Estimating body composition using CT scans of cross-bred lambs fed at two feeding levels and two stages of maturity to inform predictive growth models1.

Livestock producers would benefit from more precise predictions of the growth response from nutrients consumed. Previously published models are often limited by the realities of data collection and are unable to account for alterations to body composition, due in part to the response of visceral organs to an alternate diet. The computerized tomography (CT) scanning of lambs enables the analysis of changes in body composition of individual animals over time, potentially supporting better model development and testing. The aim of this experiment was to develop a repeatable method for the analysis of live lamb body composition using CT scans. A secondary aim was to compare the data collected from CT scanning during a feeding trial to two predictive lamb growth models. Cross-bred lambs were fed two feeding levels at two stages of maturity, with CT scans at the beginning and end of each eight-week feeding period. The CT scan derived values for body composition taken at the beginning of feeding periods were used as inputs for two existing lamb growth models. Predictions of body composition were compared with CT scan derived values at the end of feeding periods. The CT scan analysis method used a proportion of images from each lamb to reduce manual image editing. The method was developed by comparing the estimated mass and volume of empty body components using all available CT scans to estimated values using a reduced number of scans from 12 lambs. The CT scan derived lean tissue mass aligned with model predictions at the end of each feeding period, however, CT scan derived fat mass was greater than predictions by both models especially for the high feeding level at the later stage of maturity. These results highlight that the analysis of body composition using CT scans requires further validation, particularly for the viscera, and that models likely require refinement to better predict the efficiency of energy utilisation by different tissues. The use of live animal CT scans can provide more accurate predictions of the growth of saleable products than measuring liveweight alone and will enable ruminant growth models to better adapt to different genetics and changing diets than comparative slaughter. To replicate the current data using comparative slaughter would require four times the animals, as individual lambs were CT scanned four times in this study, demonstrating the potential value of CT scanning in live animal research.

Nemolizumab with concomitant topical therapy in adolescents and adults with moderate-to-severe atopic dermatitis (ARCADIA 1 and ARCADIA 2): results from two replicate, double-blind, randomised controlled phase 3 trials.

BACKGROUND: Nemolizumab, an interleukin (IL)-31 receptor subunit α antagonist, inhibits the IL-31 pathway of itch and skin inflammation in atopic dermatitis. Two international phase 3 studies were done to assess the efficacy and safety of nemolizumab in atopic dermatitis. In this Article we report results for the 16-week initial treatment period of both trials. METHODS: ARCADIA 1 and ARCADIA 2 were identical 48-week randomised, double-blind, placebo-controlled phase 3 trials in adult and adolescent participants (aged ≥12 years) with moderate-to-severe atopic dermatitis, associated pruritus, and inadequate response to topical steroids. Participants were enrolled from 281 clinics, hospitals, and academic centres in 22 countries across both trials, and were randomly assigned (2:1) to receive nemolizumab 30 mg subcutaneously (baseline loading dose 60 mg) or matching placebo once every 4 weeks with background topical corticosteroids (TCS) with or without topical calcineurin inhibitors (TCI; ie, TCS-TCI background treatment). Randomisation was done via interactive response technology and stratified by baseline disease and pruritus severity. Study staff and participants were masked throughout the study, with outcome assessors masked until database lock. Coprimary endpoints at week 16 post-baseline were Investigator's Global Assessment (IGA) success (score of 0 [clear skin] or 1 [almost clear skin] with a ≥2-point improvement from baseline) and at least 75% improvement in Eczema Area and Severity Index score from baseline (EASI-75 response). Outcome rates were compared between groups with the Cochran-Mantel-Haenszel test adjusting for randomisation strata. The key secondary endpoints were the proportion of participants with Peak Pruritus Numerical Rating Scale (PP-NRS) score improvement of at least 4 points at weeks 1, 2, 4, and 16; PP-NRS score below 2 at weeks 4 and 16; Sleep Disturbance Numerical Rating Scale score improvement of at least 4 points at week 16; EASI-75 response plus PP-NRS score improvement of at least 4 points at week 16; and IGA success plus PP-NRS score improvement of at least 4 points at week 16. Efficacy analyses were done on an intention-to-treat basis; safety analyses included all participants who received one dose of nemolizumab or placebo. Both studies are completed (ClinicalTrials.gov: ARCADIA 1, NCT03985943 and ARCADIA 2, NCT03989349). FINDINGS: Between Aug 9, 2019, and Nov 2, 2022, 1728 participants were enrolled across both trials: 1142 were allocated to nemolizumab plus TCS-TCI (620 in ARCADIA 1 and 522 in ARCADIA 2) and 586 to placebo plus TCS-TCI (321 in ARCADIA 1 and 265 in ARCADIA 2). ARCADIA 1 included 500 (53%) male participants and 441 (47%) female participants, and ARCADIA 2 included 381 (48%) male participants and 406 (52%) female participants. Mean age ranged from 33·3 (SD 15·6) years to 35·2 (17·0) years across the treatment groups. Both trials met the coprimary endpoints; at week 16, a greater proportion of participants receiving nemolizumab plus TCS-TCI versus placebo plus TCS-TCI had IGA success (ARCADIA 1: 221 [36%] of 620 vs 79 [25%] of 321, adjusted percentage difference 11·5% [97·5% CI 4·7-18·3], p=0·0003; ARCADIA 2: 197 [38%] of 522 vs 69 [26%] of 265, adjusted difference 12·2% [4·6-19·8], p=0·0006) and an EASI-75 response (ARCADIA 1: 270 [44%] vs 93 [29%], adjusted difference 14·9% [7·8-22·0], p<0·0001; ARCADIA 2: 220 [42%] vs 80 [30%], adjusted difference 12·5% [4·6-20·3], p=0·0006). Significant benefits were observed with nemolizumab for all key secondary endpoints including improvement in itch, as early as week 1, and sleep improvement by week 16. The safety profile was similar between nemolizumab plus TCS-TCI and placebo plus TCS-TCI. In the safety sets, 306 (50%) of 616 participants (ARCADIA 1) and 215 (41%) of 519 participants (ARCADIA 2) who received nemolizumab plus TCS-TCI had at least one treatment-emergent adverse event (serious treatment-emergent adverse events in six [1%] and 13 [3%], respectively); and 146 (45%) of 321 (ARCADIA 1) and 117 (44%) of 263 (ARCADIA 2) who received placebo plus TCS-TCI had at least one treatment-emergent adverse event (serious treatment-emergent adverse events in four [1%] and three [1%], respectively). Ten serious treatment-emergent adverse events possibly related to nemolizumab were reported in five (1%) participants in ARCADIA 2. No deaths occurred. INTERPRETATION: Nemolizumab plus TCS-TCI was efficacious and showed statistically and clinically significant improvements in inflammation and itch in adults and adolescents with moderate-to-severe atopic dermatitis. Nemolizumab might offer a valuable extension of current therapies if approved. FUNDING: Galderma.

Recent strides in macromolecular targeted photodynamic therapy for cancer.

The recent approval of Akalux® for antibody-targeted photodynamic therapy (PDT) in Japan (also known as photoimmunotherapy), and the recent approval of Cytalux® for folate-specific image guided surgery by the FDA have motivated the continued development of macromolecular targeted PDT for cancer management. This review spotlights some of the most recent advances in macromolecular targeted PDT since 2021, exploring the latest advances in protein engineering, adaptive macromolecular constructs and nanotechnology, adoption of immune checkpoint inhibitors, and targeting using biomimetic membranes. These strategies summarized here attempt to expand the functionality, benefit, and success of macromolecular targeting for PDT to advance the technology beyond what has already entered into the clinical realm.

Endoscopic Tissue Expander Implantation in Post-Burn Reconstruction: A Review of the Literature.

BACKGROUND: Tissue expansion generates new tissue that can be used in post-burn reconstruction. Expanders are placed through small incisions, requiring difficult and sometimes blind dissection to prepare an adequate pocket. Recently, the use of endoscopy to assist in expander placement has been described, which may improve intraoperative visualization and allow for a smaller incision. In this review, we summarize the existing literature of endoscopic tissue expander (TE) placement in post-burn reconstruction and highlight areas for future research. METHODS: The Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines were utilized to conduct this review. The following databases were queried for the initial search of relevant articles: PubMed, Embase, Scopus, Cochrane, and Web of Science. The data was assessed qualitatively due to the heterogeneity in reporting between the studies. RESULTS: Our literature query yielded 1,023 studies. Sixteen manuscripts underwent full-text review, and seven met inclusion criteria. All studies demonstrated that the endoscopic approach led to successful tissue expansion. Four articles performed a comparative analysis between the open and endoscopic approach, all of which found a significant reduction in complications like seroma, hematoma formation, and device exposure with endoscopic TE implantation. Two studies demonstrated that the use of endoscopy significantly reduced operative time and length of stay. CONCLUSION: Endoscopy is a safe and effective tool in tissue expansion for post-burn reconstruction. Further prospective research should include evaluating the cost-benefit of this approach and long-term outcomes for patients who have undergone endoscopic-assisted tissue expander placement.

The Silent Threat of Hypokalemia after High Voltage Electrical Injuries: A Case Study and Review of the Literature.

High-voltage electrical injuries, especially from lightning strikes, can cause life-threatening complications due to extreme temperature and voltage exposure. While burns and cardiac complications have been widely described, the documentation of metabolic imbalances, particularly hypokalemia, has not been as prevalent. This report focuses on a patient with profound transient hypokalemia following a lightning strike, alongside a review of three similar cases of transient hypokalemia from the literature. Our patient, a previously healthy young man, was struck by lightning and subsequently suffered transient hypokalemia with lower extremity sensory changes, which resolved after the normalization of serum potassium levels. While the exact underlying mechanisms of transient hypokalemia following high-voltage electrical injuries are unknown, we propose a multifactorial mechanism, which includes massive intracellular shifts of potassium due to elevated epinephrine levels and the prevention of potassium efflux through the electrical disruption of voltage-gated potassium channels. Our report underscores the importance of recognizing hypokalemia in patients with high-voltage electrical injuries and contributes to the understanding of the complex mechanisms involved. Further research is necessary to understand the connection between cellular changes induced by high-voltage exposure and their effects on metabolism, particularly in relation to hypokalemia.

Using Bibliometric Analysis to Map the Impact of a Simulation Program's Published Scholarly Works.

SUMMARY STATEMENT: Bibliometrics quantitatively evaluates the targeted literature sources and can help define research and scholarly publications' impact and demonstrate connections for authors, departments, or universities. This article presents a methodology for simulation programs to evaluate their influence in terms of both impact and scope of their published simulation-based healthcare scholarly output. Using the authors' home university and healthcare system as an example, the article outlines a methodology to map research and scholarly works networks within the systems, identify and map connections outside the system, and quantifiably score the overall impact of the simulation program's scholarly output using a common scoring metric, the h-index. This generates an objective measure of impact, rather than a subjective opinion of an organization's research and scholarly impact. The combination of an institutional h-index with mapping of simulation-based healthcare scholarly output provides a full, objective description of the institution's output and provides a benchmark for other simulation programs for comparison.

A General Mechanism for the General Stress Response in Bacteria.

The General Stress Response promotes survival of bacteria in adverse conditions, but how sensor proteins transduce species-specific signals to initiate the response is not known. The serine/threonine phosphatase RsbU initiates the General Stress Response in B. subtilis upon binding a partner protein (RsbT) that is released from sequestration by environmental stresses. We report that RsbT activates RsbU by inducing otherwise flexible linkers of RsbU to form a short coiled-coil that dimerizes and activates the phosphatase domains. Importantly, we present evidence that related coiled-coil linkers and phosphatase dimers transduce signals from diverse sensor domains to control the General Stress Response and other signaling across bacterial phyla. These results additionally resolve the mystery of how shared sensory domains control serine/threonine phosphatases, diguanylate cyclases and histidine kinases, revealing a common coiled-coil linker transduction mechanism. We propose that this provides bacteria with a modularly exchangeable toolkit for the evolution of diverse signaling pathways.

Bibliometric Indices As Indicators of Research Output: Analyzing Anesthesiologists as a Paradigm for Surgical Disciplines.

Anesthesiology is one of the increasingly competitive surgical specialties with a growing emphasis on scholarly activity. A metric of productivity and citation influence, the Hirsch index (h-index), can help identify mentors capable of guiding postgraduate trainees toward successful academic achievements. This study sought to determine associations between h-indices or m-quotients and manuscript publication in anesthesiology. Using the American Society of Anesthesiologists (ASA) website, accepted abstracts from the ASA Annual Meetings from 2019 to 2021 were screened (n=2146). The first author (FAHi) and senior author (SAHi) h-indices, as well as the first author (FAMq) and senior author (SAMq) m-quotients, were collected for each abstract using the Scopus database. Whether an accepted abstract was subsequently published as a manuscript in a peer-reviewed journal was also noted, along with the number of days between ASA presentation and publication date. Linear and logistic regression models were used for statistical analyses. In total, 348 (34.4%) of the 1012 eligible abstracts were published as manuscripts. Mean FAHi, SAHi, FAMq, and SAMq, were significantly higher for accepted ASA abstracts that were later published in peer-reviewed journals compared to accepted abstracts that were not published (p<0.001). FAHi, SAHi, FAMq, and SAMq had significant positive associations with odds of publication (p=0.002; p<0.001; p=0.006; p<0.001, respectively). There was no statistical significance between FAHi, SAHi, FAMq, or SAMq and the number of days between ASA presentation and publication. Our study uniquely demonstrates the positive, direct association between h-indices and m-quotients with the probability of publication in anesthesiology. We propose that bibliometric indices are adapted to provide a refined perspective of a physician-scientist's capabilities. Postgraduate trainees can use these indices to discern research mentors primed to foster academic excellence.

Targeted Temperature Management for Patients with Acute Ischemic Stroke: A Literature Review.

Despite significant advances in medical imaging, thrombolytic therapy, and mechanical thrombectomy, acute ischemic strokes (AIS) remain a major cause of mortality and morbidity globally. Targeted temperature management (TTM) has emerged as a potential therapeutic intervention, aiming to mitigate neuronal damage and improve outcomes. This literature review examines the efficacy and challenges of TTM in the context of an AIS. A comprehensive literature search was conducted using databases such as PubMed, Cochrane, Web of Science, and Google Scholar. Studies were selected based on relevance and quality. We identified key factors influencing the effectiveness of TTM such as its timing, depth and duration, and method of application. The review also highlighted challenges associated with TTM, including increased pneumonia rates. The target temperature range was typically between 32 and 36 °C, with the duration of cooling from 24 to 72 h. Early initiation of TTM was associated with better outcomes, with optimal results observed when TTM was started within the first 6 h post-stroke. Emerging evidence indicates that TTM shows considerable potential as an adjunctive treatment for AIS when implemented promptly and with precision, thereby potentially mitigating neuronal damage and enhancing overall patient outcomes. However, its application is complex and requires the careful consideration of various factors.

Subdomain dynamics enable chemical chain reactions in non-ribosomal peptide synthetases.

Many peptide-derived natural products are produced by non-ribosomal peptide synthetases (NRPSs) in an assembly-line fashion. Each amino acid is coupled to a designated peptidyl carrier protein (PCP) through two distinct reactions catalysed sequentially by the single active site of the adenylation domain (A-domain). Accumulating evidence suggests that large-amplitude structural changes occur in different NRPS states; yet how these molecular machines orchestrate such biochemical sequences has remained elusive. Here, using single-molecule Förster resonance energy transfer, we show that the A-domain of gramicidin S synthetase I adopts structurally extended and functionally obligatory conformations for alternating between adenylation and thioester-formation structures during enzymatic cycles. Complementary biochemical, computational and small-angle X-ray scattering studies reveal interconversion among these three conformations as intrinsic and hierarchical where intra-A-domain organizations propagate to remodel inter-A-PCP didomain configurations during catalysis. The tight kinetic coupling between structural transitions and enzymatic transformations is quantified, and how the gramicidin S synthetase I A-domain utilizes its inherent conformational dynamics to drive directional biosynthesis with a flexibly linked PCP domain is revealed.

Development and Evaluation of Aeyeconsult: A Novel Ophthalmology Chatbot Leveraging Verified Textbook Knowledge and GPT-4.

OBJECTIVE: There has been much excitement on the use of large language models (LLMs) such as ChatGPT in ophthalmology. However, LLMs are limited in that they are trained on unverified information and do not cite their sources. This paper highlights a new methodology to create a generative AI chatbot to answer eye care related questions which uses only verified ophthalmology textbooks as data and cites its sources. SETTING: Yale School of Medicine Department of Ophthalmology and Visual Science. DESIGN/METHODS: Aeyeconsult, an ophthalmology chatbot, was developed using GPT-4 (the LLM used to power the publicly available chatbot ChatGPT-4), LangChain, and Pinecone. Ophthalmology textbooks were processed into embeddings and stored in Pinecone. User queries were similarly converted, compared to stored embeddings, and GPT-4 generated responses. The interface was adapted from public code. Both Aeyeconsult and ChatGPT-4 were tested on the same 260 questions from OphthoQuestions.com, with the first response from Aeyeconsult and ChatGPT-4 recorded as the answer. RESULTS: Aeyeconsult outperformed ChatGPT-4 on the OKAP dataset, with 83.4% correct answers compared to 69.2% (p = 0.0118). Aeyeconsult also had fewer instances of no answer and multiple answers. Both systems performed best in General Medicine, with Aeyeconsult achieving 96.2% accuracy. Aeyeconsult's weakest performance was in Clinical Optics at 68.1%, but it still outperformed ChatGPT-4 in this category (45.5%). CONCLUSION: LLMs may be useful in answering ophthalmology questions but their trustworthiness and accuracy is limited due to training on unverified internet data and lack of source citation. We used a new methodology, using verified ophthalmology textbooks as source material and providing citations, to mitigate these issues, resulting in a chatbot more accurate than ChatGPT-4 in answering OKAPs style questions.

Data science competition for cross-site individual tree species identification from airborne remote sensing data.

Data on individual tree crowns from remote sensing have the potential to advance forest ecology by providing information about forest composition and structure with a continuous spatial coverage over large spatial extents. Classifying individual trees to their taxonomic species over large regions from remote sensing data is challenging. Methods to classify individual species are often accurate for common species, but perform poorly for less common species and when applied to new sites. We ran a data science competition to help identify effective methods for the task of classification of individual crowns to species identity. The competition included data from three sites to assess each methods' ability to generalize patterns across two sites simultaneously and apply methods to an untrained site. Three different metrics were used to assess and compare model performance. Six teams participated, representing four countries and nine individuals. The highest performing method from a previous competition in 2017 was applied and used as a baseline to understand advancements and changes in successful methods. The best species classification method was based on a two-stage fully connected neural network that significantly outperformed the baseline random forest and gradient boosting ensemble methods. All methods generalized well by showing relatively strong performance on the trained sites (accuracy = 0.46-0.55, macro F1 = 0.09-0.32, cross entropy loss = 2.4-9.2), but generally failed to transfer effectively to the untrained site (accuracy = 0.07-0.32, macro F1 = 0.02-0.18, cross entropy loss = 2.8-16.3). Classification performance was influenced by the number of samples with species labels available for training, with most methods predicting common species at the training sites well (maximum F1 score of 0.86) relative to the uncommon species where none were predicted. Classification errors were most common between species in the same genus and different species that occur in the same habitat. Most methods performed better than the baseline in detecting if a species was not in the training data by predicting an untrained mixed-species class, especially in the untrained site. This work has highlighted that data science competitions can encourage advancement of methods, particularly by bringing in new people from outside the focal discipline, and by providing an open dataset and evaluation criteria from which participants can learn.

Structural and functional studies reveal the molecular basis of substrate promiscuity of a glycosyltransferase originating from a major agricultural pest.

The two-spotted spider mite, Tetranychus urticae, is a major cosmopolitan pest that feeds on more than 1100 plant species. Its genome contains an unprecedentedly large number of genes involved in detoxifying and transporting xenobiotics, including 80 genes that code for UDP glycosyltransferases (UGTs). These enzymes were acquired via horizontal gene transfer from bacteria after loss in the Chelicerata lineage. UGTs are well-known for their role in phase II metabolism; however, their contribution to host adaptation and acaricide resistance in arthropods, such as T. urticae, is not yet resolved. TuUGT202A2 (Tetur22g00270) has been linked to the ability of this pest to adapt to tomato plants. Moreover, it was shown that this enzyme can glycosylate a wide range of flavonoids. To understand this relationship at the molecular level, structural, functional, and computational studies were performed. Structural studies provided specific snapshots of the enzyme in different catalytically relevant stages. The crystal structure of TuUGT202A2 in complex with UDP-glucose was obtained and site-directed mutagenesis paired with molecular dynamic simulations revealed a novel lid-like mechanism involved in the binding of the activated sugar donor. Two additional TuUGT202A2 crystal complexes, UDP-(S)-naringenin and UDP-naringin, demonstrated that this enzyme has a highly plastic and open-ended acceptor-binding site. Overall, this work reveals the molecular basis of substrate promiscuity of TuUGT202A2 and provides novel insights into the structural mechanism of UGTs catalysis.

Propofol-Based Anesthesia Maintenance and/or Volatile Anesthetics during Intracranial Aneurysm Repair: A Comparative Analysis of Neurological Outcomes.

BACKGROUND: Volatile and intravenous anesthetics have substantial effects on physiological functions, notably influencing neurological function and susceptibility to injury. Despite the importance of the anesthetic approach, data on its relative risks or benefits during surgical clipping or endovascular treatments for unruptured intracranial aneurysms (UIAs) remains scant. We investigated whether using volatile anesthetics alone or in combination with propofol infusion yields superior neurological outcomes following UIA obliteration. METHODS: We retrospectively reviewed 1001 patients who underwent open or endovascular treatment for UIA, of whom 596 had short- and long-term neurological outcome data (modified Rankin Scale) recorded. Multivariable ordinal regression analysis was performed to examine the association between the anesthetic approach and outcomes. RESULTS: Of 1001 patients, 765 received volatile anesthetics alone, while 236 received propofol infusion and volatile anesthetics (combined anesthetic group). Short-term neurological outcome data were available for 619 patients and long-term data for 596. No significant correlation was found between the anesthetic approach and neurologic outcomes, irrespective of the type of procedure (open craniotomy or endovascular treatment). The combined anesthetic group had a higher rate of ICU admission (p < 0.001) and longer ICU and hospital length of stay (LOS, p < 0.001). Similarly, a subgroup analysis revealed longer ICU and hospital LOS (p < 0.0001 and p < 0.001, respectively) in patients who underwent endovascular UIA obliteration under a combined anesthetic approach (n = 678). CONCLUSIONS: The addition of propofol to volatile anesthetics during UIA obliteration does not provide short- or long-term benefits to neurologic outcomes. Compared to volatile anesthetics alone, the combination of propofol and volatile anesthetics may be associated with an increased rate of ICU admission, as well as longer ICU and hospital LOS.

INDIVIDUAL ARTICLE: NECOM 3: A Practical Algorithm for the Management of Radiation Therapy-Related Acute Radiation Dermatitis.

BACKGROUND: In the Nordic European countries in 2020, cancer diagnoses accounted for 175,925 patients. About 50% of cancer patients receive radiation therapy (RT), which may lead to radiation dermatitis (RD). Notably, patients with breast, head, neck, and anal cancers may be prone to developing RD. However, few algorithms exist for the prevention and treatment of RD. METHODS: The Nordic European Cutaneous Oncodermatology Management (NECOM) project aims to improve cancer patient outcomes by offering tools to prevent and treat cancer therapy-related cutaneous adverse events (cAEs). The first 2 NECOM papers presented various cAEs and skincare regimens involving hygiene, moisturization, sun protection, and camouflage products for preventing and managing cAEs. The NECOM 3 practical algorithm for preventing and managing acute RD (ARD) is intended to promote healthy skin and reduce RT-related ARD, improving cancer patient outcomes.  Results: The NECOM advisors discussed the results of a systematic literature review and obtained consensus on the evidence and opinion-based practical algorithm for ARD to support all stakeholders in the Nordic European healthcare setting. The algorithm starts with skin-preserving therapy, followed by skin condition assessment and patient-specific interventions based on the grade of RD present.  Conclusion: ARD may lead to symptoms of pruritus and pain, decreased QoL and morbidity, and treatment interruptions. Patient education on the prevention of RD and treatment recommendations given in the NECOM 3 algorithm may help prevent and manage RD and improve the overall care of patients receiving RT. J Drugs Dermatol. 2023;22:11(Suppl 2):s3-s10.

The PALB2 DNA-binding domain is an intrinsically disordered recombinase.

The Partner and Localizer of BRCA2 (PALB2) tumor suppressor is a scaffold protein that links BRCA1 with BRCA2 to initiate homologous recombination (HR). PALB2 interaction with DNA strongly enhances HR efficiency. The PALB2 DNA-binding domain (PALB2-DBD) supports DNA strand exchange, a complex multistep reaction supported by only a few protein families such as RecA-like recombinases or Rad52. The mechanisms of PALB2 DNA binding and strand exchange are unknown. We performed circular dichroism, electron paramagnetic spectroscopy, and small-angle X-ray scattering analyses and determined that PALB2-DBD is intrinsically disordered, even when bound to DNA. The intrinsically disordered nature of this domain was further supported by bioinformatics analysis. Intrinsically disordered proteins (IDPs) are prevalent in the human proteome and have many important biological functions. The complexity of the strand exchange reaction significantly expands the functional repertoire of IDPs. The results of confocal single-molecule FRET indicated that PALB2-DBD binding leads to oligomerization-dependent DNA compaction. We hypothesize that PALB2-DBD uses a chaperone-like mechanism to aid formation and resolution of complex DNA and RNA multichain intermediates during DNA replication and repair. Since PALB2-DBD alone or within the full-length PALB2 is predicted to have strong liquid-liquid phase separation (LLPS) potential, protein-nucleic acids condensates are likely to play a role in complex functionality of PALB2-DBD. Similar DNA-binding intrinsically disordered regions may represent a novel class of functional domains that evolved to function in eukaryotic nucleic acid metabolism complexes.

Autoimmune PaneLs as PrEdictors of Toxicity in Patients TReated with Immune Checkpoint InhibiTors (ALERT).

BACKGROUND: Immune-checkpoint inhibitors (ICI) can lead to immune-related adverse events (irAEs) in a significant proportion of patients. The mechanisms underlying irAEs development are mostly unknown and might involve multiple immune effectors, such as T cells, B cells and autoantibodies (AutoAb). METHODS: We used custom autoantigen (AutoAg) microarrays to profile AutoAb related to irAEs in patients receiving ICI. Plasma was collected before and after ICI from cancer patients participating in two clinical trials (NCT03686202, NCT02644369). A one-time collection was obtained from healthy controls for comparison. Custom arrays with 162 autoAg were used to detect IgG and IgM reactivities. Differences of median fluorescent intensity (MFI) were analyzed with Wilcoxon sign rank test and Kruskal-Wallis test. MFI 500 was used as threshold to define autoAb reactivity. RESULTS: A total of 114 patients and 14 healthy controls were included in this study. irAEs of grade (G) ≥ 2 occurred in 37/114 patients (32%). We observed a greater number of IgG and IgM reactivities in pre-ICI collections from patients versus healthy controls (62 vs 32 p < 0.001). Patients experiencing irAEs G ≥ 2 demonstrated pre-ICI IgG reactivity to a greater number of AutoAg than patients who did not develop irAEs (39 vs 33 p = 0.040). We observed post-treatment increase of IgM reactivities in subjects experiencing irAEs G ≥ 2 (29 vs 35, p = 0.021) and a decrease of IgG levels after steroids (38 vs 28, p = 0.009). CONCLUSIONS: Overall, these results support the potential role of autoAb in irAEs etiology and evolution. A prospective study is ongoing to validate our findings (NCT04107311).

Biomimetic human skin model patterned with rete ridges.

Rete ridges consist of undulations between the epidermis and dermis that enhance the mechanical properties and biological function of human skin. However, most human skin models are fabricated with a flat interface between the epidermal and dermal layers. Here, we report a micro-stamping method for producing human skin models patterned with rete ridges of controlled geometry. To mitigate keratinocyte-induced matrix degradation, telocollagen-fibrin matrices with and without crosslinks enable these micropatterned features to persist during longitudinal culture. Our human skin model exhibits an epidermis that includes the following markers: cytokeratin 14, p63, and Ki67 in the basal layer, cytokeratin 10 in the suprabasal layer, and laminin and collagen IV in the basement membrane. We demonstrated that two keratinocyte cell lines, one from a neonatal donor and another from an adult diabetic donor, are compatible with this model. We tested this model using an irritation test and showed that the epidermis prevents rapid penetration of sodium dodecyl sulfate. Gene expression analysis revealed differences in keratinocytes obtained from the two donors as well as between 2D (control) and 3D culture conditions. Our human skin model may find potential application for drug and cosmetic testing, disease and wound healing modeling, and aging studies.