OMIP-103: A 35-marker imaging mass cytometry panel for the co-detection of HIV and immune cell populations in human formalin fixed paraffin embedded intestinal tissue.

We introduce a 35-marker imaging mass cytometry (IMC) panel for a detailed examination of immune cell populations and HIV RNA in formalin fixed paraffin embedded (FFPE) human intestinal tissue. The panel has broad cell type coverage and particularly excels in delineating subsets of mononuclear phagocytes and T cells. Markers for key tissue structures are included, enabling identification of epithelium, blood vessels, lymphatics, and musculature. The described method for HIV RNA detection can be generalized to other low abundance RNA targets, whether endogenous or pathogen derived. As such, the panel presented here is useful for high parameter spatial mapping of intestinal immune cells and their interactions with pathogens such as HIV.

Integrative multi-region molecular profiling of primary prostate cancer in men with synchronous lymph node metastasis.

Localized prostate cancer is frequently composed of multiple spatially distinct tumors with significant inter- and intra-tumoral molecular heterogeneity. This genomic diversity gives rise to many competing clones that may drive the biological trajectory of the disease. Previous large-scale sequencing efforts have focused on the evolutionary process in metastatic prostate cancer, revealing a potential clonal progression to castration resistance. However, the clonal origin of synchronous lymph node (LN) metastases in primary disease is still unknown. Here, we perform multi-region, targeted next generation sequencing and construct phylogenetic trees in men with prostate cancer with synchronous LN metastasis to better define the pathologic and molecular features of primary disease most likely to spread to the LNs. Collectively, we demonstrate that a combination of histopathologic and molecular factors, including tumor grade, presence of extra-prostatic extension, cellular morphology, and oncogenic genomic alterations are associated with synchronous LN metastasis.

Sports participation after craniosynostosis repair: the critical role of post-operative guidance in parental decision-making.

PURPOSE: Despite previous research supporting patient safety in sports after craniosynostosis surgery, parental anxiety remains high. This study sought to evaluate the role of healthcare providers in guiding patients and families through the decision-making process. METHODS: Parents of children with repaired craniosynostosis were asked to assess sports involvement and parental decision-making in children ages 6 and older. Questions were framed primarily on 5-point Likert scales. Sport categorizations were made in accordance with the American Academy of Pediatrics. Chi-squared, linear regression, and Pearson correlation tests were used to analyze associations between the questions. RESULTS: Forty-three complete parental responses were recorded. Mean ages at surgery and time of sports entry were 7.93 ± 4.73 months and 4.76 ± 2.14 years, respectively. Eighty-two percent of patients participated in a contact sport. Discussions with the primary surgeon were more impactful on parental decisions about sports participation than those with other healthcare providers (4.04 ± 1.20 vs. 2.69 ± 1.32). Furthermore, children whose parents consulted with the primary surgeon began participating in sports at a younger age (4.0 ± 1.0 vs. 5.8 ± 2.7 years, p = 0.034). The mean comfort level with contact sports (2.8 ± 1.4) was lower than that with limited-contact (3.8 ± 1.1, p = 0.0001) or non-contact (4.4 ± 1.3, p < 0.0001) sports. CONCLUSION: This study underscores the critical role that healthcare professionals, primarily surgeons, have in guiding families through the decision-making process regarding their children's participation in contact sports.

The Correlation of Surgical Setting With Perioperative Opioid Prescriptions for Wide-Awake Carpal Tunnel Release.

BACKGROUND: Prior studies have compared perioperative opioid prescriptions between carpal tunnel release (CTR) performed wide-awake and with traditional anesthetic techniques, but the association of opioid prescriptions with surgical setting has not been fully explored. The current study assessed the association of opioid prescriptions with surgical setting (office or operating room) for wide-awake CTR. METHODS: Patients with open CTR were identified in an administrative claims database (PearlDiver). Exclusion criteria included age less than 18 years, preoperative data less than 6 months, postoperative data less than 1 month, bilateral surgery, concomitant hand surgery, and traditional anesthesia (general anesthesia, sedation, or regional block). Patients were stratified by surgical setting (office or operating room) and matched by age, sex, Elixhauser Comorbidity Index, and geographic region. Prior opioid prescriptions, opioid dependence/abuse, substance use disorder, back/neck pain, generalized anxiety, and major depression were identified. Opioid prescriptions within 7 days before and 30 days after surgery were characterized. RESULTS: Each matched cohort included 5713 patients. Compared with patients with surgery in the operating room, fewer patients with office-based surgery filled opioid prescriptions (45% vs 62%), and those prescriptions had lower morphine milligram equivalents (MMEs, median 130 vs 188). These findings were statistically significant on univariate and multivariate analysis. CONCLUSIONS: Following office-based CTR, fewer patients filled opioid prescriptions, and filled prescriptions had lower MME. This likely reflects patient and provider attitudes about pain control and opioid utilization. Further patient- and provider-level investigation may provide additional insights that could aid in efforts to reduce perioperative opioid utilization across surgical settings.

Functionalizing Yeast Lipid Droplets as Versatile Biomaterials.

Lipid droplets (LD) are dynamic cellular organelles of ≈1 µm diameter in yeast where a neutral lipid core is surrounded by a phospholipid monolayer and attendant proteins. Beyond the storage of lipids, opportunities for LD engineering remain underdeveloped but they show excellent potential as new biomaterials. In this research, LD from yeast Saccharomyces cerevisiae is engineered to display mCherry fluorescent protein, Halotag ligand binding protein, plasma membrane binding v-SNARE protein, and carbonic anhydrase enzyme via linkage to oleosin, an LD anchoring protein. Each protein-oleosin fusion is coded via a single gene construct. The expressed fusion proteins are specifically displayed on LD and their functions can be assessed within cells by fluorescence confocal microscopy, TEM, and as isolated materials via AFM, flow cytometry, spectrophotometry, and by enzyme activity assay. LD isolated from the cell are shown to be robust and stabilize proteins anchored into them. These engineered LD function as reporters, bind specific ligands, guide LD and their attendant proteins into union with the plasma membrane, and catalyze reactions. Here, engineered LD functions are extended well beyond traditional lipid storage toward new material applications aided by a versatile oleosin platform anchored into LD and displaying linked proteins.

Imaging Mass Cytometry for In Situ Immune Profiling.

The complexities and cellular heterogeneity associated with tissues necessitate the concurrent detection of markers beyond the limitations of conventional imaging approaches in order to spatially resolve the relationships between immune cell populations and their environments. This is a necessary complement to single-cell suspension-based methods to inform a better understanding of the events that may underlie pathological conditions. Imaging mass cytometry is a high-dimensional imaging modality that allows for the concurrent detection of up to 40 protein markers of interest across tissues at subcellular resolution. Here, we present an optimized staining protocol for imaging mass cytometry with modifications that integrate RNAscope. This unique addition enables combined protein and single-molecule RNA detection, thereby expanding the utility of imaging mass cytometry to researchers investigating low abundance or noncoding targets. In general, the procedure described is broadly applicable for comprehensive immune profiling of host-pathogen interactions, tumor microenvironments and inflammatory conditions, all within the tissue contexture.

Artificial Methylotrophic Cells via Bottom-Up Integration of a Methanol-Utilizing Pathway.

Methanol has gained substantial attention as a substrate for biomanufacturing due to plentiful stocks and nonreliance on agriculture, and it can be sourced renewably. However, due to inevitable complexities in cell metabolism, microbial methanol conversion requires further improvement before industrial applicability. Here, we present a novel, parallel strategy using artificial cells to provide a simplified and well-defined environment for methanol utilization as artificial methylotrophic cells. We compartmentalized a methanol-utilizing enzyme cascade, including NAD-dependent methanol dehydrogenase (Mdh) and pyruvate-dependent aldolase (KHB aldolase), in cell-sized lipid vesicles using the inverted emulsion method. The reduction of cofactor NAD+ to NADH was used to quantify the conversion of methanol within individual artificial methylotrophic cells via flow cytometry. Compartmentalization of the reaction cascade in liposomes led to a 4-fold higher NADH production compared with bulk enzyme experiments, and the incorporation of KHB aldolase facilitated another 2-fold increase above the Mdh-only reaction. This methanol-utilizing platform can serve as an alternative route to speed up methanol biological conversion, eventually shifting sugar-based bioproduction toward a sustainable methanol bioeconomy.

A disrupted compartment boundary underlies abnormal cardiac patterning and congenital heart defects.

Failure of septation of the interventricular septum (IVS) is the most common congenital heart defect (CHD), but mechanisms for patterning the IVS are largely unknown. We show that a Tbx5+/Mef2cAHF+ progenitor lineage forms a compartment boundary bisecting the IVS. This coordinated population originates at a first- and second heart field interface, subsequently forming a morphogenetic nexus. Ablation of Tbx5+/Mef2cAHF+ progenitors cause IVS disorganization, right ventricular hypoplasia and mixing of IVS lineages. Reduced dosage of the CHD transcription factor TBX5 disrupts boundary position and integrity, resulting in ventricular septation defects (VSDs) and patterning defects, including Slit2 and Ntn1 misexpression. Reducing NTN1 dosage partly rescues cardiac defects in Tbx5 mutant embryos. Loss of Slit2 or Ntn1 causes VSDs and perturbed septal lineage distributions. Thus, we identify essential cues that direct progenitors to pattern a compartment boundary for proper cardiac septation, revealing new mechanisms for cardiac birth defects.

A diffusion tensor imaging comparison of white matter development in nonsyndromic craniosynostosis to neurotypical infants.

PURPOSE: Nonsyndromic craniosynostosis (NSC) is associated with neurocognitive deficits, and intervention at infancy is standard of care to limit the negative effects of NSC on brain development. In this study, diffusion tensor imaging (DTI) was implemented to investigate white matter microstructure in infants with NSC undergoing cranial vault remodeling, and a comparison was made with white matter development in neurotypical controls. METHODS: Infants presenting with NSC (n = 12) underwent DTI scans before and after cranial vault remodeling. Neurotypical infants (n = 5), age matched to NSC patients at preoperative scans, were compared to preoperative DTI scans. Pre- and postoperative NSC scans were compared in aggregate, and the sagittal synostosis (n = 8) patients were evaluated separately. Finally, neurotypical infants from the University of North Carolina/University of New Mexico Baby Connectome Project (BCP), who underwent DTI scans at timepoints matching the NSC pre- and postoperative DTI scans, were analyzed (n = 9). Trends over the same time period were compared between NSC and BCP scans. RESULTS: No significant differences were found between preoperative NSC scans and controls. White matter development was more limited in NSC patients than in BCP patients, with microstructural parameters of the corpus body and genu and inferior and superior longitudinal fasciculi consistently lagging behind developmental changes observed in healthy patients. CONCLUSION: Infant white matter development appears more limited in NSC patients undergoing cranial vault remodeling relative to that in neurotypical controls. Further investigation is needed to explore these differences and the specific effects of early surgical intervention.

Cross-feeding promotes heterogeneity within yeast cell populations.

Cellular heterogeneity in cell populations of isogenic origin is driven by intrinsic factors such as stochastic gene expression, as well as external factors like nutrient availability and interactions with neighbouring cells. Heterogeneity promotes population fitness and thus has important implications in antimicrobial and anticancer treatments, where stress tolerance plays a significant role. Here, we study plasmid retention dynamics within a population of plasmid-complemented ura3∆0 yeast cells, and show that the exchange of complementary metabolites between plasmid-carrying prototrophs and plasmid-free auxotrophs allows the latter to survive and proliferate in selective environments. This process also affects plasmid copy number in plasmid-carrying prototrophs, further promoting cellular functional heterogeneity. Finally, we show that targeted genetic engineering can be used to suppress cross-feeding and reduce the frequency of plasmid-free auxotrophs, or to exploit it for intentional population diversification and division of labour in co-culture systems.

Perceived Age and Gender Perception Using Facial Recognition Software Following Facial Feminization Surgery.

Measures of success for facial feminization surgery (FFS) have previously included improved rates of external gender perception as female and patient-reported outcome measures. In this study, we used artificial intelligence facial recognition software to objectively evaluate the effects of FFS on both perceived gender and age among male-to-female transgender patients, as well as their relationship with patient facial satisfaction. Standardized frontal preoperative and postoperative images of 27 transgender women undergoing FFS were analyzed by Amazon's AI facial recognition software to determine gender, femininity confidence score, and perceived age. Female gender-typing, improvement in gender-typing (preoperatively to postoperatively), and femininity confidence scores were analyzed. To assess patient satisfaction, FACE-Q modules were completed postoperatively. Preoperatively, FFS images were perceived as female 48.1% of the time, and postoperatively, this improved to 74.1% ( P =0.05). Femininity confidence scores improved from a mean score of 0.04 preoperatively to 0.39 postoperatively ( P =0.003). FFS was associated with a decrease in perceived age relative to the patient's true age (-2.4 y, P <0.001), with older patients experiencing greater reductions. Pearson correlation matrix found no significant relationship between improved female gender typing and patient facial satisfaction. Undergoing surgery at a younger age was associated with higher overall facial satisfaction ( r =-0.6, P =0.01). Transfeminine patients experienced improvements in satisfaction with facial appearance, perceived gender, and decreases in perceived age following FFS. Notably, patient satisfaction was not directly associated with improved AI-gender typing, suggesting that other factors may influence patient satisfaction.

Quantitative Analysis of Morphometric Changes in Feminization Rhinoplasty Utilizing a Standardized Forehead-Rhinoplasty Technique.

Background: Rhinoplasty is one of the most commonly performed facial gender-affirming surgeries (FGASs) for transgender females, but well-established morphometric parameters describing feminizing nasal changes do not exist. Objectives: Describe the author's technique for feminization rhinoplasty, analyze the changes in 3-dimensional nasal anthropomorphic parameters, and describe patient-reported outcomes. Methods: Three-dimensional photogrammetric evaluation was performed both preoperatively and postoperatively in transgender female patients who underwent FGAS. Measurements assessed included the nasofrontal angle, nasolabial angle, dorsal height, mid-dorsal width, alar width, nasal tip width, and tip projection. Patients were surveyed preoperatively and postoperatively using the FACE-Q Nose module. Paired t-tests were utilized to assess changes in postoperative measurements and FACE-Q Nose satisfaction scores. Results: Twenty patients underwent FGAS during the study period. The average time between surgery and postoperative 3-dimensional images was 13.6 ± 6.8 months. The nasofrontal angle increased by 8.2° (148.0 ± 7.4° to 156.1 ± 6.7°, P < .001) and tip projection increased by 0.017 (0.58 ± 0.03 to 0.60 ± 0.04, P < .01). Dorsal height, mid-dorsal width, and tip width all decreased significantly (P < .05). There were significant improvements in patients' "Satisfaction with Nose," "Satisfaction with Facial Appearance Overall," "Psychological Function," and "Social Function" on FACE-Q. One revision rhinoplasty was performed, and no documented surgical complications were reported. Conclusions: There were statistically significant changes in the nasofrontal angle, tip projection, dorsal height, mid-dorsal width, and tip width in patients receiving feminization rhinoplasty. These data may help surgeons with preoperative planning and intraoperative decision making.

RPL22 is a tumor suppressor in MSI-high cancers and a key splicing regulator of MDM4.

Microsatellite instability high (MSI-H) tumors are malignant tumors that, despite harboring a high mutational burden, often have intact TP53. One of the most frequent mutations in MSI-H tumors is a frameshift mutation in RPL22, a ribosomal protein. Here, we identified RPL22 as a modulator of MDM4 splicing through an alternative splicing switch in exon 6. RPL22 loss increases MDM4 exon 6 inclusion, cell proliferation, and augments resistance to the MDM inhibitor Nutlin-3a. RPL22 represses expression of its paralog, RPL22L1, by mediating the splicing of a cryptic exon corresponding to a truncated transcript. Therefore, damaging mutations in RPL22 drive oncogenic MDM4 induction and reveal a common splicing circuit in MSI-H tumors that may inform therapeutic targeting of the MDM4-p53 axis and oncogenic RPL22L1 induction.

Quantifying Facial Feminization Surgery's Impact: Focus on Patient Facial Satisfaction.

Background: Facial feminization surgery (FFS) has been associated with improving gender dysphoria in transgender patients. This study aimed to quantify the impact of surgery on patient facial satisfaction, using the FACE-Q and a quality-of-life (QoL) survey. Methods: Transgender female patients were recruited to complete the FACE-Q and the World Health Organization's QoL Scale-Short Form (WHOQOL-BREF) if they were planning to or had undergone FFS at our institution. FACE-Q modules completed included "Satisfaction with Facial Appearance Overall," individual facial attributes (forehead/eyebrows, nose, cheeks, cheekbone, chin, jawline, and neck), and the WHOQOL-BREF, which assesses patient QoL through four domains (physical, psychological, social relations, and environment). Both matched and unmatched analyses of preoperative versus postoperative cohorts were performed. Results: Overall, 48 patients participated in our study and completed 31 FACE-Q surveys preoperatively and 37 postoperatively. On average, patients were 37.2 ± 12.5 years old. FACE-Q scores increased significantly for all facial attributes and for Satisfaction with Facial Appearance Overall between cohorts (P < 0.05). The facial attribute with the greatest increase in satisfaction was the jawline, followed by the nose. The WHOQOL-BREF's psychological and physical domains both improved significantly (P < 0.05). Wait time for surgery of less than 6 months (b = 22.42, P = 0.02) was associated with higher overall facial satisfaction, whereas age at surgery (b = -1.04, P < 0.01) was associated with lower overall facial satisfaction. Conclusions: Transgender female patients experienced significant improvements in facial satisfaction and QoL after FFS. Undergoing surgery at a younger age and shorter wait times for surgery were associated with increased overall facial satisfaction.

Lipofuscin-like autofluorescence within microglia and its impact on studying microglial engulfment.

Engulfment of cellular material and proteins is a key function for microglia, a resident macrophage of the central nervous system (CNS). Among the techniques used to measure microglial engulfment, confocal light microscopy has been used the most extensively. Here, we show that autofluorescence (AF) likely due to lipofuscin (lipo-AF) and typically associated with aging, can also be detected within microglial lysosomes in the young mouse brain by light microscopy. This lipo-AF signal accumulates first within microglia and it occurs earliest in white versus gray matter. Importantly, in gray matter, lipo-AF signal can confound the interpretation of antibody-labeled synaptic material within microglia in young adult mice. We further show that there is an age-dependent accumulation of lipo-AF inside and outside of microglia, which is not affected by amyloid plaques. We finally implement a robust and cost-effective strategy to quench AF in mouse, marmoset, and human brain tissue.

Interpretation of Cardiac Standstill in Children Using Point-of-Care Ultrasound.

STUDY OBJECTIVE: This study aimed to determine the level of agreement among pediatric emergency medicine (PEM) physicians in whether various point-of-care ultrasound (POCUS) video clips represent cardiac standstill in children and to highlight the factors that may be associated with the lack of agreement. METHODS: A single, online, cross-sectional, convenience sample survey was administered to PEM attendings and fellows with variable ultrasound experience. PEM attendings with an experience of 25 cardiac POCUS scans or more were the primary subgroup based on ultrasound proficiency set by the American College of Emergency Physicians. The survey contained 11 unique, 6-second video clips of cardiac POCUS performed during pulseless arrest in pediatric patients and asked the respondent if the video clip represented a cardiac standstill. The level of interobserver agreement was determined using the Krippendorff's α (Kα) coefficient across the subgroups. RESULTS: A total of 263 PEM attendings and fellows completed the survey (9.9% response rate). Of the 263 total responses, 110 responses were from the primary subgroup of experienced PEM attendings with at least 25 previously seen cardiac POCUS scans. Across all video clips, PEM attendings with 25 scans or more had an acceptable agreement (Kα=0.740; 95% CI 0.735 to 0.745). The agreement was the highest for video clips wherein the wall motion corresponded to the valve motion. However, the agreement fell to unacceptable levels (Kα=0.304; 95% CI 0.287 to 0.321) across video clips wherein the wall motion occurred without the valve motion. CONCLUSION: There is an overall acceptable interobserver agreement when interpreting cardiac standstill among PEM attendings with an experience of at least 25 previously reported cardiac POCUS scans. However, factors that may influence the lack of agreement include discordances between the wall and valve motion, suboptimal views, and the lack of a formal reference standard. More specific consensus reference standards of pediatric cardiac standstill may help to improve interobserver agreement moving forward and should include more specific details regarding the wall and valve motion.

Flux estimation analysis systematically characterizes the metabolic shifts of the central metabolism pathway in human cancer.

Introduction: Glucose and glutamine are major carbon and energy sources that promote the rapid proliferation of cancer cells. Metabolic shifts observed on cell lines or mouse models may not reflect the general metabolic shifts in real human cancer tissue. Method: In this study, we conducted a computational characterization of the flux distribution and variations of the central energy metabolism and key branches in a pan-cancer analysis, including the glycolytic pathway, production of lactate, tricarboxylic acid (TCA) cycle, nucleic acid synthesis, glutaminolysis, glutamate, glutamine, and glutathione metabolism, and amino acid synthesis, in 11 cancer subtypes and nine matched adjacent normal tissue types using TCGA transcriptomics data. Result: Our analysis confirms the increased influx in glucose uptake and glycolysis and decreased upper part of the TCA cycle, i.e., the Warburg effect, in almost all the analyzed cancer. However, increased lactate production and the second half of the TCA cycle were only seen in certain cancer types. More interestingly, we failed to detect significantly altered glutaminolysis in cancer tissues compared to their adjacent normal tissues. A systems biology model of metabolic shifts through cancer and tissue types is further developed and analyzed. We observed that (1) normal tissues have distinct metabolic phenotypes; (2) cancer types have drastically different metabolic shifts compared to their adjacent normal controls; and (3) the different shifts in tissue-specific metabolic phenotypes result in a converged metabolic phenotype through cancer types and cancer progression. Discussion: This study strongly suggests the possibility of having a unified framework for studies of cancer-inducing stressors, adaptive metabolic reprogramming, and cancerous behaviors.

Three recent sex chromosome-to-autosome fusions in a Drosophila virilis strain with high satellite DNA content.

The karyotype, or number and arrangement of chromosomes, has varying levels of stability across both evolution and disease. Karyotype changes often originate from DNA breaks near the centromeres of chromosomes, which generally contain long arrays of tandem repeats or satellite DNA. Drosophila virilis possesses among the highest relative satellite abundances of studied species, with almost half its genome composed of three related 7 bp satellites. We discovered a strain of D. virilis that we infer recently underwent three independent chromosome fusion events involving the X and Y chromosomes, in addition to one subsequent fission event. Here, we isolate and characterize the four different karyotypes we discovered in this strain which we believe demonstrates remarkable genome instability. We discovered that one of the substrains with an X-autosome fusion has an X-to-Y chromosome nondisjunction rate 20 × higher than the D. virilis reference strain (21% vs 1%). Finally, we found an overall higher rate of DNA breakage in the substrain with higher satellite DNA compared to a genetically similar substrain with less satellite DNA. This suggests that satellite DNA abundance may play a role in the risk of genome instability. Overall, we introduce a novel system consisting of a single strain with four different karyotypes, which we believe will be useful for future studies of genome instability, centromere function, and sex chromosome evolution.

Lipofuscin-like autofluorescence within microglia and its impact on studying microglial engulfment.

Engulfment of cellular material and proteins is a key function for microglia, a resident macrophage of the central nervous system (CNS). Among the techniques used to measure microglial engulfment, confocal light microscopy has been used the most extensively. Here, we show that autofluorescence (AF), likely due to lipofuscin and typically associated with aging, can also be detected within microglial lysosomes in the young mouse brain by light microscopy. This lipofuscin-AF signal accumulates first within microglia and increases with age, but it is not exacerbated by amyloid beta-related neurodegeneration. We further show that this lipofuscin-AF signal within microglia can confound the interpretation of antibody-labeled synaptic material within microglia in young adult mice. Finally, we implement a robust strategy to quench AF in mouse, marmoset, and human brain tissue.