyrB (Thr442→Asn, Gly470→Ala, and Ile487→Pro) and previously reported substitutions in GyrB (Asp489→Glu) and ParC (Thr105→Pro).

Freespace estimation in an autonomous wheelchair using a stereoscopic camera system.

This paper is concerned with the estimation of the height and width of freespace based on a Bayesian Recursive (BR) algorithm for an autonomous wheelchair using a stereoscopic camera system for disabled people. A 2D distance map for the purpose of freespace estimation is converted from a 3D point map using geometric projection and computation. The comparison of this 2D map to a 2D map obtained from Laser is carried out. Moreover, freespaces in the 2D map are estimated using a BR algorithm based on uncertainty information and control data. Given the average probability, a possible movement decision is then made for the mobile wheelchair. Experimental results obtained in an indoor environment prove the effectiveness of this estimation algorithm.

Multiple kernel learning for classification of diffuse lung disease using HRCT lung images.

A novel algorithm is presented for classification of four patterns of diffuse lung disease: normal, emphysema, honeycombing and ground glass opacity, on the basis of textural analysis of high resolution computed tomography (HRCT) lung images. The algorithm incorporates scale-space features based on Gaussian derivative filters and multi-dimensional multi-scale features based on wavelet and contourlet transforms of the original images. The mean, standard deviation, skewness and kurtosis along with generalized Gaussian density are used to model the output of filters and transforms, and construct feature vectors. Multi-class multiple kernel learning (m-MKL) classifier is used to evaluate the performance of the feature extraction scheme. The method is tested on a collection of 89 slices from 38 patients, each slice of size 512×512, 16 bits/pixel in DICOM format. The dataset contains 70,000 ROIs from slices already marked by experienced radiologists. The average sensitivity and specificity achieved is 94.16% and 98.68%, respectively.

Mycobacterial biofilms facilitate horizontal DNA transfer between strains of Mycobacterium smegmatis.

Conjugal transfer of chromosomal DNA between strains of Mycobacterium smegmatis occurs by a novel mechanism. In a transposon mutagenesis screen, three transfer-defective insertions were mapped to the lsr2 gene of the donor strain mc(2)155. Because lsr2 encodes a nonspecific DNA-binding protein, mutations of lsr2 give rise to a variety of phenotypes, including an inability to form biofilms. In this study, we show that efficient DNA transfer between strains of M. smegmatis occurs in a mixed biofilm and that the process requires expression of lsr2 in the donor but not in the recipient strain. Testing cells from different strata of standing cultures showed that transfer occurred predominantly at the biofilm air-liquid interface, as other strata containing higher cell densities produced very few transconjugants. These data suggest that the biofilm plays a role beyond mere facilitation of cell-cell contact. Surprisingly, we found that under standard assay conditions the recipient strain does not form a biofilm. Taking these results together, we conclude that for transfer to occur, the recipient strain is actively recruited into the biofilm. In support of this idea, we show that donor and recipient cells are present in almost equal numbers in biofilms that produce transconjugants. Our demonstration of genetic exchange between mycobacteria in a mixed biofilm suggests that conjugation occurs in the environment. Since biofilms are considered to be the predominant natural microhabitat for bacteria, our finding emphasizes the importance of studying biological and physical processes that occur between cells in mixed biofilms.