Structural Remodeling of the Human Colonic Mesenchyme in Inflammatory Bowel Disease.

Intestinal mesenchymal cells play essential roles in epithelial homeostasis, matrix remodeling, immunity, and inflammation. But the extent of heterogeneity within the colonic mesenchyme in these processes remains unknown. Using unbiased single-cell profiling of over 16,500 colonic mesenchymal cells, we reveal four subsets of fibroblasts expressing divergent transcriptional regulators and functional pathways, in addition to pericytes and myofibroblasts. We identified a niche population located in proximity to epithelial crypts expressing SOX6, F3 (CD142), and WNT genes essential for colonic epithelial stem cell function. In colitis, we observed dysregulation of this niche and emergence of an activated mesenchymal population. This subset expressed TNF superfamily member 14 (TNFSF14), fibroblastic reticular cell-associated genes, IL-33, and Lysyl oxidases. Further, it induced factors that impaired epithelial proliferation and maturation and contributed to oxidative stress and disease severity in vivo. Our work defines how the colonic mesenchyme remodels to fuel inflammation and barrier dysfunction in IBD.

Climate change impacts on respiratory health: exposure, vulnerability, and risk.

Anthropogenic climate change adversely impacts human health. In this perspective, we examine the impact of climate change on respiratory health risk. We describe five respiratory health threats-heat, wildfires, pollen, extreme weather events, and viruses-and discuss their impact on health outcomes in a warming climate. The risk of experiencing an adverse health outcome occurs at the intersection of exposure and vulnerability, consisting of sensitivity and adaptive capacity. Exposed individuals and communities most at risk are those with high sensitivity and low adaptive capacity, as influenced by the social determinants of health. We call for the implementation of a transdisciplinary strategy for accelerating respiratory health research, practice, and policy in the context of climate change.

Becalming Type 17 Inflammation in Ulcerative Colitis.

Genome-wide association studies in ulcerative colitis point to a role for FcγRIIA, a receptor for IgG. Castro-Dopico et al. (2019) find a profound induction of anti-commensal IgG in the colonic mucosa of UC patients and outline a pathway whereby FcγR activation by IgG triggers IL-1ß production, type 17 immunity, and the exacerbation of inflammation.

Colonic epithelial cell diversity in health and inflammatory bowel disease.

The colonic epithelium facilitates host-microorganism interactions to control mucosal immunity, coordinate nutrient recycling and form a mucus barrier. Breakdown of the epithelial barrier underpins inflammatory bowel disease (IBD). However, the specific contributions of each epithelial-cell subtype to this process are unknown. Here we profile single colonic epithelial cells from patients with IBD and unaffected controls. We identify previously unknown cellular subtypes, including gradients of progenitor cells, colonocytes and goblet cells within intestinal crypts. At the top of the crypts, we find a previously unknown absorptive cell, expressing the proton channel OTOP2 and the satiety peptide uroguanylin, that senses pH and is dysregulated in inflammation and cancer. In IBD, we observe a positional remodelling of goblet cells that coincides with downregulation of WFDC2-an antiprotease molecule that we find to be expressed by goblet cells and that inhibits bacterial growth. In vivo, WFDC2 preserves the integrity of tight junctions between epithelial cells and prevents invasion by commensal bacteria and mucosal inflammation. We delineate markers and transcriptional states, identify a colonic epithelial cell and uncover fundamental determinants of barrier breakdown in IBD.

Clinicopathologic Features of Gastrointestinal Tract Langerhans Cell Histiocytosis.

Gastrointestinal (GI) tract involvement by Langerhans cell histiocytosis (LCH) is rare and its clinicopathologic characteristics have only been described in case reports and small series. We reviewed hematoxylin and eosin and CD1a, S100, and Langerin immunohistochemical-stained slides from 47 patients with well-documented demographic and clinical findings. Our cases included 8 children and 39 adults, with a mean follow-up of 63 months. All pediatric patients had concurrent multisystem LCH, presented with GI symptoms, and showed nonpolypoid lesions. Seven (88%) showed multifocal GI disease, including 5 with multiple GI organ involvement. All sampled lesions from children exhibited infiltrative growth. More than half had died of the disease or manifested persistent LCH at last follow-up. Twenty-five of 39 (64%) adults had LCH involving only the GI tract (single system), with the remaining 14 (36%) exhibiting multisystem disease. Adult single-system GI LCH was typically encountered incidentally on screening/surveillance endoscopy (72%). Most exhibited isolated colorectal involvement (88%) as a solitary polyp (92%), with a well-demarcated/noninfiltrative growth pattern (70%), and excellent prognosis (100%). In comparison, adult patients with multisystem LCH more frequently presented with GI symptoms (92%, P < .001), noncolorectal GI site involvement (50%, P = .02), multifocal GI lesions (43%, P = .005), nonpolypoid lesions (71%, P < .001), infiltrative histologic growth pattern (78%, P = .04), and persistent disease (57%, P < .001). Adult patients with multisystem LCH appear to exhibit similar clinicopathologic features to those of pediatric patients. These results demonstrated that adults with single-system LCH involving the GI tract have an excellent prognosis, whereas multisystem LCH occurring at any age carries an unfavorable prognosis. High-risk features of GI LCH include pediatric age, GI symptomatology, noncolorectal GI involvement, multifocal GI disease, nonpolypoid lesions, and infiltrative growth pattern.

Outcomes of surgical resection and vagus nerve stimulation in patients with medically refractory epilepsy and glutamic acid decarboxylase 65 antibody positivity.

Epilepsy associated with high-titer glutamic acid decarboxylase 65 (GAD65) IgG is often refractory to immunotherapies and antiseizure medication. This study sought to determine the efficacy of vagus nerve stimulation (VNS) and surgical resection in patients with drug-resistant epilepsy associated with GAD65-IgG. We retrospectively identified 15 patients with drug-resistant epilepsy and high serum GAD65 antibody titers (>20 nmol·L-1) who underwent VNS implantation (n = 6), surgical resection (n = 7), or both (n = 2). A responder to VNS was defined as someone with a ≥50% reduction in seizure frequency, and a favorable surgical outcome was defined as Engel I-II. Of the eight patients who underwent VNS implantation, three (37.5%) were initially responders, but this was not sustained in two. Of the nine patients who underwent surgical resection, three (33.3%) had a favorable outcome; however, only one patient was seizure-free at last follow-up. Pathology was available in six patients, and only one had evidence of inflammation; this patient had seizure onset 1 year prior to surgery. Favorable seizure outcome correlated with older age at time of resective surgery, with a trend favoring later age of seizure onset. Taken together, surgical resection and VNS implantation may have limited efficacy in this patient population but can be considered in carefully selected cases.

Factor structure of psychosis screening questionnaire in Ugandan adults.

BACKGROUND: Psychotic disorders are common and contribute significantly to morbidity and mortality of people with psychiatric diseases. Therefore, early screening and detection may facilitate early intervention and reduce adverse outcomes. Screening tools that lay persons can administer are particularly beneficial in low resource settings. However, there is limited research evaluating the validity of psychosis screening instruments in Uganda. We aimed to assess the construct validity and psychometric properties of the Psychosis Screening Questionnaire (PSQ) in Uganda in a population with no history of a psychotic disorder. METHODS: The sample consisted of 2101 Ugandan adults participating as controls in a larger multi-country case-control study on psychiatric genetics who were recruited between February 2018 and March 2020. Participants were individuals seeking outpatient general medical care, caretakers of individuals seeking care, and staff or students recruited from five medical facilities that were age 18 years or older and able to provide consent. Individuals were excluded who had acute levels of alcohol or substance use, including being under inpatient hospitalization or acute medical care for one of these conditions. We used confirmatory factor analysis (CFA) and item response theory (IRT) to evaluate the factor structure and item properties of the PSQ. RESULTS: The overall prevalence screening positive for psychotic symptoms was 13.9% 95% CI (12.4,15.4). "Strange experiences" were the most endorsed symptoms 6.6% 95% CI (5.6,7.8). A unidimensional model seemed to be a good model or well-fitting based on fit indices including the root mean square error of approximation (RMSEA of 0.00), comparative fit index (CFI of 1.000), and Tucker-Lewis Index (TLI of 1.000). The most discriminating items along the latent construct of psychosis were items assessing thought disturbance followed by items assessing paranoia, with a parameter (discrimination) value of 2.53 and 2.40, respectively. CONCLUSION: The PSQ works well in Uganda as an initial screening tool for moderate to high-level of psychotic symptoms.

Diagnosis and treatment of patients with suspected mucinous cystic neoplasms of the liver: a retrospective cohort study.

PURPOSE: Mucinous cystic neoplasms of the liver (MCN-L) are hepatic cysts with a low malignant potential. The recent European Association for the Study of the Liver (EASL) guidelines provide guidance on the imaging features and surgical management of MCN-L, yet are hampered by a lack of studies adhering to the revised World Health Organization (WHO) criteria. This study attempted to validate the new 2022 EASL-guidelines in a retrospective cohort study of patients who underwent surgery for suspected MCN-L. METHODS: Patients undergoing surgery for suspected MCN-L in a single center between 2010 and 2020 were included. Imaging features were assessed according to the EASL guidelines and were compared to final pathological diagnoses, according to the WHO criteria. RESULTS: In total, 35 patients were included. In three patients, there were no worrisome imaging features, yet final pathological diagnosis showed MCN-L. Contrarily, six patients with worrisome imaging features did not have MCN-L. Five patients were diagnosed with MCN-L on final pathology. The sensitivity of the EASL-guidelines for the diagnosis of MCN-L was 40% (95%CI: 5.3-85%) and the specificity was 80% (95% CI: 61-92%). CONCLUSION: Although the new EASL-guidelines provide some guidance, they could not reliably distinguish MCN-L from other cysts in our series. Thus, preoperative diagnosis of MCN-L remains challenging and we should be careful in selecting surgical strategies based on these criteria.

Housing instability patterns among low-income, urban Black young adults in California and associations with mental health outcomes: baseline data from a randomized waitlist-controlled trial.

BACKGROUND: Deep-rooted racial residential segregation and housing discrimination have given rise to housing disparities among low-income Black young adults in the US. Most studies have focused on single dimensions of housing instability, and thus provide a partial view of how Black young adults experience multiple, and perhaps overlapping, experiences of housing instability including homelessness, frequent moves, unaffordability, or evictions. We aimed to illuminate the multiple forms of housing instability that Black young adults contend with and examine relationships between housing instability and mental health outcomes. METHODS: Using baseline data from the Black Economic Equity Movement (BEEM) guaranteed income trial with 300 urban low-income Black young adults (aged 18-24), we conducted a three-stage latent class analysis using nine housing instability indicators. We identified distinct patterns by using fit indices and theory to determine the optimal number of latent classes. We then used multinomial logistic regression to identify subpopulations disproportionately represented within unstable housing patterns. Finally, we estimated associations between housing experience patterns and mental health outcomes: depression, anxiety, and hope. RESULTS: We found high prevalence of housing instability with 27.3% of participants reporting experiences of homelessness in the prior year and 39.0% of participants reporting multiple measures of housing instability. We found the 4-class solution to be the best fitting model for the data based on fit indices and theory. Latent classes were characterized as four housing experience patterns: 1) more stably housed, 2) unaffordable and overcrowded housing, 3) mainly unhoused, and 4) multiple dimensions of housing instability. Those experiencing unaffordable and overcrowded housing and being mainly unhoused were more than four times as likely to have symptoms of depression (Unaffordable: aOR = 4.57, 95% CI: 1.64, 12.72; Unhoused: aOR = 4.67, 95% CI:1.18, 18.48) and more than twice as likely to report anxiety (Unaffordable: aOR = 2.28, 95% CI: 1.03, 5.04; Unhoused: aOR = 3.36, 95% CI: 1.12, 10.05) compared to the more stably housed pattern. We found that hope scores were similarly high across patterns. CONCLUSIONS: High prevalence of housing instability and mental health challenges among low-income Black young adults demands tailored interventions to reduce instability, given widening racial disparities and implications for future well-being into adulthood.

Adapting and testing measures of organizational context in primary care clinics in KwaZulu-Natal, South Africa.

BACKGROUND: Implementation science frameworks situate intervention implementation and sustainment within the context of the implementing organization and system. Aspects of organizational context such as leadership have been defined and measured largely within US health care settings characterized by decentralization and individual autonomy. The relevance of these constructs in other settings may be limited by differences like collectivist orientation, resource constraints, and hierarchical power structures. We aimed to adapt measures of organizational context in South African primary care clinics. METHODS: We convened a panel of South African experts in social science and HIV care delivery and presented implementation domains informed by existing frameworks and prior work in South Africa. Based on panel input, we selected contextual domains and adapted candidate items. We conducted cognitive interviews with 25 providers in KwaZulu-Natal Province to refine measures. We then conducted a cross-sectional survey of 16 clinics with 5-20 providers per clinic (N = 186). We assessed reliability using Cronbach's alpha and calculated interrater agreement (awg) and intraclass correlation coefficient (ICC) at the clinic level. Within clinics with moderate agreement, we calculated correlation of clinic-level measures with each other and with hypothesized predictors - staff continuity and infrastructure - and a clinical outcome, patient retention on antiretroviral therapy. RESULTS: Panelists emphasized contextual factors; we therefore focused on elements of clinic leadership, stress, cohesion, and collective problem solving (critical consciousness). Cognitive interviews confirmed salience of the domains and improved item clarity. After excluding items related to leaders' coordination abilities due to missingness and low agreement, all other scales demonstrated individual-level reliability and at least moderate interrater agreement in most facilities. ICC was low for most leadership measures and moderate for others. Measures tended to correlate within facility, and higher stress was significantly correlated with lower staff continuity. Organizational context was generally more positively rated in facilities that showed consistent agreement. CONCLUSIONS: As theorized, organizational context is important in understanding program implementation within the South African health system. Most adapted measures show good reliability at individual and clinic levels. Additional revision of existing frameworks to suit this context and further testing in high and low performing clinics is warranted.

Machine Learning Effectively Diagnoses Mandibular Deformity Using Three-Dimensional Landmarks.

BACKGROUND: Jaw deformity diagnosis requires objective tests. Current methods, like cephalometry, have limitations. However, recent studies have shown that machine learning can diagnose jaw deformities in two dimensions. Therefore, we hypothesized that a multilayer perceptron (MLP) could accurately diagnose jaw deformities in three dimensions (3D). PURPOSE: Examine the hypothesis by focusing on anomalous mandibular position. We aimed to: (1) create a machine learning model to diagnose mandibular retrognathism and prognathism; and (2) compare its performance with traditional cephalometric methods. STUDY DESIGN, SETTING, SAMPLE: An in-silico experiment on deidentified retrospective data. The study was conducted at the Houston Methodist Research Institute and Rensselaer Polytechnic Institute. Included were patient records with jaw deformities and preoperative 3D facial models. Patients with significant jaw asymmetry were excluded. PREDICTOR VARIABLES: The tests used to diagnose mandibular anteroposterior position are: (1) SNB angle; (2) facial angle; (3) mandibular unit length (MdUL); and (4) MLP model. MAIN OUTCOME VARIABLE: The resultant diagnoses: normal, prognathic, or retrognathic. COVARIATES: None. ANALYSES: A senior surgeon labeled the patients' mandibles as prognathic, normal, or retrognathic, creating a gold standard. Scientists at Rensselaer Polytechnic Institute developed an MLP model to diagnose mandibular prognathism and retrognathism using the 3D coordinates of 50 landmarks. The performance of the MLP model was compared with three traditional cephalometric measurements: (1) SNB, (2) facial angle, and (3) MdUL. The primary metric used to assess the performance was diagnostic accuracy. McNemar's exact test tested the difference between traditional cephalometric measurement and MLP. Cohen's Kappa measured inter-rater agreement between each method and the gold standard. RESULTS: The sample included 101 patients. The diagnostic accuracy of SNB, facial angle, MdUL, and MLP were 74.3, 74.3, 75.3, and 85.2%, respectively. McNemar's test shows that our MLP performs significantly better than the SNB (P = .027), facial angle (P = .019), and MdUL (P = .031). The agreement between the traditional cephalometric measurements and the surgeon's diagnosis was fair. In contrast, the agreement between the MLP and the surgeon was moderate. CONCLUSION AND RELEVANCE: The performance of the MLP is significantly better than that of the traditional cephalometric measurements.

ChatGPT Provides Unsatisfactory Responses to Frequently Asked Questions Regarding Anterior Cruciate Ligament Reconstruction.

PURPOSE: To determine whether the free online artificial intelligence platform ChatGPT could accurately, adequately, and appropriately answer questions regarding anterior cruciate ligament (ACL) reconstruction surgery. METHODS: A list of 10 questions about ACL surgery was created based on a review of frequently asked questions that appeared on websites of various orthopaedic institutions. Each question was separately entered into ChatGPT (version 3.5), and responses were recorded, scored, and graded independently by 3 authors. The reading level of the ChatGPT response was calculated using the WordCalc software package, and readability was assessed using the Flesch-Kincaid grade level, Simple Measure of Gobbledygook index, Coleman-Liau index, Gunning fog index, and automated readability index. RESULTS: Of the 10 frequently asked questions entered into ChatGPT, 6 were deemed as unsatisfactory and requiring substantial clarification; 1, as adequate and requiring moderate clarification; 1, as adequate and requiring minor clarification; and 2, as satisfactory and requiring minimal clarification. The mean DISCERN score was 41 (inter-rater reliability, 0.721), indicating the responses to the questions were average. According to the readability assessments, a full understanding of the ChatGPT responses required 13.4 years of education, which corresponds to the reading level of a college sophomore. CONCLUSIONS: Most of the ChatGPT-generated responses were outdated and failed to provide an adequate foundation for patients' understanding regarding their injury and treatment options. The reading level required to understand the responses was too advanced for some patients, leading to potential misunderstanding and misinterpretation of information. ChatGPT lacks the ability to differentiate and prioritize information that is presented to patients. CLINICAL RELEVANCE: Recognizing the shortcomings in artificial intelligence platforms may equip surgeons to better set expectations and provide support for patients considering and preparing for ACL reconstruction.

Improving Image Segmentation with Contextual and Structural Similarity.

Deep learning models for medical image segmentation are usually trained with voxel-wise losses, e.g., cross-entropy loss, focusing on unary supervision without considering inter-voxel relationships. This oversight potentially leads to semantically inconsistent predictions. Here, we propose a contextual similarity loss (CSL) and a structural similarity loss (SSL) to explicitly and efficiently incorporate inter-voxel relationships for improved performance. The CSL promotes consistency in predicted object categories for each image sub-region compared to ground truth. The SSL enforces compatibility between the predictions of voxel pairs by computing pair-wise distances between them, ensuring that voxels of the same class are close together whereas those from different classes are separated by a wide margin in the distribution space. The effectiveness of the CSL and SSL is evaluated using a clinical cone-beam computed tomography (CBCT) dataset of patients with various craniomaxillofacial (CMF) deformities and a public pancreas dataset. Experimental results show that the CSL and SSL outperform state-of-the-art regional loss functions in preserving segmentation semantics.

Measuring people's views on health system performance: Design and development of the People's Voice Survey.

Todd Lewis and co-authors discuss development and use of the People's Voice Survey for health system assessment.

Into the unknown: The end of the coronavirus disease 2019 public health emergency in the United States and potential impacts on organ transplant recipients.

The public health emergency for coronavirus disease 2019 ended on April 11, 2023, 1 month earlier than its planned termination. The hasty cessation of emergency measures may negatively impact the care of solid organ transplant recipients and other immunosuppressed hosts. Accelerated pathways for drug and vaccine approvals, research funding, and insurance coverage for medical therapies and diagnostic testing are likely to be affected. Health care disparities that characterized the early pandemic may again be intensified. It is imperative that the transplant community promptly anticipate the impact of these changes and prepare accordingly to avoid disruptions in care for the most vulnerable patients.

ADAM8 is expressed widely in breast cancer and predicts poor outcome in hormone receptor positive, HER-2 negative patients.

BACKGROUND: Breast malignancies are the predominant cancer-related cause of death in women. New methods of diagnosis, prognosis and treatment are necessary. Previously, we identified the breast cancer cell surface protein ADAM8 as a marker of poor survival, and a driver of Triple-Negative Breast Cancer (TNBC) growth and spread. Immunohistochemistry (IHC) with a research-only anti-ADAM8 antibody revealed 34.0% of TNBCs (17/50) expressed ADAM8. To identify those patients who could benefit from future ADAM8-based interventions, new clinical tests are needed. Here, we report on the preclinical development of a highly specific IHC assay for detection of ADAM8-positive breast tumors. METHODS: Formalin-fixed paraffin-embedded sections of ADAM8-positive breast cell lines and patient-derived xenograft tumors were used in IHC to identify a lead antibody, appropriate staining conditions and controls. Patient breast cancer samples (n = 490) were used to validate the assay. Cox proportional hazards models assessed association between survival and ADAM8 expression. RESULTS: ADAM8 staining conditions were optimized, a lead anti-human ADAM8 monoclonal IHC antibody (ADP2) identified, and a breast staining/scoring control cell line microarray (CCM) generated expressing a range of ADAM8 levels. Assay specificity, reproducibility, and appropriateness of the CCM for scoring tumor samples were demonstrated. Consistent with earlier findings, 36.1% (22/61) of patient TNBCs expressed ADAM8. Overall, 33.9% (166/490) of the breast cancer population was ADAM8-positive, including Hormone Receptor (HR) and Human Epidermal Growth Factor Receptor-2 (HER2) positive cancers, which were tested for the first time. For the most prevalent HR-positive/HER2-negative subtype, high ADAM8 expression identified patients at risk of poor survival. CONCLUSIONS: Our studies show ADAM8 is widely expressed in breast cancer and provide support for both a diagnostic and prognostic value of the ADP2 IHC assay. As ADAM8 has been implicated in multiple solid malignancies, continued development of this assay may have broad impact on cancer management.

Psychometric properties of the mini international neuropsychiatric interview (MINI) psychosis module: a Sub-Saharan Africa cross country comparison.

BACKGROUND: The Mini International Neuropsychiatric Inventory 7.0.2 (MINI-7) is a widely used tool and known to have sound psychometric properties; but very little is known about its use in low and middle-income countries (LMICs). This study aimed to examine the psychometric properties of the MINI-7 psychosis items in a sample of 8609 participants across four countries in Sub-Saharan Africa. METHODS: We examined the latent factor structure and the item difficulty of the MINI-7 psychosis items in the full sample and across four countries. RESULTS: Multiple group confirmatory factor analyses (CFAs) revealed an adequate fitting unidimensional model for the full sample; however, single group CFAs at the country level revealed that the underlying latent structure of psychosis was not invariant. Specifically, although the unidimensional structure was an adequate model fit for Ethiopia, Kenya, and South Africa, it was a poor fit for Uganda. Instead, a 2-factor latent structure of the MINI-7 psychosis items provided the optimal fit for Uganda. Examination of item difficulties revealed that MINI-7 item K7, measuring visual hallucinations, had the lowest difficulty across the four countries. In contrast, the items with the highest difficulty were different across the four countries, suggesting that MINI-7 items that are the most predictive of being high on the latent factor of psychosis are different for each country. CONCLUSIONS: The present study is the first to provide evidence that the factor structure and item functioning of the MINI-7 psychosis vary across different settings and populations in Africa.

Estimating the Prevalence of over- and Under-Reporting in HIV Testing, Status and Treatment in Rural Northeast South Africa: A Comparison of a Survey and Clinic Records.

We assess the accuracy of self-reported testing, HIV status, and treatment responses compared to clinical records in Ehlanzeni District, South Africa. We linked a 2018 population-based survey of adults 18-49 years old with clinical data at local primary healthcare facilities from 2014 to 2018. We calculated self-reported testing, HIV status, and treatment, and triangulated findings with clinic record data. We adjusted testing estimates for known gaps in HIV test documentation. Of 2089 survey participants, 1657 used a study facility and were eligible for analysis. Half of men and 84% of women reported an HIV test in the past year. One third of reported tests could be confirmed in clinic data within 1 year and an additional 13% within 2 years; these fractions increased to 57% and 22% respectively limiting to participants with a verified clinic file. After accounting for gaps in clinic documentation, we found that prevalence of recent HIV testing was closer to 15% among men and 51% in women. Estimated prevalence of known HIV was 16.2% based on self-report vs. 27.6% with clinic documentation. Relative to clinical records among confirmed clinic users, self report of HIV testing and of current treatment were highly sensitive but non-specific (sensitivity 95.5% and 98.8%, specificity 24.2% and 16.1% respectively), while self report of HIV status was highly specific but not sensitive (sensitivity 53.0%, specificity 99.3%). While clinical records are imperfect, survey-based measures should be interpreted with caution in this rural South African setting.

Anionic Phospholipids Stabilize RecA Filament Bundles in Escherichia coli.

We characterize the interaction of RecA with membranes in vivo and in vitro and demonstrate that RecA binds tightly to the anionic phospholipids cardiolipin (CL) and phosphatidylglycerol (PG). Using computational models, we identify two regions of RecA that interact with PG and CL: (1) the N-terminal helix and (2) loop L2. Mutating these regions decreased the affinity of RecA to PG and CL in vitro. Using 3D super-resolution microscopy, we demonstrate that depleting Escherichia coli PG and CL altered the localization of RecA foci and hindered the formation of RecA filament bundles. Consequently, E. coli cells lacking aPLs fail to initiate a robust SOS response after DNA damage, indicating that the membrane acts as a scaffold for nucleating the formation of RecA filament bundles and plays an important role in the SOS response.

Anisotropic dynamics of an interfacial enzyme active site observed using tethered substrate analogs and ultrafast 2D IR spectroscopy.

Enzymes accelerate the rates of biomolecular reactions by many orders of magnitude compared to bulk solution, and it is widely understood that this catalytic effect arises from a combination of polar pre-organization and electrostatic transition state stabilization. A number of recent reports have also implicated ultrafast (femtosecond-picosecond) timescale motions in enzymatic activity. However, complications arising from spatially-distributed disorder, the occurrence of multiple substrate binding modes, and the influence of hydration dynamics on solvent-exposed active sites still confound many experimental studies. Here we use ultrafast two-dimensional infrared (2D IR) spectroscopy and covalently-tethered substrate analogs to examine dynamical properties of the promiscuous Pyrococcus horikoshii ene-reductase (PhENR) active site in two binding configurations mimicking proposed "inactive" and "reactive" Michaelis complexes. Spectral diffusion measurements of aryl-nitrile substrate analogs reveal an end-to-end tradeoff between fast (sub-ps) and slow (>5 ps) motions. Fermi resonant aryl-azide analogs that sense interactions of coupled oscillators are described. Lineshape and quantum beat analyses of these probes reveal characteristics that correlate with aryl-nitrile frequency fluctuation correlation functions parameters, demonstrating that this anisotropy is an intrinsic property of the water-exposed active site, where countervailing gradients of fast dynamics and disorder in the reactant ground state are maintained near the hydration interface. Our results suggest several plausible factors leading to state-selective rate enhancement and promiscuity in PhENR. This study also highlights a strategy to detect perturbations to vibrational modes outside the transparent window of the mid-IR spectrum, which may be extended to other macromolecular systems.