Optimized intravital three-photon imaging of intact mouse tibia links plasma cell motility to functional states.

Intravital deep bone marrow imaging is crucial to studying cellular dynamics and functions but remains challenging, and minimally invasive methods are needed. We employed a high pulse-energy 1650 nm laser to perform three-photon microscopy in vivo, reaching ≈400 µm depth in intact mouse tibia. Repetition rates of 3 and 4 MHz allowed us to analyze motility patterns of fast and rare cells within unperturbed marrow and to identify a bi-modal migratory behavior for plasma cells. Third harmonic generation (THG) was identified as a label-free marker for cellular organelles, particularly endoplasmic reticulum, indicating protein synthesis capacity. We found a strong THG signal, suggesting high antibody secretion, in one-third of plasma cells while the rest showed low signals. We discovered an inverse relationship between migratory behavior and THG signal, linking motility to functional plasma cell states. This method may enhance our understanding of marrow microenvironment effects on cellular functions.

Transcript errors generate amyloid-like proteins in huwman cells.

Aging is characterized by the accumulation of proteins that display amyloid-like behavior. However, the molecular mechanisms by which these proteins arise remain unclear. Here, we demonstrate that amyloid-like proteins are produced in a variety of human cell types, including stem cells, brain organoids and fully differentiated neurons by mistakes that occur in messenger RNA molecules. Some of these mistakes generate mutant proteins already known to cause disease, while others generate proteins that have not been observed before. Moreover, we show that these mistakes increase when cells are exposed to DNA damage, a major hallmark of human aging. When taken together, these experiments suggest a mechanistic link between the normal aging process and age-related diseases.

Longitudinal Changes in Estimated Glomerular Filtration Rate Following Repeat Heart Transplant in Children and Young Adults.

BACKGROUND: Chronic kidney disease (CKD) is common after heart transplantation (HT). There are scarce studies looking at longitudinal changes in estimated glomerular filtration rate (eGFR) after repeat HT (RT) and risk factors for the presence of CKD3 post-RT. METHODS: First-time HT recipients (FT) were matched with RT, based on age at transplant, sex, race, and transplant era. eGFR was derived from CKiD-U25 formula using creatinine. Changes in eGFR were measured within and between patients using a mixed effects model. Logistic regression and survival analysis were performed to identify significant risk factors for CKD3 presence post-RT. RESULTS: The unmatched cohort included 393 HT recipients and 47 RT. 29 patients in both groups with at least 2 years of follow-up data underwent matching. Over 6 years, the mean eGFR for FT and RT did not significantly change overtime. For those with CKD3 or higher prior to RT, 73% improved to CKD2 or lower at 2 years post-RT. Risk factors for CKD3 post-RT included female sex (OR 17.5 [1.7-181.9], p = 0.017) and greater than two acute kidney injuries (AKI) between FT and RT (OR 1.77 [1.06-2.95], p = 0.03). Approximately 50% of females and those with greater than two AKIs maintained or developed CKD3 at 7.5 and 3 years, respectively. CONCLUSION: This study provides data on longitudinal changes in eGFR over 6 years and finds that female sex and repeat HT recipients with greater 2 AKIs between transplants are at greatest risk for CKD3 presence post-RT.

Case report: Treatment of parkinsonism secondary to ciltacabtagene autoleucel using a combination dopaminergic regimen.

We report on a patient with ciltacabtagene autoleucel-induced movement and neurocognitive toxicity, which was refractory to immunosuppression but responsive to combination dopaminergic therapy (carbidopa/levodopa, ropinirole, amantadine). Response was seen upon both initial treatment and rechallenge after unintended withdrawal. This is the first report of a successful symptomatic treatment of this well-described neurotoxic syndrome.

Level of expression of MHCI-presented neoepitopes influences tumor rejection by neoantigen-specific CD8+ T cells.

Neoantigen-targeted therapy holds an array of benefits for cancer immunotherapy, but the identification of peptide targets with tumor rejection capacity remains a limitation. To better define the criteria dictating tumor rejection potential, we examined the capacity of high-magnitude T cell responses induced towards several distinct neoantigen targets to regress MC38 tumors. Surprisingly, despite their demonstrated immunogenicity, vaccine-induced T-cell responses were unable to regress established MC38 tumors or prevent tumor engraftment in a prophylactic setting. However, T cells were functional with robust killing capacity towards neoantigen peptide-loaded cells. Furthermore, tumor cell killing was rescued in proportion to the expression level or saturation of target peptide-loaded MHCs on the cell surface. Overall, this study demonstrates a pivotal role for target protein expression levels in modulating the tumor rejection capacity of neoantigens. Thus, inclusion of this metric, in addition to immunogenicity analysis, may benefit antigen prediction techniques to ensure the full anti-tumor effect of cancer vaccines.

A multisite feasibility study of integrated cognitive-behavioral treatment for co-existing nocturia and chronic insomnia.

BACKGROUND: Nocturia (waking from sleep at night to void) and chronic insomnia frequently co-exist in older adults, contributing synergistically to sleep disturbance. Treatments typically target either nocturia or insomnia rather than simultaneously addressing shared mechanisms for these disorders. METHODS: We conducted a multisite feasibility study to: (1) test and refine a protocol for recruitment, randomization, and assessment of older adults with co-existing nocturia and insomnia; and (2) examine preliminary changes in outcome measures to inform a future larger, multisite clinical trial. Participants were men and women aged 60 years and older recruited from outpatient clinics, reporting an average of two or more nocturia episodes per night over the past 4 weeks and meeting diagnostic criteria for chronic insomnia disorder. Participants were randomized to receive either integrated cognitive-behavioral therapy for insomnia and nocturia or a health education control program involving five weekly visits with a trained nurse practitioner interventionist. Outcomes (e.g., nocturia episodes) were measured 1-week post-treatment and 4-month post-randomization. Descriptive statistics examined the feasibility of outcomes to guide preparations for a future efficacy trial. RESULTS: Of 245 adults screened, 55% were ineligible and 25% declined to participate. Sixty-one percent of 49 participants who provided informed consent were randomized. Of the 30 participants randomized (mean age = 70.6 years, 60% White), 14 were assigned to integrated cognitive-behavioral treatment and 16 to the control group. All randomized participants provided 4-month follow-up data. At 4 months, mean nightly nocturia episodes decreased by 0.9 (SD 1.0) in the integrated treatment group and by 0.2 (SD 1.2) in the control group compared with baseline. DISCUSSION: Findings demonstrate the feasibility of recruiting, randomizing, and collecting outcome data from older adults (predominantly male) assigned to an integrated cognitive-behavioral therapy for coexisting insomnia and nocturia or a health education control program.

Aqueous compatible post-synthetic on-column conjugation of nucleic acids using amino-modifiers.

Nucleic acid conjugation methodologies involves linking the nucleic acid sequence to other (bio)molecules covalently. This typically allows for nucleic acid property enhancement whether it be for therapeutic purposes, biosensing, etc. Here, we report a streamlined aqueous compatible on-column conjugation methodology using nucleic acids containing a site-specific amino-modifier. Both monophosphates and carboxylates were amenable to the conjugation strategy, allowing for the introduction of a variety of useful handles including azide, aryl, and hydrophobic groups in DNA. We find that an on-column approach is superior to post-synthetic template-directed synthesis, mainly with respect to product purification and recovery.

Examining longitudinal associations between initial perceptions and experiences with electronic nicotine delivery system (ENDS) use and use patterns among adults who smoke and recently initiated ENDS.

INTRODUCTION: Limited data exist on factors associated with concurrent use patterns of electronic nicotine delivery systems (ENDS) and cigarettes. We examined longitudinally perceptions and experiences with ENDS in relationship to concurrent use patterns among established, recent smokers who recently initiated ENDS. METHODS: Participant recruitment took place using paid digital advertisements on social media. Between December 2020 and October 2021, 303 adults aged ≥21 years from across the US who currently or recently smoked and had initiated ENDS use within the past 30 days or reinitiated ENDS use after more than one year of non-use were surveyed. Multinomial logistic regressions were conducted to analyze association between the outcome of current use pattern at follow-up at 1 month [rejectors (discontinued ENDS, continued smoking), primary smokers (concurrent users, mostly smoke), dual user (similar smoking and ENDS use), primary vapers (concurrent users, mostly vape), and switchers (discontinued smoking, continued using ENDS) or quitters (discontinued both smoking and ENDS] and perceptions of and experiences with ENDS predictors at baseline. RESULTS: At follow-up at 1 to 2 months after initiating ENDS, 20% were rejectors, 31% were primary smokers, 13% were dual users, 19% were primary vapers, and 17% were switchers/quitters. Perceiving ENDS as less harmful than smoking or being uncertain and as equally or more enjoyable smoking, experiencing a lot or complete reduction in cravings to smoking and in irritability with ENDS use, liking the taste of ENDS, and being satisfied with vaping were associated with higher odds of quitting smoking compared to rejecting ENDS or mostly smoking at follow-up at 1 month. CONCLUSIONS: Findings highlight the importance of initial ENDS perceptions and experiences when examining tobacco outcomes and potentially for developing policies and interventions targeting smoking cessation. ENDS initiators are differentiating into distinct use patterns based on these factors within a short period of time.

Incubation period of Clostridioides difficile infection in hospitalized patients and long-term care facility residents: a prospective cohort study.

Background: The incubation period for Clostridioides difficile infection (CDI) is generally considered to be less than 1 week, but some recent studies suggest that prolonged carriage prior to disease onset may be common. Objective: To estimate the incubation period for patients developing CDI after initial negative cultures. Methods: In 3 tertiary care medical centers, we conducted a cohort study to identify hospitalized patients and long-term care facility residents with negative initial cultures for C. difficile followed by a diagnosis of CDI with or without prior detection of carriage. Cases were classified as healthcare facility-onset, community-onset, healthcare facility-associated, or community-associated and were further classified as probable, possible, or unlikely CDI. A parametric accelerated failure time model was used to estimate the distribution of the incubation period. Results: Of 4,179 patients with negative enrollment cultures and no prior CDI diagnosis within 56 days, 107 (2.6%) were diagnosed as having CDI, including 19 (17.8%) with and 88 (82.2%) without prior detection of carriage. When the data were censored to only include participants with negative cultures collected within 14 days, the estimated median incubation period was 6 days with 25% and 75% of estimated incubation periods occurring within 3 and 12 days, respectively. The observed estimated incubation period did not differ significantly for patients classified as probable, possible, or unlikely CDI. Conclusion: Our findings are consistent with the previous studies that suggested the incubation period for CDI is typically less than 1 week and is less than 2 weeks in most cases.

Structural and Theoretical Assessment of Covalency in a Pu(III) Borohydride Complex.

Despite the discovery of actinide borohydride complexes over 80 years ago, no plutonium borohydride complexes have been structurally validated using single-crystal X-ray diffraction (XRD). Here we describe Pu2(H3BPtBu2BH3)6, the first example of a Pu(III) borohydride complex authenticated by XRD and NMR spectroscopy. Theoretical calculations (DFT, EDA, and QTAIM) and experimental comparisons of metal-boron distances suggest that metal-borohydride covalency in M2(H3BPtBu2BH3)6 complexes generally decreases in the order M = U(III) > Pu(III) > Ln(III).

Cardiorespiratory Fitness Is an Indicator of Arterial Stiffness and Aortic Blood Pressure in Healthy Adolescents.

Background/Objectives: We aimed to investigate the influence of cardiorespiratory fitness (CRF) and body composition on arterial stiffness. Methods: Carotid-to-femoral pulse wave velocity (cfPWV) and aortic systolic (ASBP) and diastolic (ADBP) blood pressure were compared between upper and lower tertiles of oxygen consumption at the aerobic threshold (VO2AerT), peak oxygen consumption (VO2peak), percentage of fat mass (FM%), and body mass index (BMI) in sixty adolescents (30 males and 30 females, 14.9 ± 2.1 years old). A stepwise multivariable linear regression analysis was performed to investigate the independent associations between VO2AerT and VO2peak and cfPWV, and between BMI and FM% and cfPWV with adjustments for age, sex, ASBP, and ADBP. Results: cfPWV and ADBP were lower in the second and third VO2AerT tertiles compared to the first tertile (cfPWV, 4.7 ± 0.5 and 4.7 ± 0.5 vs. 5.3 ± 0.8 m/s, p < 0.01; ADBP, 62 ± 7 and 62 ± 7 vs. 70 ± 8 mmHg, p < 0.01). ASBP was lower in the third VO2AerT tertile compared to the first tertile (94 ± 7 vs. 101 ± 12 mmHg, p = 0.05). ADBP was lower in the second VO2peak tertile compared to the first tertile (62 ± 7 vs. 68 ± 9 mmHg, p = 0.03). ASBP was lower in the first and second BMI tertiles compared to the third tertile (95 ± 8 and 95 ± 7 vs. 102 ± 11 mmHg, p = 0.02). The eight-variable model significantly contributed to the variance of cfPWV (F(8, 51) = 7.450, p < 0.01), accounting for 47% of the variance. Individually, age (p < 0.05) and ADBP (p < 0.01) significantly predicted cfPWV. Conclusions: Submaximal indicators of CRF such as VO2AerT should be considered as a part of the risk stratification of cardiovascular disease in healthy adolescents.

Harnessing the Power of Generative Artificial Intelligence in Pathology Education.

CONTEXT.­: Generative artificial intelligence (AI) technologies are rapidly transforming numerous fields, including pathology, and hold significant potential to revolutionize educational approaches. OBJECTIVE.­: To explore the application of generative AI, particularly large language models and multimodal tools, for enhancing pathology education. We describe their potential to create personalized learning experiences, streamline content development, expand access to educational resources, and support both learners and educators throughout the training and practice continuum. DATA SOURCES.­: We draw on insights from existing literature on AI in education and the collective expertise of the coauthors within this rapidly evolving field. Case studies highlight practical applications of large language models, demonstrating both the potential benefits and unique challenges associated with implementing these technologies in pathology education. CONCLUSIONS.­: Generative AI presents a powerful tool kit for enriching pathology education, offering opportunities for greater engagement, accessibility, and personalization. Careful consideration of ethical implications, potential risks, and appropriate mitigation strategies is essential for the responsible and effective integration of these technologies. Future success lies in fostering collaborative development between AI experts and medical educators, prioritizing ongoing human oversight and transparency to ensure that generative AI augments, rather than supplants, the vital role of educators in pathology training and practice.

Sleep Differences in Firefighters: Barracks vs. Home.

It has been shown that the firefighter occupation leads to poor sleep quality and sleep architecture. Disturbed sleep in these occupations can lead to deleterious outcomes including a series of chronic diseases and illnesses such as CVD. PURPOSE: The aims were (1) to quantify firefighters' sleep via polysomnography, (2) to identify differences between sleeping in the barracks versus sleeping at home, and (3) to compare firefighter data to age-matched normative data. We expected significant differences between both the home and the barrack conditions as well as significant differences when both conditions were compared to normative data. METHODS: 10 male firefighters completed 3 nights of polysomnography recordings (SleepProfilerTM (Advanced Brain Monitoring, Carlsbad, CA, USA)) counterbalanced in both their own beds or barracks. A one-way rmANOVA statistical analysis was used to determine differences in sleep values with a Bonferroni correction if a significant difference was found with significance set at p < 0.05. RESULTS: Three important variables, cortical arousals (p < 0.05), autonomic activations (p < 0.01), and spindle duration (p < 0.01), had differences that were statistically significant between sleep at home or in the barracks, with sleep in the barracks being more disturbed. Clinical differences were also observed between the home and barrack conditions and all sleep results were more deleterious when compared to normative data. CONCLUSIONS: The data demonstrates that firefighters show poor sleep quality and heavily impacted sleep architecture. This may be due to the effects of rotating shifts and occupational stress on the sleep-wake cycle. These results, when compared to age-matched normative data, show clinical manifestations of disturbed sleep in the firefighter population.

Differing Transcriptomic Responses in High Titer versus Low Titer Aedes aegypti Mosquitoes after Oral Infection with Sindbis Virus.

Oral infection of mosquitoes by arboviruses often results in a large degree of variation in the amount of infectious virus between individual mosquitoes, even when the mosquitoes are from inbred laboratory strains. This variability in arbovirus load has been shown to affect virus transmissibility. Previously, our group described population genetic and specific infectivity differences between the virus populations found in high and low titer Aedes aegypti mosquitoes that had been orally infected with Sindbis virus (SINV). In this study, we sought to investigate whether there were also differences in transcriptomic response between these high and low titer mosquitoes. Results from the transcriptomic data analysis showed that more genes involved in antiviral activity, endopeptidase activity, and methyltransferase activity were upregulated in low titer mosquitoes than in high titer mosquitoes, relative to blood-fed controls. Meanwhile, genes involved in ion transport, energy metabolism, acetylation, glycosylation, lipid metabolism, and transport tended to be upregulated in high titer mosquitoes more than in low titer mosquitoes, relative to blood-fed mosquitoes. Overall, genes involved in antiviral activities tended to be upregulated in low titer mosquitoes while genes involved in proviral activities were mostly upregulated in high titer mosquitoes. This study has identified a number of candidate mosquito genes that are putatively associated with SINV titer variability after oral infection of Ae. aegypti, and these can now be investigated in order to ascertain their roles in virus replication and their contributions to determining vector competence.

Adverse tissue reactions to ORISE gel during endoscopic mucosal resection and dissection.

BACKGROUND: Submucosal injection of lifting agents (LAs) is frequently used to facilitate endoscopic mucosal resection or endoscopic submucosal dissection. ORISE (Boston Scientific Corporation) gel, approved in 2018 by the United States Food and Drug Administration, has been commonly used until recently. Its use proliferated because of its practical prefilled syringe, prolonged lift effect, and ideal coloration. However, it has been noted to cause unexpected tissue reactions, described as "giant cell reaction" (GCR), which can obscure macroscopic and microscopic views, potentially interfering with pathologic evaluation. This study aimed to describe the adverse effects of ORISE gel. METHODS: This retrospective study analyzed pathologic specimens from all consecutive patients who received ORISE injections for attempted polyp removal and subsequently underwent segmental colon resection at our center between 2019 and 2022. A descriptive analysis was performed. RESULTS: A total of 45 patients were included, of whom 17 (38.0%) were female and the median age was 66 years. The indications for surgery included adenocarcinoma in 14 patients (31%), suspected malignancy in 3 patients (7%) who had benign GCR-induced masses, and other indications in 28 patients, such as large polyps or recurrent polyps after initial endoscopic treatment. Surgical procedures included right hemicolectomy (44%), low anterior resection (13%), left colectomy (11%), sigmoidectomy (7%), and abdominoperineal resection (4%). Histologic evidence of previous LA injection was seen in 31 patients (69%), with 24 patients exhibiting GCR. At final pathology, no residual neoplasm was found in 9 patients (18%); however, 14 patients (31%) had adenocarcinoma (T1 [7], T2 [3], T3 [3], and T4 [1]). CONCLUSION: ORISE gel interacts with various tissue layers of the colon, frequently resulting in GCR. This reaction and the potential subsequent mass effect formation can affect decision-making regarding the management of complex colorectal lesions. Further study into the causes and consequences of LA tissue reactions is warranted.

Ileoanal pouch salvage rates with endoluminal vacuum therapy for early vs late anastomotic leaks.

BACKGROUND: Anastomotic leaks after ileal pouch-anal anastomosis (IPAA) significantly compromise patient outcomes and increase healthcare resource utilization. The aim of this study was to evaluate the outcomes of endoluminal vacuum therapy (EndoVac) for pouch leaks. We hypothesized that EndoVac for early compared with late leaks was associated with a higher pouch survival rate. METHODS: We retrospectively reviewed consecutive pouch anastomotic leaks treated with EndoVac therapy at our institution between 2013 and 2023. Patients were stratified into early (≤30 days) and late (>30 days) leak groups. Anastomotic healing was defined as complete closure of the leak site and resolution of symptoms. Pouch failure was defined as a permanent ileostomy or pouch excision. The probability of pouch survival was estimated using the Kaplan-Meier method. RESULTS: A total of 14 patients with IPAA were included: median age, 34 years; 71% were males; and median body mass index, 23.46 kg/m2. The following were the diagnoses made: ulcerative colitis (n = 12) and familial adenomatous polyposis (n = 2). The median (IQR) time from pouch construction to leak was 44.5 (12-192) days; of these, 6 (43%) were early, and 8 (57%) were late. All (100%) leaks were at the anastomosis; all (100%) were diverted at the time of EndoVac therapy: 10 (71%) still diverted, and 4 (29%) rediverted. Patients underwent a median of 5.5 EndoVac changes (3-7) over a 13.5 (6-21) day period from initiation of treatment to cessation of therapy. After EndoVac therapy, healing was observed in 10 (71%) patients; 2 of whom required a minor handsewn anastomotic revision but healed completely, and 4 did not heal; 3 had pouch excision, and 1 underwent redo pouch surgery. Anastomotic healing (66.7% vs 75%, P = .7) and pouch survival (83.3% vs 75%, P = .73) were not significantly different between the early and late leak groups. The overall pouch salvage rate was 78.5%. CONCLUSION: EndoVac therapy was effective in achieving high rates of pouch salvage and anastomotic healing in patients with ileoanal pouch leaks, irrespective of the timing of intervention postoperatively. This supports the use of EndoVac as a viable treatment option for both early and late anastomotic leaks.

Priority screening of contaminants of emerging concern (CECs) in surface water: Comparing cell-based bioassays and exposure-activity ratios (EARs).

In this study, we compared a wide range of cell-based bioassays to the use of chemical analysis followed by exposure-activity ratio (EAR) and Toxicological Prioritization index (ToxPi) for prioritizing chemicals, sites, and hazard concerns in water samples. Surface water samples were collected from nine sites in three Central Pennsylvania streams and analyzed for a forty-six contaminants of emerging concern (CECs), including pesticides, personal care products, and pharmaceuticals. Cell-based reporter assays evaluated human and zebrafish molecular initiating events (MIEs) in endocrine and metabolic disruption, altered lipid metabolism, and oxidative stress. Bioassays showed that 12 out of 40 assays had at least one site with activity over the effect-based trigger (EBT) values. The receptors that exhibited the highest number of samples above the EBT that would be expected to cause toxicity were Aryl hydrocarbon receptor (AhR, human and zebrafish), Pregnane X Receptor (PXR), Estrogen Receptor-beta (ERB), and Androgen Receptor (AR). Characterizing the collection sites by their bioactivity aligned closely with the stream in which samples were collected. The sum of all EARs for each chemical indicated that the pharmaceutical Carbamazepine and the pesticides Carbaryl and Atrazine posed the greatest concern. However, predicted activity and site prioritization based on individual chemical analysis and calculated EAR were different than those measured by bioassay, indicating that biologically active chemicals are present in the samples that were not included in the targeted analytes. Taken together, these data show that chemical analysis and EAR analysis are beneficial for prioritization of chemicals, whereas mechanism-based bioassays are more inclusive of known as well as unknown chemical contaminants and thus of more use for overall water quality analysis and site prioritization.

Adeno-associated virus-mediated gene delivery of Perm1 enhances cardiac contractility in mice.

Reduced muscle contractility and mitochondrial bioenergetics are the hallmarks of systolic heart failure. There is currently no therapy targeting both. Here, we show that gene delivery of Perm1 via adeno-associated virus (AAV) simultaneously enhances cardiac contractility and mitochondrial biogenesis in C57BL6 mice. Moreover, we found that PERM1 interacts with troponin C (TnC), a key contractile protein in striated muscle, and that AAV-Perm1 led to the upregulation of TnC. This study suggests that gene delivery of Perm1 may be a novel therapeutic approach to treat systolic heart failure by simultaneously restoring cardiac contractility and mitochondrial bioenergetics.NEW & NOTEWORTHY Perm1 gene delivered with AAV9 enhances cardiac contractility in mice, and it is concomitant with the increase of mitochondrial bioenergetics and upregulation of TnC. This is the first study showing that PERM1, previously known as a striated muscle-specific mitochondrial regulator, also positively regulates cardiac contractility.