Multicolour and lineage-specific interphase chromosome Flow-FISH: method development and clinical validation.

Flow cytometry can be applied in the detection of fluorescence in situ hybridisation (FISH) signals to efficiently analyse chromosomal aberrations. However, such interphase chromosome (IC) Flow-FISH protocols are currently limited to detecting a single colour. Furthermore, combining IC Flow-FISH with conventional multicolour flow cytometry is difficult because the DNA-denaturation step in FISH assay also disrupts cellular integrity and protein structures, precluding subsequent antigen-antibody binding and hindering concurrent labeling of surface antigens and FISH signals. We developed a working protocol for concurrent multicolour flow cytometry detection of nuclear IC FISH signals and cell surface markers. The protocol was validated by assaying sex chromosome content of blood cells, which was indicative of chimerism status in patients who had received sex-mismatched allogeneic haematopoietic stem cell transplants (allo-HSCT). The method was also adapted to detect trisomy 12 in chronic lymphocytic leukaemia (CLL) subjects. We first demonstrated the feasibility of this protocol in detecting multiple colours and concurrent nuclear and surface signals with high agreement. In clinical validation experiments, chimerism status was identified in clinical samples (n=56) using the optimised IC Flow-FISH method; the results tightly corresponded to those of conventional slide-based FISH (R2=0.9649 for XX cells and 0.9786 for XY cells). In samples from patients who received sex-mismatched allo-HSCT, individual chimeric statuses in different lineages could be clearly distinguished with high flexibility in gating strategies. Furthermore, in CLL samples with trisomy 12, this method could demonstrate that enriched trisomy 12 FISH signal was present in B cells rather than in T cells. Finally, by performing combined labelling of chromosome 12, X chromosome, and surface markers, we could detect rare residual recipient CLL cells with trisomy 12 after allo-HSCT. This adaptable protocol for multicolour and lineage-specific IC Flow-FISH advances the technique to allow for its potential application in various clinical contexts where conventional FISH assays are currently being utilised.

Unusual body weight loss due to primary hyperparathyroidism: A case study with literature review.

Brown tumors (osteitis fibrosa cystica) are rare pathognomonic signs that occur in patients with primary hyperparathyroidism (PHPT). Brown tumors can exist in multiple bones and can easily be misdiagnosed as a metastatic tumor or multiple myeloma. It is also localized in the forearm, humerus, and leg. The symptoms of hypercalcemia, pathologic fracture, and bodyweight loss may increase the diagnostic difficulty of brown tumors because multiple myeloma and bone metastasis also show the same symptoms. We studied a 68-year-old woman who had experienced unusual bodyweight loss in the past 6 months (56kg-40kg) and bone pain. She went to the hospital after a fall with a complaint of bone pain. An X-ray revealed a left bubbly-like cystic change and multiple fractures at the left ulna midshaft. Upon investigation, the level of intact parathyroid hormone was ascertained to be 1800 (normal: 10-60) pg/ml. Microscopically, the tumor demonstrated a benign bone lesion and was compatible with osteitis fibrosa cystica due to PHPT. The parathyroid scan (Tc-99 m sestamibi) indicated right parathyroid hyperplasia, which was later confirmed by a parathyroidectomy. She was diagnosed with osteitis fibrosa cystica associated with PHPT due to a parathyroid adenoma. PHPT can be presented with multiple fractures, bone pain, and bodyweight loss. Therefore, if a patient presents these symptoms, PHPT should be considered.

Nanoparticles with intermediate hydrophobicity polarize macrophages to plaque-specific Mox phenotype via Nrf2 and HO-1 activation.

Mox macrophages were identified recently and are closely associated with atherosclerosis. Considering the potential health risks and the impact on macrophage modulation, this study investigated the Mox polarization of macrophages induced by nanoparticles (NPs) with tunable hydrophobicity. One nanoparticle (C4NP) with intermediate hydrophobicity efficiently upregulated the mRNA expression of Mox-related genes including HO-1, Srxn1, Txnrd1, Gsr, Vegf and Cox-2 through increased accumulation of Nrf2 at a nontoxic concentration in both resting and LPS-challenged macrophages. Additionally, C4NP impaired phagocytic capacity by 20% and significantly increased the secretion of cytokines, including TNFα, IL-6 and IL-10. Mechanistic studies indicated that intracellular reactive oxygen species (ROS) were elevated by 1.5-fold and 2.6-fold in resting and LPS-challenged macrophages respectively. Phosphorylated p62 was increased by 2.5-fold in resting macrophages and maintained a high level in LPS-challenged ones, both of which partially accounted for the significant accumulation of Nrf2 and HO-1. Notably, C4NP depolarized mitochondrial membrane potential by more than 50% and switched macrophages from oxidative phosphorylation-based aerobic metabolism to glycolysis for energy supply. Overall, this study reveals a novel molecular mechanism potentially involving ROS-Nrf2-p62 signaling in mediating macrophage Mox polarization, holding promise in ensuring safer and more efficient use of nanomaterials.

Elevated Free Thyroxine Levels Might Alter the Effect of the Lipid Profile on Insulin Resistance in Type 2 Diabetes Mellitus.

Previous studies have shown that hyperthyroidism is associated with heightened insulin resistance and dyslipidemia. Therefore, in this study, we aim to explore the relationship between elevated thyroid hormone levels and the lipid profile in insulin resistance in patients with type 2 diabetes mellitus (T2DM) with hyperthyroidism. A total of 177 participants were included and grouped according to diagnosis. The serum test results demonstrated that free thyroxine (FT4) increased the insulin resistance index (HOMA-IR) by positively correlating with triglyceride (TG) levels (p = 0.005, r2 = 0.35). In patients with T2DM with hyperthyroidism, the decreasing high-density lipoprotein levels showed an association with HOMA-IR (p = 0.005). Among all the patients, with different levels of FT4, the areas under the ROC curve (AUCs) of the TG level, TG/high-density lipoprotein ratio, and HOMA-IR were 0.620 (95% CI: 0.536 to 0.698), 0.614 (95% CI: 0.530 to 0.692), and 0.722 (95% CI: 0.645 to 0.791), respectively. Our results suggest that elevated FT4 levels due to hyperthyroidism could alter the association with the lipid profile and insulin resistance in patients with T2DM. We also suggest that among all the included patients with T2DM, irrespective of the presence of hyperthyroidism, FT4 levels are positively correlated with insulin resistance.

Non-viral vaccination through cationic guanidium polymer-pDNA polyplex mediated gene transfer.

Vaccination through cellular transfection of nucleotide-based vaccines is a powerful approach to combatting disease. Plasmid DNA (pDNA) vaccines are particularly promising vectors for non-viral immunomodulation that afford high degrees of potency and flexibility. Versatile guanidinium-functionalized poly(oxanorbornene)imide (PONI-Guan) homopolymers were used to facilitate non-disruptive pDNA condensation into discrete polyplexes, enabling efficient in vitro transfection of endothelial cells and HD-11 macrophages. Translation of these vectors for vaccination of white leghorn chickens against Newcastle disease virus (NDV) elicited strong humoral immune responses against the virus. This approach presents a highly versatile method for targeted immunomodulation in vivo, with the potential for translatability as a non-viral vaccine platform.

Engineered Polymer-siRNA Polyplexes Provide Effective Treatment of Lung Inflammation.

Uncontrolled inflammation is responsible for acute and chronic diseases in the lung. Regulating expression of pro-inflammatory genes in pulmonary tissue using small interfering RNA (siRNA) is a promising approach to combatting respiratory diseases. However, siRNA therapeutics are generally hindered at the cellular level by endosomal entrapment of delivered cargo and at the organismal level by inefficient localization in pulmonary tissue. Here we report efficient anti-inflammatory activity in vitro and in vivo using polyplexes of siRNA and an engineered cationic polymer (PONI-Guan). PONI-Guan/siRNA polyplexes efficiently deliver siRNA cargo to the cytosol for highly efficient gene knockdown. Significantly, these polyplexes exhibit inherent targeting to inflamed lung tissue following intravenous administration in vivo. This strategy achieved effective (>70%) knockdown of gene expression in vitro and efficient (>80%) silencing of TNF-α expression in lipopolysaccharide (LPS)-challenged mice using a low (0.28 mg/kg) siRNA dosage.

Porous Polymerized High Internal Phase Emulsions Prepared Using Proteins and Essential Oils for Antimicrobial Applications.

High internal phase emulsions (HIPEs) provide a versatile platform for encapsulating large volumes of therapeutics that are immiscible in water. A stable scaffold is obtained by polymerizing the external phase, resulting in polyHIPEs. However, fabrication of polyHIPEs usually requires using a considerable quantity of surfactants along with nonbiocompatible components, which hinders their biological applications, e.g., drug-eluting devices. We describe here a straightforward method for generating porous biomaterials by using proteins as both the emulsifier and the building blocks for the fabrication of polyHIPEs. We demonstrate the versatility of this method by using different essential oils as the internal phase. After the gelation of protein building blocks is triggered by the addition of reducing agents, a stable protein hydrogel containing essential oils can be formed. These oils can be either extracted to obtain protein-based porous scaffolds or slowly released for antimicrobial applications.

Probiotic Lactiplantibacillus plantarum Tana Isolated from an International Weightlifter Enhances Exercise Performance and Promotes Antifatigue Effects in Mice.

Exercise causes changes in the gut microbiota, and in turn, the composition of the gut microbiota affects exercise performance. In addition, the supplementation of probiotics is one of the most direct ways to change the gut microbiota. In recent years, the development and application of human-origin probiotics has gradually attracted attention. Therefore, we obtained intestinal Lactiplantibacillus plantarum "Tana" from a gold-medal-winning weightlifter, who has taken part in various international competitions such as the World Championships and the Olympic Games, to investigate the benefits of Tana supplementation for improving exercise performance and promoting antifatigue effects in mice. A total of 40 male Institute of Cancer Research (ICR) mice were divided into four groups (10 mice/group): (1) vehicle (0 CFU/mice/day), (2) Tana-1× (6.15 × 107 CFU/mice/day), (3) Tana-2× (1.23 × 108 CFU /mice/day), and (4) Tana-5× (3.09 × 108 CFU/mice/day). After four weeks of Tana supplementation, we found that the grip strength, endurance exercise performance, and glycogen storage in the liver and muscle were significantly improved compared to those in the vehicle group (p < 0.05). In addition, supplementation with Tana had significant effects on fatigue-related biochemical markers; lactate, ammonia, and blood urea nitrogen (BUN) levels and creatine kinase (CK) activity were significantly lowered (p < 0.05). We also found that the improved exercise performance and antifatigue benefits were significantly dose-dependent on increasing doses of Tana supplementation (p < 0.05), which increased the abundance and ratio of beneficial bacteria in the gut. Taken together, Tana supplementation for four weeks was effective in improving the gut microbiota, thereby enhancing exercise performance, and had antifatigue effects. Furthermore, supplementation did not cause any physiological or histopathological damage.

Clinical significance of hepatic function in Graves disease with type 2 diabetic mellitus: A single-center retrospective cross-sectional study in Taiwan.

Graves disease (GD) and type 2 diabetes mellitus (T2DM) both impair liver function; we therefore explored the possibility of a relationship among diabetic control, thyroid function, and liver function. This retrospective, cross-sectional study compared serum liver function biomarkers of primary GD patients in a single center between 2016 and 2020, derived from clinical databases, and clarified the correlation of liver function in GD patients with or without T2DM. Furthermore, the diabetes mellitus group was divided into glycated hemoglobin A1C (HbA1C) <6.5% group and ≥6.5% group to further analyze the effect by disease control in patients. Statistical differences between groups were assessed using independent t tests to clarify the association of serum biomarkers between GD with T2DM. Pearson test was applied to assess within-group statistical correlation of serum biomarkers. The correlation of factors in each group was demonstrated by using the Kendall tau-b method and stepwise regression analysis. A total of 77 patients were included in the study. In the study population, glutamate pyruvate transaminase (GPT) was significantly correlated with thyroid-stimulating hormone, and HbA1C was significantly correlated with alkaline phosphatase (ALK-P), glutamate oxaloacetate transaminase (GOT), and GPT. An examination of GOT, GPT, free thyroxine (FT4), and HbA1C levels revealed a significant difference between the non-T2DM and T2DM groups. GPT also exhibited a significant correlation with triiodothyronine in the T2DM group. The T2DM group was further divided into groups: HbA1C <6.5% and ≥6.5%. The results demonstrated that ALK-P, GOT, GPT, and FT4 levels were significantly different between the groups. A significant correlation between ALK-P and thyroid-stimulating hormone and between GOT and FT4 was also identified in the HbA1C <6.5% group. Our single-center study revealed that diabetes affects liver function in patients with GD. For patients with T2DM, when liver function becomes impaired, thyroid function control deteriorates. GPT was correlated with triiodothyronine but not with FT4, which indicated the impairment of deiodination in the liver. This phenomenon was not observed in the non-T2DM population. The early detection of abnormal liver function in patients with GD and T2DM may help limit the development of comorbidities and improve disease management.

Cytosolic Protein Delivery Using Modular Biotin-Streptavidin Assembly of Nanocomposites.

Current strategies for the delivery of proteins into cells face general challenges of endosomal entrapment and concomitant degradation of protein cargo. Efficient delivery directly to the cytosol overcomes this obstacle: we report here the use of biotin-streptavidin tethering to provide a modular approach to the generation of nanovectors capable of a cytosolic delivery of biotinylated proteins. This strategy uses streptavidin to organize biotinylated protein and biotinylated oligo(glutamate) peptide into modular complexes that are then electrostatically self-assembled with a cationic guanidinium-functionalized polymer. The resulting polymer-protein nanocomposites demonstrate efficient cytosolic delivery of six biotinylated protein cargos of varying size, charge, and quaternary structure. Retention of protein function was established through efficient cell killing via delivery of the chemotherapeutic enzyme granzyme A. This platform represents a versatile and modular approach to intracellular delivery through the noncovalent tethering of multiple components into a single delivery vector.

Phlebosclerotic colitis: an analysis of clinical and CT findings in 29 patients with long-term follow-up.

BACKGROUND: Phlebosclerotic colitis (PC) is a rare form of nonthrombotic colonic ischemia. This retrospective study analyzed the clinical findings and temporal CT changes in 29 PC patients with long-term follow-up. METHODS: Twenty-nine patients with characteristic CT features of PC collected between 1997 and 2020 were stratified into the acute abdomen group (AA-group) (n = 10), chronic-progressive group (CP-group) (n = 14) and chronic-stable group (CS-group) (n = 5). Clinical and CT changes during follow-up, comorbidities and final outcomes were compared. RESULTS: The AA-group exhibited a significantly thicker colonic wall and more involved segments and pericolic inflammation than the CP-group and CS-group on initial CT (p = < 0.001-0.031). Seven patients in the AA-group who underwent right hemicolectomy had no recurrence during follow-up (mean ± SD, 7.1 ± 3.3 years), and the remaining three patients with renal or hepatic comorbidities who underwent conservative treatment died within 14 days. The CP-group showed significantly higher frequencies of chronic renal failure, urinary tract malignancies and liver cirrhosis than the AA-group (p = 0.005-0.008). In addition, CT follow-up (7.9 ± 4.3 years) showed significant increases in mesenteric venous calcifications, colonic wall thickening and involved colonic segments (p = 0.001-0.008) but conservative treatments were effective. The CS-group remained unchanged for years (8.2 ± 3.9 years). CONCLUSIONS: Early surgery offered excellent prognosis in PC-related acute abdomen denoted by marked right colonic wall thickening and pericolic inflammation on CT. Conservative treatments with a wait-and-watch strategy were appropriate for CP-PC and CS-PC, albeit CP-PC harbored significant increases in calcifications, colonic wall thickening and affected segments in long-term CT follow-up.

Supramolecular arrangement of protein in nanoparticle structures predicts nanoparticle tropism for neutrophils in acute lung inflammation.

This study shows that the supramolecular arrangement of proteins in nanoparticle structures predicts nanoparticle accumulation in neutrophils in acute lung inflammation (ALI). We observed homing to inflamed lungs for a variety of nanoparticles with agglutinated protein (NAPs), defined by arrangement of protein in or on the nanoparticles via hydrophobic interactions, crosslinking and electrostatic interactions. Nanoparticles with symmetric protein arrangement (for example, viral capsids) had no selectivity for inflamed lungs. Flow cytometry and immunohistochemistry showed NAPs have tropism for pulmonary neutrophils. Protein-conjugated liposomes were engineered to recapitulate NAP tropism for pulmonary neutrophils. NAP uptake in neutrophils was shown to depend on complement opsonization. We demonstrate diagnostic imaging of ALI with NAPs; show NAP tropism for inflamed human donor lungs; and show that NAPs can remediate pulmonary oedema in ALI. This work demonstrates that structure-dependent tropism for neutrophils drives NAPs to inflamed lungs and shows NAPs can detect and treat ALI.

CT characteristics and clinical implications of acute type A aortic intramural hematoma.

Objectives: Computed tomography (CT) has been increasingly used in the diagnosis of acute aortic syndrome, and a number of high-risk CT imaging features have been reported. We aimed to identify CT imaging findings suggesting high-risk for acute aortic syndrome by examining clinical outcomes of patients with acute type A aortic intramural hematoma (TAIMH). Methods: This retrospective study analyzed the relationship of clinical patient characteristics and imaging features with mortality and aortic events in 63 patients receiving initial medical treatment for TAIMH. Multivariate regression analysis was used to determine the predictors of aortic events, and the Kaplan-Meier method was used to analyze survival and aortic events. Results: During a median follow-up of 4.2 years, 25 patients experienced aortic events and 40% of these occurred within 7 days of admission. In total, 12 patients experienced aortic death and 12 patients underwent open aortic surgery or endovascular stenting for aortic disease. In multivariate regression analysis, penetrating atherosclerotic ulcers (PAUs) or ulcer-like projections (ULPs) (P = 0.04) and pericardial effusion (P = 0.03) were independent predictors of aortic events. In the Cox regression model, PAUs/ULPs (P = 0.04) and pericardial effusion (P = 0.04) were independently associated with lower aortic event-free survival. Conclusion: Identification of high-risk CT features is important for clinical decision-making during TAIMH treatment. Early and frequent CT imaging follow-up is required in patients receiving medical treatment. PAUs/ULP and pericardial effusion were the strongest predictors of adverse aortic events.

lncExplore: a database of pan-cancer analysis and systematic functional annotation for lncRNAs from RNA-sequencing data.

Over the past few years, with the rapid growth of deep-sequencing technology and the development of computational prediction algorithms, a large number of long non-coding RNAs (lncRNAs) have been identified in various types of human cancers. Therefore, it has become critical to determine how to properly annotate the potential function of lncRNAs from RNA-sequencing (RNA-seq) data and arrange the robust information and analysis into a useful system readily accessible by biological and clinical researchers. In order to produce a collective interpretation of lncRNA functions, it is necessary to integrate different types of data regarding the important functional diversity and regulatory role of these lncRNAs. In this study, we utilized transcriptomic sequencing data to systematically observe and identify lncRNAs and their potential functions from 5034 The Cancer Genome Atlas RNA-seq datasets covering 24 cancers. Then, we constructed the 'lncExplore' database that was developed to comprehensively integrate various types of genomic annotation data for collective interpretation. The distinctive features in our lncExplore database include (i) novel lncRNAs verified by both coding potential and translation efficiency score, (ii) pan-cancer analysis for studying the significantly aberrant expression across 24 human cancers, (iii) genomic annotation of lncRNAs, such as cis-regulatory information and gene ontology, (iv) observation of the regulatory roles as enhancer RNAs and competing endogenous RNAs and (v) the findings of the potential lncRNA biomarkers for the user-interested cancers by integrating clinical information and disease specificity score. The lncExplore database is to our knowledge the first public lncRNA annotation database providing cancer-specific lncRNA expression profiles for not only known but also novel lncRNAs, enhancer RNAs annotation and clinical analysis based on pan-cancer analysis. lncExplore provides a more complete pathway to highly efficient, novel and more comprehensive translation of laboratory discoveries into the clinical context and will assist in reinterpreting the biological regulatory function of lncRNAs in cancer research. Database URL http://lncexplore.bmi.nycu.edu.tw.

Mucormycosis causing massive lower gastrointestinal bleeding: a case report.

BACKGROUND: Lower gastrointestinal bleeding (LGIB) is very common in the hospital setting. Most bleedings stop spontaneously, but rare infectious causes of LGIB may lead to rapid and serious complications if left untreated and are sometimes very difficult to diagnose preoperatively. CASE PRESENTATION: We described a young man with poorly controlled Type I diabetes mellitus and chronic alcohol abuse who presented with acute altered mental status. During his hospitalization for treatment of diabetic ketoacidosis, acute renal failure, and sepsis, he suddenly developed massive hematochezia of 1500 mL. Colonoscopy was performed and a deep ulcer covered with mucus with peripheral elevation was noted at the transverse colon. Biopsy of the ulcer later revealed nonpigmented, wide (5-20 µm in diameter), thin-walled, ribbon-like hyphae with few septations and right-angle branching suggestive of mucormycosis demonstrated by Periodic acid-Schiff stain. He received 2 months of antifungal treatment. Follow up colonoscopy post-treatment was normal with no ulcer visualized. CONCLUSIONS: Early diagnosis and treatment of gastrointestinal (GI) mucormycosis infection is critical but can be challenging, especially in the setting of massive hematochezia. Therefore, clinical awareness for immunocompromised patients and prompt antifungal prophylaxis in cases with high suspicion of infection are essential.

Protein Delivery: If Your GFP (or Other Small Protein) Is in the Cytosol, It Will Also Be in the Nucleus.

Intracellular protein delivery is a transformative tool for biologics research and medicine. Delivery into the cytosol allows proteins to diffuse throughout the cell and access subcellular organelles. Inefficient delivery caused by endosomal entrapment is often misidentified as cytosolic delivery. This inaccuracy muddles what should be a key checkpoint in assessing delivery efficiency. Green fluorescent protein (GFP) is a robust cargo small enough to passively diffuse from the cytosol into the nucleus. Fluorescence of GFP in the nucleus is a direct readout for cytosolic access and effective delivery. Here, we highlight recent examples from the literature for the accurate assessment of cytosolic protein delivery using GFP fluorescence in the cytosol and nucleus.

Regulation of Proteins to the Cytosol Using Delivery Systems with Engineered Polymer Architecture.

Intracellular protein delivery enables selective regulation of cellular metabolism, signaling, and development through introduction of defined protein quantities into the cell. Most applications require that the delivered protein has access to the cytosol, either for protein activity or as a gateway to other organelles such as the nucleus. The vast majority of delivery vehicles employ an endosomal pathway however, and efficient release of entrapped protein cargo from the endosome remains a challenge. Recent research has made significant advances toward efficient cytosolic delivery of proteins using polymers, but the influence of polymer architecture on protein delivery is yet to be investigated. Here, we developed a family of dendronized polymers that enable systematic alterations of charge density and structure. We demonstrate that while modulation of surface functionality has a significant effect on overall delivery efficiency, the endosomal release rate can be highly regulated by manipulating polymer architecture. Notably, we show that large, multivalent structures cause slower sustained release, while rigid spherical structures result in rapid burst release.