Dual-Omics Approach Unveils Novel Perspective on the Quality Control of Genetically Engineered Exosomes.

Exosomes, nanoscale vesicles derived from human cells, offer great promise for targeted drug delivery. However, their inherent diversity and genetic modifications present challenges in terms of ensuring quality in clinical use. To explore solutions, we employed advanced gene fusion and transfection techniques in human 293T cells to generate two distinct sets of genetically engineered samples. We used dual-omics analysis, combining transcriptomics and proteomics, to comprehensively assess exosome quality by comparing with controls. Transcriptomic profiling showed increased levels of engineering scaffolds in the modified groups, confirming the success of genetic manipulation. Through transcriptomic analysis, we identified 15 RNA species, including 2008 miRNAs and 13,897 mRNAs, loaded onto exosomes, with no significant differences in miRNA or mRNA levels between the control and engineered exosomes. Proteomics analysis identified changes introduced through genetic engineering and over 1330 endogenous exosome-associated proteins, indicating the complex nature of the samples. Further pathway analysis showed enrichment in a small subset of cellular signaling pathways, aiding in our understanding of the potential biological impacts on recipient cells. Detection of over 100 cow proteins highlighted the effectiveness of LC-MS for identifying potential contaminants. Our findings establish a dual-omics framework for the quality control of engineered exosome products, facilitating their clinical translation and therapeutic applications in nanomedicine.

A Novel Dual-Reporter System Reveals Distinct Characteristics of Exosome-Mediated Protein Secretion in Human Cells.

BACKGROUND: Exosomes, a special subtype of extracellular vesicles derived from human cells, serve as vital mediators of intercellular communication by transporting diverse bioactive cargos, including proteins and enzymes. However, the underlying mechanisms governing exosome secretion and regulation remain poorly understood. In this study, we employed a dual-reporter system consisting of bioluminescent Gaussia luciferase and fluorescent proteins to investigate the dynamics and regulation of exosome secretion in cultured human cells. RESULTS: Our results demonstrated that the engineered dual-reporters effectively monitored both exosome-mediated and ER-Golgi-mediated secretory pathways in a specific and quantitative manner. Notably, we observed distinct characteristics of exosome-mediated protein secretion, including significantly lower capacity and different dynamics compared to the ER-Golgi pathway. This phenomenon was observed in human kidney 293T cells and liver HepG2 cells, emphasizing the conserved nature of exosome-mediated secretion across cell types. Furthermore, we investigated the impact of brefeldin A (BFA), an inhibitor of ER-to-Golgi membrane trafficking, on protein secretion. Interestingly, BFA inhibited protein secretion via the ER-Golgi pathway while stimulating exosome-mediated protein secretion under same experimental conditions. CONCLUSIONS: Collectively, our study highlights the utility of the dual-reporter system for real-time monitoring and quantitative analysis of protein secretion through conventional ER-Golgi and unconventional exosome pathways. Moreover, our findings unveil distinct features of exosome-mediated protein secretion, shedding light on its differential capacity, dynamics, and regulatory mechanisms compared to ER-Golgi-mediated proteins in human cells.

Hydroxyzine-induced priapism.

Priapism is a severe urologic condition requiring emergency management. Ischemic priapism is the most common subtype which is characterized by a long-lasting, painful, and rigid erection which can be caused by medications with alpha-adrenergic properties such as hydroxyzine. Typically, medication-induced priapism is reported at therapeutic doses and few case reports exist implicating medication overdose as the cause. We report a case of a patient taking hypercompliant doses of hydroxyzine hydrochloride for worsening insomnia (200-600 mg), including the night before admission. Blood-gas analysis of blood from the right corpora was completed and revealed a pH of 6.736, pCO2 of 147, HCO3 of 18.6 and a base excess of 17.7. The patient required aspiration and 560 µg of intracavernosal phenylephrine to achieve sustained detumescence. Emergency physicians should be aware of this risk as priapism is a medical emergency and this is the first report with hydroxyzine after an intentional overdose to our knowledge.

Divergent low-density lipoprotein receptor (LDLR) linked to low VSV G-dependent viral infectivity and unique serum lipid profile in zebra finches.

The low-density lipoprotein receptor (LDLR) is key to cellular cholesterol uptake and is also the main receptor for the vesicular stomatitis virus glycoprotein (VSV G). Here we show that in songbirds LDLR is highly divergent and lacks domains critical for ligand binding and cellular trafficking, inconsistent with universal structure conservation and function across vertebrates. Linked to the LDLR functional domain loss, zebra finches show inefficient infectivity by lentiviruses (LVs) pseudotyped with VSV G, which can be rescued by the expression of human LDLR. Finches also show an atypical plasma lipid distribution that relies largely on high-density lipoprotein (HDL). These findings provide insights into the genetics and evolution of viral infectivity and cholesterol transport mechanisms in vertebrates.

The vocal organ of hummingbirds shows convergence with songbirds.

How sound is generated in the hummingbird syrinx is largely unknown despite their complex vocal behavior. To fill this gap, syrinx anatomy of four North American hummingbird species were investigated by histological dissection and contrast-enhanced microCT imaging, as well as measurement of vocalizations in a heliox atmosphere. The placement of the hummingbird syrinx is uniquely located in the neck rather than inside the thorax as in other birds, while the internal structure is bipartite with songbird-like anatomical features, including multiple pairs of intrinsic muscles, a robust tympanum and several accessory cartilages. Lateral labia and medial tympaniform membranes consist of an extracellular matrix containing hyaluronic acid, collagen fibers, but few elastic fibers. Their upper vocal tract, including the trachea, is shorter than predicted for their body size. There are between-species differences in syrinx measurements, despite similar overall morphology. In heliox, fundamental frequency is unchanged while upper-harmonic spectral content decrease in amplitude, indicating that syringeal sounds are produced by airflow-induced labia and membrane vibration. Our findings predict that hummingbirds have fine control of labia and membrane position in the syrinx; adaptations that set them apart from closely related swifts, yet shows convergence in their vocal organs with those of oscines.

Case Series of Reproductive Outcomes after Laparoscopic Radiofrequency Ablation of Symptomatic Myomas.

STUDY OBJECTIVE: To analyze pregnancy delivery and safety outcomes after patient receipt of percutaneous, laparoscopic intra-abdominal ultrasound-guided radiofrequency ablation (Lap-RFA) for symptomatic uterine myomas. DESIGN: Case series (2010-2017); evidence was obtained from 2 randomized, controlled trials (level I), 6 cohort studies (level II-2), and in commercial settings (level II-3). SETTING: Multiple sites in the United States, Canada, Europe, and Latin America (university hospitals, community hospitals, and stand-alone surgery centers). Commercial cases were United States based and followed US Food and Drug Administration clearance of Lap-RFA. PATIENTS: Premenopausal adult women with symptomatic uterine myoma types 1 through 6. INTERVENTIONS: The Lap-RFA procedure was conducted under general anesthesia with laparoscopic and intra-abdominal ultrasound guidance. MEASUREMENTS AND MAIN RESULTS: Safety unknowns included the safety of a full-term pregnancy for mother and baby, rates of spontaneous abortion, preterm delivery, postpartum hemorrhage, placental abnormalities, intrauterine growth restriction, and vaginal versus cesarean delivery. A total of 28 women (mean age = 35.0 ± 3.4 years) conceived a total of 30 times after Lap-RFA, either as part of a clinical study or in commercial settings. The number of myomas treated per patient ranged from 1 to 7. The diameter of treated myomas ranged from 0.9 to 11.0 cm. Most patients had 1 or 2 myomas, and most myomas were ≤5.5 cm in maximal diameter. The 30 pregnancies resulted in 26 full-term live births (86.7%), all healthy infants, with an equal distribution of vaginal and cesarean deliveries. Four (13.3%) spontaneous abortions occurred. No cases of preterm delivery, uterine rupture, placental abruption, placenta accreta, or intrauterine growth restriction were reported. One event each of placenta previa and postpartum hemorrhage were reported. CONCLUSION: Conception and safe, full-term pregnancy are achievable after Lap-RFA of symptomatic myomas. Additional large, rigorous, multivariate prospective studies that adjust for confounders and report pregnancy outcomes after symptomatic myoma treatment are needed.

Seasonal changes in the song control nuclei of the Rufous-bellied Thrush, Turdus rufiventris (Oscine, Passeriformes, and Turdidae).

In vocal learning birds, memorization and song production rely on a set of telencephalic nuclei referred to as the song control system. Seasonal changes in song production are correlated with changes in the volume of the song control nuclei and are influenced by photoperiodic conditions and hormonal cues. The seasonal volume changes in the avian brain that controls singing are thought to involve regulation of neuronal replacement, which is a striking example of neuronal plasticity. The Rufous-bellied Thrush (Turdus rufiventris) is a seasonally breeding bird that actively sings during the spring and summer (breeding season) and is relatively silent in the fall, yet possible mechanisms behind the periodic changes in song production remain unknown. Here, we have examined two song control nuclei: High vocal center (HVC) and robust nucleus of arcopallium (RA) in fall males, spring males, and fall females of Rufous-bellied Thrush. The cytoarchitectonic organization was analyzed and quantified from Nissl-stained sections, and gene expression of song nuclei markers was examined by in situ hybridization during breeding and nonbreeding seasons. We observed a reduction in HVC volume and reductions in parvalbumin, and RGS4 expression in HVC and RA in males during the nonbreeding season. These findings provide evidence of seasonal changes in the song system of a representative tropical-breeding Turdidae species that does not maintain territories or mate bonding, setting the histological and molecular groundwork for future studies aimed at better understanding of song nuclei changes in seasonally breeding songbirds.

Black Jacobin hummingbirds vocalize above the known hearing range of birds.

Hummingbirds are a fascinating group of birds, but some aspects of their biology are poorly understood, such as their highly diverse vocal behaviors. We show here that the predominant vocalization of black jacobins (Florisuga fusca), a hummingbird prevalent in the mountains of the Brazilian Atlantic Forest, consists of a triplet of syllables with high fundamental frequency (mean F0 ∼11.8 kHz), rapid frequency oscillations and strong ultrasonic harmonics and no detectable elements below ∼10 kHz. These are the most common vocalizations of these birds, and their frequency range is above the known hearing range of any bird species recorded to date, including hearing specialists such as owls. These observations suggest that black jacobins either have an atypically high frequency hearing range, or alternatively their primary vocalization has a yet unknown function unrelated to vocal communication. Black jacobin vocalizations challenge current notions about vocal communication in birds.

Digital Colposcopy With Dynamic Spectral Imaging for Detection of Cervical Intraepithelial Neoplasia 2+ in Low-Grade Referrals: The IMPROVE-COLPO Study.

OBJECTIVE: The aim of the study was to determine, in a wide "real-world" setting, whether digital colposcopy with adjunctive dynamic spectral imaging (DSI) mapping increases the detection of women with high-grade cervical intraepithelial neoplasia (CIN). MATERIALS AND METHODS: A multicenter, two-arm, observational, cross-sectional study that recruited women 21 years and older, having colposcopy after a low-grade abnormality screening result. The prospective arm collected outcomes of digital colposcopy with DSI used for identifying biopsy sites at the colposcopists' discretion. The retrospective control arm (number of subjects matched 1:1 per colposcopist) collected outcomes of standard colposcopy. The primary outcome was histopathological detection of women with CIN 2+ by colposcopic biopsy. RESULTS: The study included 1,788 women in the retrospective and 1,857 in the prospective arm from 39 US community-based clinics. Subject characteristics were comparable. A total of 71.6% of the women in the retrospective and 71.5% in the prospective arm underwent biopsy. The average number of biopsies increased from 1.032 (retrospective) to 1.256 (prospective). The yield of CIN 2+ patients was 7.21% in the retrospective and 9.48% in the prospective arm, a 2.27% difference (95% confidence interval = 0.47%-4.07%, p = .014) and 31.4% relative increase. The yield of CIN 3+ patients was 2.07% in the retrospective and 3.23% in the prospective arm, a 1.16% (95% confidence interval = 0.12%-2.24%, p = .031) absolute difference and 56.1% relative increase. The false-positive rates for biopsied patients were comparable (64.43% vs 62.04%, p = .139). CONCLUSIONS: Digital colposcopy with the adjunctive DSI increased CIN 2+ and CIN 3+ detection in low-grade referrals compared with standard colposcopy, with a similar number of women undergoing biopsy.

Noise and diffusion of a vibrated self-propelled granular particle.

Granular materials are an important physical realization of active matter. In vibration-fluidized granular matter, both diffusion and self-propulsion derive from the same collisional forcing, unlike many other active systems where there is a clean separation between the origin of single-particle mobility and the coupling to noise. Here we present experimental studies of single-particle motion in a vibrated granular monolayer, along with theoretical analysis that compares grain motion at short and long time scales to the assumptions and predictions, respectively, of the active Brownian particle (ABP) model. Our results show that despite the unique relation between noise and propulsion, a variety of granular particles are correctly described by the ABP model. Additionally, our scheme of analysis for validating the inputs and outputs of the model can be applied to other granular and non-granular active systems.

Increased detection of precancerous cervical lesions with adjunctive dynamic spectral imaging.

OBJECTIVE: To validate, in US community-based colposcopy clinics, previous reports of increased detection of high-grade cervical intraepithelial neoplasia (CIN2+) with biopsies selected using dynamic spectral imaging (DSI) mapping after standard colposcopy. STUDY DESIGN: Cross-sectional observational study of 26 colposcopists across nine clinics recruiting consecutive colposcopy patients. Standard assessment with biopsy selections was completed before seeing the DSI map which was subsequently interpreted and used for additional biopsies per clinical judgment. Primary measure was the number of women with CIN2+ detected by DSI-assisted biopsies, over those detected by standard colposcopy biopsies. RESULTS: A total of 887 women were recruited. After exclusions, 881 women and 1,189 biopsies were analyzed. Standard biopsy detected 78 women with CIN2+ and DSI-assisted biopsies another 34, increasing the detection rate from 8.85% to 12.71% (p=0.00016). This was achieved with 16.16% of DSI-assisted biopsies finding CIN2+ compared to 13.24% for the preceding standard biopsies. For secondary specificity analysis, 431 women had only

Dynamic gene expression in the song system of zebra finches during the song learning period.

The brain circuitry that controls song learning and production undergoes marked changes in morphology and connectivity during the song learning period in juvenile zebra finches, in parallel to the acquisition, practice and refinement of song. Yet, the genetic programs and timing of regulatory change that establish the neuronal connectivity and plasticity during this critical learning period remain largely undetermined. To address this question, we used in situ hybridization to compare the expression patterns of a set of 30 known robust molecular markers of HVC and/or area X, major telencephalic song nuclei, between adult and juvenile male zebra finches at different ages during development (20, 35, 50 days post-hatch, dph). We found that several of the genes examined undergo substantial changes in expression within HVC or its surrounds, and/or in other song nuclei. They fit into broad patterns of regulation, including those whose expression within HVC during this period increases (COL12A1, COL 21A1, MPZL1, PVALB, and CXCR7) or decreases (e.g., KCNT2, SAP30L), as well as some that show decreased expression in the surrounding tissue with little change within song nuclei (e.g. SV2B, TAC1). These results reveal a broad range of molecular changes that occur in the song system in concert with the song learning period. Some of the genes and pathways identified are potential modulators of the developmental changes associated with the emergence of the adult properties of the song control system, and/or the acquisition of learned vocalizations in songbirds.

Drinking songs: alcohol effects on learned song of zebra finches.

Speech impairment is one of the most intriguing and least understood effects of alcohol on cognitive function, largely due to the lack of data on alcohol effects on vocalizations in the context of an appropriate experimental model organism. Zebra finches, a representative songbird and a premier model for understanding the neurobiology of vocal production and learning, learn song in a manner analogous to how humans learn speech. Here we show that when allowed access, finches readily drink alcohol, increase their blood ethanol concentrations (BEC) significantly, and sing a song with altered acoustic structure. The most pronounced effects were decreased amplitude and increased entropy, the latter likely reflecting a disruption in the birds' ability to maintain the spectral structure of song under alcohol. Furthermore, specific syllables, which have distinct acoustic structures, were differentially influenced by alcohol, likely reflecting a diversity in the neural mechanisms required for their production. Remarkably, these effects on vocalizations occurred without overt effects on general behavioral measures, and importantly, they occurred within a range of BEC that can be considered risky for humans. Our results suggest that the variable effects of alcohol on finch song reflect differential alcohol sensitivity of the brain circuitry elements that control different aspects of song production. They also point to finches as an informative model for understanding how alcohol affects the neuronal circuits that control the production of learned motor behaviors.

Long-distance retinoid signaling in the zebra finch brain.

All-trans retinoic acid (ATRA), the main active metabolite of vitamin A, is a powerful signaling molecule that regulates large-scale morphogenetic processes during vertebrate embryonic development, but is also involved post-natally in regulating neural plasticity and cognition. In songbirds, it plays an important role in the maturation of learned song. The distribution of the ATRA-synthesizing enzyme, zRalDH, and of ATRA receptors (RARs) have been described, but information on the distribution of other components of the retinoid signaling pathway is still lacking. To address this gap, we have determined the expression patterns of two obligatory RAR co-receptors, the retinoid X receptors (RXR) α and γ, and of the three ATRA-degrading cytochromes CYP26A1, CYP26B1, and CYP26C1. We have also studied the distribution of zRalDH protein using immunohistochemistry, and generated a refined map of ATRA localization, using a modified reporter cell assay to examine entire brain sections. Our results show that (1) ATRA is more broadly distributed in the brain than previously predicted by the spatially restricted distribution of zRalDH transcripts. This could be due to long-range transport of zRalDH enzyme between different nuclei of the song system: Experimental lesions of putative zRalDH peptide source regions diminish ATRA-induced transcription in target regions. (2) Four telencephalic song nuclei express different and specific subsets of retinoid-related receptors and could be targets of retinoid regulation; in the case of the lateral magnocellular nucleus of the anterior nidopallium (lMAN), receptor expression is dynamically regulated in a circadian and age-dependent manner. (3) High-order auditory areas exhibit a complex distribution of transcripts representing ATRA synthesizing and degrading enzymes and could also be a target of retinoid signaling. Together, our survey across multiple connected song nuclei and auditory brain regions underscores the prominent role of retinoid signaling in modulating the circuitry that underlies the acquisition and production of learned vocalizations.

Two-photon absorption in CdSe colloidal quantum dots compared to organic molecules.

We discuss fundamental differences in electronic structure as reflected in one- and two-photon absorption spectra of semiconductor quantum dots and organic molecules by performing systematic experimental and theoretical studies of the size-dependent spectra of colloidal quantum dots. Quantum-chemical and effective-mass calculations are used to model the one- and two-photon absorption spectra and compare them with the experimental results. Currently, quantum-chemical calculations are limited to only small-sized quantum dots (nanoclusters) but allow one to study various environmental effects on the optical spectra such as solvation and various surface functionalizations. The effective-mass calculations, on the other hand, are applicable to the larger-sized quantum dots and can, in general, explain the observed trends but are insensitive to solvent and ligand effects. Careful comparison of the experimental and theoretical results allows for quantifying the range of applicability of theoretical methods used in this work. Our study shows that the small clusters can be in principle described in a manner similar to that used for organic molecules. In addition, there are several important factors (quality of passivation, nature of the ligands, and intraband/interband transitions) affecting optical properties of the nanoclusters. The larger-size quantum dots, on the other hand, behave similarly to bulk semiconductors, and can be well described in terms of the effective-mass models.

Proper care, husbandry, and breeding guidelines for the zebra finch, Taeniopygia guttata.

The zebra finch Taeniopygia guttata castanotis is a songbird commonly used in the laboratory, particularly for studies of vocal learning, neurobiology, and physiology. Within the laboratory, it is important to adopt careful husbandry practices that allow for normal development of the birds. For example, their song is a learned trait, passed culturally from adult males to juveniles, and thus its learning can be influenced by the health and social conditions of the birds present in the laboratory. Here we present guidelines for the successful maintenance and breeding of captive zebra finches.

Chemical bonding and magnetic exchange in two-dimensional [M(TCNE)(NCMe)2]X (M = Fe, Mn; X = FeCl4, SbF6) magnets: a pressure study.

Pressure-dependent X-ray diffraction studies reveal the bulk modulus and compression anisotropy of the 2D magnet [Mn(TCNE)(NCMe)(2)]SbF(6). The Raman response of this and the similar [Fe(TCNE)(NCMe)(2)]FeCl(4) layered magnet, shows that the evolution of the a(g) ν(C=C) frequency correlates well with the magnetic exchange and T(c) variations of these materials under pressure. There is a significantly more complex correlation between the a(g) ν(C≡N) frequency and T(c) despite the fact that some unpaired π* electron density (~0.125 e) is localized on each of TCNE nitrile N≡C group. The shortening of the M-NC bond with pressure (<0.5 GPa) does not result in a T(c) increase, which suggests a more complex bond length magnetic exchange relationship.

Spectroscopic study of (two-dimensional) molecule-based magnets: [M(II)(TCNE)(NCMe)2][SbF6] (M = Fe, Mn, Ni).

The M-[TCNE] (M = 3d metal; TCNE = tetracyanoethylene) system is one of the most interesting classes of molecule-based magnets, exhibiting a plethora of compositions and structures (inorganic polymer chains, 2D layers, 3D networks, and amorphous solids) with a wide range of magnetic ordering temperatures (up to 400 K). A systematic study of vibrational (both infrared and, for the first time, Raman) properties of the family of new TCNE-based magnets of M(II)(TCNE) (NCMe)(2)[SbF(6)] [M = Mn, Fe, Ni] composition is discussed in conjunction with their magnetic behavior and newly reso-lved crystal structures. The vibrational properties of the isolated TCNE(●-) anion in the paramagnetic Bu(4)N [TCNE(●-)] salt and recently characterized 2D layered magnet Fe(II)(TCNE)(NCMe)(2)[FeCl(4)] are also reported for comparison. Additionally, a linear correlation between ν(C=C) (a(g)) frequency of the TCNE ligand and its formal charge Z (the spin density on the π* orbital), Z = [1571 - ν(C=C) (a(g))]/154.5 [e], is presented. It is shown that monitoring Z by Raman spectroscopy is of great use in providing information that allows understanding the peculiarity of the superexchange interaction in M-[TCNE] magnets and establishing the structure-magnetic properties correlations in this class of magnetic material.

Singing under the influence: examining the effects of nutrition and addiction on a learned vocal behavior.

The songbird model is widely established in a number of laboratories for the investigation of the neurobiology and development of vocal learning. While vocal learning is rare in the animal kingdom, it is a trait that songbirds share with humans. The neuroanatomical and physiological organization of the brain circuitry that controls learned vocalizations has been extensively characterized, particularly in zebra finches (Taeniopygia guttata). Recently, several powerful molecular and genomic tools have become available in this organism, making it an attractive choice for neurobiologists interested in the neural and genetic basis of a complex learned behavior. Here, we briefly review some of the main features of vocal learning and associated brain structures in zebra finches and comment on some examples that illustrate how themes related to nutrition and addiction can be explored using this model organism.