High-Fidelity Assay Based on Turn-Off Fluorescence to Detect the Perturbations of Cellular Proteostasis.

The persistence of neurodegenerative diseases has necessitated the development of new strategies to monitor protein homeostasis (proteostasis). Previous efforts in our laboratory have focused on the development of fluorogenic strategies to observe the onset and progression of proteostatic stress. These works utilized solvatochromic and viscosity sensitive fluorophores to sense protein folded states, enabling stressor screening with an increase in the emission intensity upon aggregation. In this work, we present a novel, high-fidelity assay to detect perturbations of cellular proteostasis, where the fluorescence intensity decreases with the onset of proteostatic stress. Utilizing a fluorogenic, hydroxymethyl silicon-rhodamine probe to differentiate between protein folded states, we establish the validity of this technology in living cells by demonstrating a two-fold difference in fluorescence intensity between unstressed and stressed conditions.

Characterization of Transthyretin Mutation G47V Associated with Hereditary Cardiac Amyloidosis.

INTRODUCTION: Amyloidosis caused by TTR mutations (ATTRv) is a rare inherited and autosomal dominant disease. More than 150 mutants of TTR have been reported, whereas some of them remain to be investigated. METHODS: A 52-year-old male presented with heart failure and clinically diagnosed ATTR cardiac amyloidosis (ATTR-CA) was recruited. Whole exome sequencing (WES) was performed. Biochemical and biophysical experiments characterized protein stability using urea-mediated tryptophan fluorescence. Drug response was analyzed by fibril formation assay. Finally, tetramer TTR concentration in patient' serum sample was measured by ultra-performance liquid chromatography (UPLC). RESULTS: For the proband, whole exome sequencing revealed a mutation (c.200G>T; p.Gly67Val and referred to as G47V) in TTR gene. Biochemical and biophysical kinetics study showed that the thermodynamic stability of G47V-TTR (Cm = 2.4 M) was significantly lower than that of WT-TTR (Cm = 3.4 M) and comparable to that of L55P-TTR (Cm = 2.3 M), an early age-of-onset mutation. G47V:WT-TTR heterozygous tetramers kinetic stability (t1/2 = 1.4 h) was further compromised compared to that of the homozygous G47V-TTR (t1/2 = 3.1 h). Among three small molecule stabilizers, AG10 exhibited the best inhibition of the fibrillation of G47V-TTR homozygous protein. Using a UPLC assay, nearly 40% of TTR in this patient was calculated to be non-tetrameric. CONCLUSION: In this work, we reported a patient presented early onset of clinically typical ATTR-CM due to G47V-TTR mutation. Our work not only for the first time characterized the biochemical properties of G47V-TTR mutation, but also provided hints for the pathogenicity of this mutation.

An exposome atlas of serum reveals the risk of chronic diseases in the Chinese population.

Although adverse environmental exposures are considered a major cause of chronic diseases, current studies provide limited information on real-world chemical exposures and related risks. For this study, we collected serum samples from 5696 healthy people and patients, including those with 12 chronic diseases, in China and completed serum biomonitoring including 267 chemicals via gas and liquid chromatography-tandem mass spectrometry. Seventy-four highly frequently detected exposures were used for exposure characterization and risk analysis. The results show that region is the most critical factor influencing human exposure levels, followed by age. Organochlorine pesticides and perfluoroalkyl substances are associated with multiple chronic diseases, and some of them exceed safe ranges. Multi-exposure models reveal significant risk effects of exposure on hyperlipidemia, metabolic syndrome and hyperuricemia. Overall, this study provides a comprehensive human serum exposome atlas and disease risk information, which can guide subsequent in-depth cause-and-effect studies between environmental exposures and human health.

Theabrownins in dark tea form complexes with tea polysaccharide conjugates.

BACKGROUND: Theabrownins (TBs) are one of most important quality components in dark tea, but have not been produced industrially. In this study, the aqueous extract was obtained from Pu-erh ripe tea, one kind of dark tea. Caffeine, theaflavin, catechin and saponin were removed by trichloromethane, ethyl acetate and n-butanol in turn to obtain a TB isolate. The TB isolate was subjected to column chromatography using a macroporous resin HPD-750 and eluted with a gradient of 0-700 g kg-1 ethanol aqueous solution. Four fractions were obtained, and named as TBs-FC1, TBs-FC2, TBs-FC3 and TBs-FC4. RESULTS: These four fractions contained polysaccharides and no small molecules such as catechins, caffeine and theaflavins as well as average molecular weights of 123.000 kDa, 23.380 kDa, 89.870 kDa and 106.600 kDa. It was revealed that they were complexes of TBs and tea polysaccharide conjugates (TPCs). Ultraviolet-visible (UV-visible) and infrared (IR) spectra showed the properties of TBs and TPCs. Their zeta potentials ranged from -13.40 mV to -38.80 mV in aqueous solutions at pH 3.0-9.0. CONCLUSION: This study reveals that TBs do not exist in free state but in combined state in dark tea, which provide the theoretical basis for the industrialization of TBs. © 2024 Society of Chemical Industry.

Time-Resolved Ultraviolet Photodissociation Mass Spectrometry Probes the Mutation-Induced Alterations in Protein Stability and Unfolding Dynamics.

How mutations impact protein stability and structure dynamics is crucial for understanding the pathological process and rational drug design. Herein, we establish a time-resolved native mass spectrometry (TR-nMS) platform via a rapid-mixing capillary apparatus for monitoring the acid-initiated protein unfolding process. The molecular details in protein structure unfolding are further profiled by a 193 nm ultraviolet photodissociation (UVPD) analysis of the structure-informative photofragments. Compared with the wild-type dihydrofolate reductase (WT-DHFR), the M42T/H114R mutant (MT-DHFR) exhibits a significant stability decrease in TR-nMS characterization. UVPD comparisons of the unfolding intermediates and original DHFR forms indicate the special stabilization effect of cofactor NADPH on DHFR structure, and the M42T/H114R mutations lead to a significant decrease in NADPH-DHFR interactions, thus promoting the structure unfolding. Our study paves the way for probing the mutation-induced subtle changes in the stability and structure dynamics of drug targets.

Suberoylanilide hydroxamic acid attenuates cognitive impairment in offspring caused by maternal surgery during mid-pregnancy.

Some pregnant women have to experience non-obstetric surgery during pregnancy under general anesthesia. Our previous studies showed that maternal exposure to sevoflurane, isoflurane, propofol, and ketamine causes cognitive deficits in offspring. Histone acetylation has been implicated in synaptic plasticity. Propofol is commonly used in non-obstetric procedures on pregnant women. Previous studies in our laboratory showed that maternal propofol exposure in pregnancy impairs learning and memory in offspring by disturbing histone acetylation. The present study aims to investigate whether HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) could attenuate learning and memory deficits in offspring caused by maternal surgery under propofol anesthesia during mid-pregnancy. Maternal rats were exposed to propofol or underwent abdominal surgery under propofol anesthesia during middle pregnancy. The learning and memory abilities of the offspring rats were assessed using the Morris water maze (MWM) test. The protein levels of histone deacetylase 2 (HDAC2), phosphorylated cAMP response-element binding (p-CREB), brain-derived neurotrophic factor (BDNF), and phosphorylated tyrosine kinase B (p-TrkB) in the hippocampus of the offspring rats were evaluated by immunofluorescence staining and western blot. Hippocampal neuroapoptosis was detected by TUNEL staining. Our results showed that maternal propofol exposure during middle pregnancy impaired the water-maze learning and memory of the offspring rats, increased the protein level of HDAC2 and reduced the protein levels of p-CREB, BDNF and p-TrkB in the hippocampus of the offspring, and such effects were exacerbated by surgery. SAHA alleviated the cognitive dysfunction and rescued the changes in the protein levels of p-CREB, BDNF and p-TrkB induced by maternal propofol exposure alone or maternal propofol exposure plus surgery. Therefore, SAHA could be a potential and promising agent for treating the learning and memory deficits in offspring caused by maternal nonobstetric surgery under propofol anesthesia.

Unraveling Intracellular Protein Corona Components of Nanoplastics via Photocatalytic Protein Proximity Labeling.

Bioaccumulation of nanoplastic particles has drawn increasing attention regarding environmental sustainability and biosafety. How nanoplastic particles interact with the cellular milieu still remains elusive. Herein, we exemplify a general approach to profile the composition of a "protein corona" interacting with nanoparticles via the photocatalytic protein proximity labeling method. To enable photocatalytic proximity labeling of the proteome interacting with particles, iodine-substituted BODIPY (I-BODIPY) is selected as the photosensitizer and covalently conjugated onto amino-polystyrene nanoparticles as a model system. Next, selective proximity labeling of interacting proteins is demonstrated using I-BODIPY-labeled nanoplastic particles in both Escherichia coli lysate and live alpha mouse liver 12 cells. Mechanistic studies reveal that the covalent modifications of proteins by an aminoalkyne substrate are conducted via a reactive oxygen species photosensitization pathway. Further proteomic analysis uncovers that mitochondria-related proteins are intensively involved in the protein corona, indicating substantial interactions between nanoplastic particles and mitochondria. In addition, proteostasis network components are also identified, accompanied by consequent cellular proteome aggregation confirmed by fluorescence imaging. Together, this work exemplifies a general strategy to interrogate the composition of the protein corona of nanomaterials by endowing them with photooxidation properties to enable photocatalytic protein proximity labeling function.

Abdominal surgery under ketamine anesthesia during second trimester impairs hippocampal learning and memory of offspring by regulating dendrite spine remodeling in rats.

Recent evidence showed that general anesthesia produces long-term neurotoxicity and cognitive dysfunction. However, it remains unclear whether maternal non-obstetric surgery under ketamine anesthesia during second trimester causes cognitive impairment in offspring. The present study assigned pregnant rats into three groups: 1) normal control group receiving no anesthesia and no surgery, 2) ketamine group receiving ketamine anesthesia for 2 h on the 14th day of gestation but no surgery, and 3) surgery group receiving abdominal surgery under ketamine anesthesia on the 14th day of gestation. On postnatal day 1, the offspring rats in Ketamine group and surgery group were assigned to receive intra-peritoneal injection of Senegenin (15 mg/kg), once per day for consecutive 14 days. The offspring's spatial perception, anxiety-like behavior, and learning and memory were evaluated. Then the offspring's hippocampal tissues were collected. The offspring of the surgery group were impaired in the spatial perception in the cliff avoidance test and the spatial learning and memory in the Morris water maze test. Accordingly, the activity of histone deacetylases increased, the protein levels of NEDD9, BDNF, p-TrkB, Syn and PSD-95 decreased, and the density of dendritic spines reduced in the hippocampus of the offspring of the surgery group, and such effects were not seen in the offspring of the ketamine group, neither in the offspring of control group. Senegenin alleviated the learning and memory impairment, and increased the protein levels of NEDD9, BDNF, p-TrkB, Syn and PSD-95 and the density of dendritic spines in the offspring of the surgery group. ketamine anesthesia plus surgery during second trimester impairs hippocampus-dependent learning and memory, and the deficits could be rescued by treatment with Senegenin.

Biochemical and biophysical properties of a rare TTRA81V mutation causing mild transthyretin amyloid cardiomyopathy.

AIMS: We conducted a presentation on an 84-year-old male patient who has been diagnosed with TTRA81V (p. TTRA101V) hereditary transthyretin cardiac amyloidosis (hATTR-CM). In order to establish its pathogenicity, we extensively investigated the biochemical and biophysical properties of the condition. METHODS AND RESULTS: Transthyretin amyloid cardiomyopathy (ATTR-CM) is an increasingly acknowledged progressive infiltrative cardiomyopathy that leads to heart failure and potentially fatal arrhythmias. Gaining a comprehensive understanding of the biochemical and biophysical characteristics of genetically mutated TTR proteins serves as the fundamental cornerstone for delivering precise medical care to individuals affected by ATTR. Laboratory assessments indicated a brain natriuretic peptide of 200.12 ng/L (normal range: 0-100 ng/L) and high-sensitivity cardiac troponin I of 0.189 µg/L (normal range: 0-0.1 µg/L). Echocardiography identified left atrial enlargement, symmetrical left ventricular hypertrophy (16 mm septal and 16 mm posterior wall), and a left ventricular ejection fraction of 56%. Cardiac-enhanced magnetic resonance imaging revealed subendocardial late gadolinium enhancement. Tc-99m-PYP nuclear scintigraphy confirmed grade 3 myocardial uptake, showing an increased heart-to-contralateral ratio (H/CL = 2.33). Genetic testing revealed a heterozygous missense mutation in the TTR gene (c.302C>T), resulting in an alanine-to-valine residue change (p. Ala81Val, following the first 20 residues of signal sequence nomenclature). Biochemical analysis of this variant displayed compromised kinetic stability in both the TTRA81V:WT (wild-type) heterozygote protein (half-life, t1/2  = 21 h) and the TTRA81V homozygote protein (t1/2  = 17.5 h). The kinetic stability fell between that of the TTRWT (t1/2  = 42 h) and the early-onset TTRL55P mutation (t1/2  = 4.4 h), indicating the patient's late-onset condition. Kinetic stabilizers (Tafamidis, Diflunisal, and AG10) all exhibited the capacity to inhibit TTRA81V acid- and mechanical force-induced fibril formation, albeit less effectively than with TTRWT. Chromatographic assessment of the patient's serum TTR tetramers indicated a slightly lower concentration (3.0 µM) before oral administration of Tafamidis compared with the normal range (3.6-7.2 µM). CONCLUSIONS: We identified a patient with hATTR-CM who possesses a rare TTRA81V mutation solely associated with cardiac complications. The slightly reduced kinetic stability of this mutation indicates its late-onset nature and contributes to the gradual progression of the disease.

An ultra performance liquid chromatography method for transthyretin variants screening and heart failure assisting diagnosis.

BACKGROUND: Transthyretin (TTR) gene mutations are associated with hereditary amyloidosis (ATTR) caused by mutant TTR protein dissociation, misfolding, aggregation, and insoluble fibrils deposition. Herein, we reported a chromatographic approach for quantification and identification of TTR tetramer in human blood serum by ultra performance liquid chromatography (UPLC). METHODS: TTR proteins and serum were incubated with a fluorescent TTR tetramer sensor (A2). The A2 sensor specifically reacted with tetrameric TTR and released stoichiometric fluorescence that was detected by fluorescence detector coupled to UPLC. The external standard was used for quantification, the chromatographic peak parameters were used to identification certain mutation types. RESULTS: UPLC correctly distinguished 18 types of mutant TTR proteins from wild type. The results were consistent with follow-up analysis of two ATTR patients' blood serum samples. In addition, the tetrameric TTR of 30 heart failure (HF) patients showed strongly correlation (r = -0.63, p < 0.00) with NT-proBNP, a HF clinical biomarker. CONCLUSIONS: UPLC method has sufficient accuracy to eliminate the necessity of sequencing for certain types of TTR mutations and allows for facile initial screening of ATTR amyloidosis patients, carriers, and healthy individuals for time-saving and economical purposes. TTR tetramer may serve as a diagnostic biomarker to evaluate the risk of HF diseases.

Effects of after school exercise service on physical fitness in primary school students / 中国学校卫生

Objective@#To investigate the effects of after school exercise service (referred to as the "after school ES") on physical health, so as to provide evidence for possible beneficial effect of after school ES.@*Methods@#Students in the fourth grade of primary school were recruited from September 2021 to July 2022 in Changsha City and divided into the control group ( n =220) and the after school ES group ( n =220). The control group only participated in the regular physical education activities of the school. The after school ES group received after school ES for one academic year, 4 times a week, 40-50 minutes per time, for a total of 32 weeks. Body shape indicators such as height, weight and percentage of body fat, as well as physical fitness indicators such as 50 meter running, grip strength and progressive aerobic cardiovascular endurance run (PACER) were measured in September to October 2021 and June to July 2022, respectively. Independent sample t-test, Chi square test and two factors repeated measurement analysis of variance were used for statistical analysis of the data.@*Results@#After one academic year, compared with the control group [(13.52±2.30)kg], muscle mass of primary school students in the after school ES group [(13.76±2.32)kg] significantly increased, while waist to hip ratio [(0.95±0.16)] and percentage of body fat [(20.17±7.43)%] significantly decreased compared to the control group [(1.01±0.21), (22.02±12.34)%]( F=330.70, 6.85, 4.33, P <0.05). The proportion of overweight and obesity in after school ES group decreased significantly from 19.5% to 12.3% ( χ 2=4.35, P <0.05). Compared with the control group, the scores of 50 meter running [(10.00±1.06, 10.21±0.83)s], 1 minute sit up [(33.25±8.24, 30.76±9.34)times], sitting and flexion [(14.53±7.50, 8.59±6.32)cm], 1 minute rope skipping [(125.01±30.50, 115.97±32.09)times], eyes closed and single legged standing [(30.00±34.72, 25.72±23.82)s], selective response time [(635.66±91.72, 652.79±120.42)ms] and VO 2max [(45.31± 1.02 , 43.67±0.85)mL/(kg〖 ·min)] in the after school ES group were significantly improved, with statistical significance ( F= 5.32 , 443.14, 97.23, 814.07, 36.49, 6.11, 396.91, P <0.05).@*Conclusions@#After school ES can improve body shape of primary school students, reduce the risk of overweight and obesity and enhance physical fitness. It is recommended that schools should appropriately increase after school ES to promote physical fitness of students.

Two New Compounds from the Endophytic Fungi of Dryopteris crassirhizoma and Their Antimicrobial Activities.

Two endophytic fungi Trichoderma afroharzianum (HP-3) and Alternaria alstroemeriae (HP-7) were isolated and purified from the fresh root of Dryopteris crassirhizoma. Chemical investigation of the two fungi resulted in the isolation of two new phenols 2,4-dihydroxy-3-farnesyl-5-methoxy benzoic acid (1) and 2-hydroxyphenethyl 2-phenylacetate (2), together with 22 known compounds. Their structures were elucidated by NMR, UV, IR, HRESIMS, and comparison to the literature data. Compounds 15 and 16 showed significant antibacterial activity against Micrococcus lysodeikticus with MIC value of 6.25 µg/mL, while 8 and 14 displayed moderate inhibitory activities against several plant pathogenic fungi and clinically important bacterial strains. This is the first study to report the isolation, identification, and antimicrobial properties of metabolites from endophytic fungi of D. crassirhizoma. Our findings may provide lead compounds for the development of new antibacterial agents.

A Functional Variant Alters the Binding of Bone morphogenetic protein 2 to the Transcription Factor NF-κB to Regulate Bone morphogenetic protein 2 Gene Expression and Chicken Abdominal Fat Deposition.

In this study, we employed a dual-luciferase reporter assay and electrophoretic mobility shift analysis (EMSA) in vitro to explore whether a 12-base pair (bp) insertion/deletion (InDel) variant (namely g.14798187_14798188insTCCCTGCCCCCT) within intron 2 of the chicken BMP2 gene, which was significantly associated with chicken abdominal fat weight and abdominal fat percentage, is a functional marker and its potential regulatory mechanism. The reporter analysis demonstrated that the luciferase activity of the deletion allele was extremely significantly higher than that of the insertion allele (p < 0.01). A bioinformatics analysis revealed that compared to the deletion allele, the insertion allele created a transcription factor binding site of nuclear factor-kappa B (NF-κB), which exhibited an inhibitory effect on fat deposition. A dual-luciferase reporter assay demonstrated that the inhibitory effect of NF-κB on the deletion allele was stronger than that on the insertion allele. EMSA indicated that the binding affinity of NF-κB for the insertion allele was stronger than that for the deletion allele. In conclusion, the 12-bp InDel chicken BMP2 gene variant is a functional variant affecting fat deposition in chickens, which may partially regulate BMP2 gene expression by affecting the binding of transcription factor NF-κB to the BMP2 gene.

Ultrasound-triggered release of miR-199a-3p from liposome nanobubbles for enhanced hepatocellular carcinoma treatment.

This study was aimed to develop an efficient tumour-targeted liposome nanobubbles (LNBs) system using ultrasound-targeted nanobubble destruction for enhanced release and transfection of miRNA-199a-3p in hepatocellular carcinoma (HCC) therapy. The prepared LNBs comprised a polyethylene glycol-modified liposome shell and a perfluoropentane (PFP) core. MiRNA-199a-3p was attached to the nanocomposite surface via electrostatic adsorption, while RGD peptide functionalized the LNBs surface for enhanced HCC cell targeting, namely PFP@miR-RGD-LNBs. The LNBs were spherical with a narrow size distribution. The gene-loaded LNBs effectively condensed miR-199a-3p and protected it from enzymatic degradation. Low-intensity focused ultrasound (LIFU) promoted a fast release of miR-199a-3p from the prepared LNBs, thereby enhancing therapeutic effects. The combined application of PFP@miR-RGD-LNBs and LIFU exhibited a more potent inhibitory effect on HepG2 cells than the other groups, potentially due to LIFU promoting rapid and efficient gene release at the target site and increasing cell membrane permeability. Quantitative reverse transcription-polymerase chain reaction analysis revealed significantly increased mRNA expression levels of key apoptosis markers (Bad, Bax, Caspase-9 and Caspase-3) in the PFP@miR-RGD-LNBs + LIFU group compared to other groups. These findings suggest that the prepared LNBs are highly likely to be promising candidates for further exploration of HCC gene delivery and therapy.

Extracellular vesicle-derived circHIPK3: Novel diagnostic biomarker for lung cancer.

PURPOSE: Lung cancer (LC) is a common malignancy worldwide. A great number of circular RNAs (circRNAs) have been identified that serve crucial roles in cancer development. Extracellular vesicles (EVs) and their contents have been shown to be biomarkers for the diagnosis and prognosis of LC. Thus, we intended to clarify the functional role of EVs-derived circRNA homology domain interacting protein kinase 3 (EVs-circHIPK3) and its underlying mechanism of action. MATERIAL AND METHODS: Bioinformatics analysis was performed to validate the potential of partially circulating HIPK3 in LC diagnosis. EVs were isolated by polyethylene glycol (PEG) precipitation from plasma of 52 LC patients and 30 healthy controls. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was employed to evaluate the expressions of candidate circRNAs (circHIPK3) and microRNA-637 (miR-637, a target of circHIPK3). RESULTS: CircHIPK3 is significantly up-regulated in LC, while miR-637 expression is significantly reduced (p â€‹< â€‹0.05). Receiver operating characteristic (ROC) curve analysis, based on the expression of EVs-circHIPK3, allowed us to distinguish LC from healthy controls (area under the curve, AUC 0.897). CONCLUSIONS: Taken together, our study shows that EV-derived circHIPK3 can serve as a promising biomarker for LC patient diagnosis. However, the downstream mRNA of the circHIPK3/miR-637 axis requires further exploration to enrich our understanding of circHIPK3's mechanism in LC.

Transaxillary Single-Port Endoscopic Nipple-Sparing Mastectomy with Immediate Implant-based Breast Reconstruction in Breast Cancer Patients Receiving Neoadjuvant Chemotherapy or Not: A Comparative Study with Analysis of Surgical Complications and Patient-Reported Outcomes.

BACKGROUND: In most cases, transaxillary single-port endoscopic nipple-sparing mastectomy with immediate implant-based breast reconstruction (E-NSM-IIBR) is conducted in patients with early-stage breast cancer, ensuring surgical safety while achieving improved breast aesthetics. However, whether E-NSM-IIBR is appropriate in patients undergoing neoadjuvant chemotherapy (NAC) is still unclear. The aim of this study was to report the surgical safety and patient-reported outcomes (PROs) of breast cancer patients who underwent E-NSM-IIBR with NAC in comparison to those who did not receive NAC. METHODS: A retrospective cohort study was conducted on patients who underwent E-NSM-IIBR with or without NAC at a single center between January 2021 and July 2022. Patient demographics, postoperative complications, and PROs evaluated using the BREAST-Q version 2.0 questionnaire were compared between the two groups. Factors associated with PROs at 9 months after surgery were assessed with linear regression analysis. RESULTS: A total of 92 patients who underwent E-NSM-IIBR were included in the study, with 27 patients receiving NAC and 65 patients not receiving NAC. There was no significant difference in the incidence of postoperative complications between the two groups. The BREAST-Q version 2.0 questionnaire was completed by 24 out of 27 patients (88.9%) in the NAC group and 59 out of 65 patients (90.8%) in the non-NAC group at 9 months after surgery. The patient-reported outcomes in various domains of the BREAST-Q did not show a significant difference between the two cohorts. The results of the multiple linear regression analysis indicated that in the both groups age (ß = - 0.985, 95% CI - 1.598 to - 0.371, p = 0.003 in the NAC group; ß = - 0.510, - 1.011 to - 0.009, p = 0.046 in the non-NAC group) and rippling (ß = - 21.862, - 36.768 to - 6.955, p = 0.006 in the NAC group; ß = - 7.787, - 15.151 to - 0.423, p = 0.039 in the non-NAC group) significantly impacted the patients' satisfaction with breasts, and PMRT was negatively associated with patients' physical well-being of chest (ß = - 13.813, - 26.962 to - 0.664, p = 0.040 in the NAC group; ß = - 18.574, - 30.661 to - 6.487, p = 0.003 in the non-NAC group). Our findings revealed that patients with larger implant volumes had higher scores in psychosocial well-being (ß = 0.082, 0.001 to 0.162, p = 0.047), whereas implant displacement (ß = - 14.937, - 28.175 to - 1.700, p=0.028) had a negative impact on patients' psychological well-being in the non-NAC group. However, our results did not demonstrate any significant influencing factors on patients' psychosocial well-being within the NAC group. CONCLUSION: Our preliminary experiences confirm that E-NSM-IIBR is a safe option for selected patients even after NAC, with favorable patient-reported outcomes comparable with those in the primary surgery setting. The postoperative long-term outcomes of patients who undergo radiation therapy after NAC merit further investigation in the future. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Plastic nanoparticles cause proteome stress and aggregation by compromising cellular protein homeostasis ex vivo and in vivo.

Decomposition of plastic materials into minuscule particles and their long-term uptake pose increasing concerns on environmental sustainability and biosafety. Besides common cell viability and cytotoxicity evaluations, how plastic nanoparticles interfere with different stress response pathways and affect cellular fitness has been less explored. Here, we provided the first piece of evidence to demonstrate plastic nanoparticles potentially can deteriorate proteome stability, compromise cellular protein homeostasis, and consequently cause global proteome misfolding and aggregation. Polystyrene (PS) nanoparticles of different sizes and surface charges were exploited as model plastic materials. In cell lysate and human blood plasma, naked PS nanoparticles with hydrophobic surface deteriorated proteome thermodynamic stability and exaggerated its aggregation propensity. While no cell viability ablation was observed in cells treated with PS nanoparticles up to 200 µg·mL-1, global proteome aggregation and stress was detected by a selective proteome aggregation sensor. Further proteomics analysis revealed how protein homeostasis network was remodeled by positively charged PS nanoparticles via differential expression of key proteins to counteract proteome stress. In mice model, size-dependent liver accumulation of positively charged PS nanoparticles induced hepatocellular proteome aggregation and compromised protein homeostasis network capacity that were invisible to standard alanine transaminase and aspartate transaminase (ALT/AST) liver function as-say and histology. Meanwhile, long-term liver accumulation of plastic nanoparticles deteriorated liver metabolism and saturated liver detoxification capacity of overdosed acetaminophen. This work highlighted the impact of nanoplastics on cellular proteome integrity and cellular fitness that are invisible to current biochemical assays and clinical tests.

Tailoring the Amphiphilicity of Fluorescent Protein Chromophores to Detect Intracellular Proteome Aggregation in Diverse Biological Samples.

The formation of amorphous misfolded and aggregated proteins is a hallmark of proteome stress in diseased cells. Given its lack of defined targeting sites, the rational design of intracellular proteome aggregation sensors has been challenging. Herein, we modulate the amphiphilicity of fluorescent protein chromophores to enable selective detection of aggregated proteins in different biological samples, including recombinant proteins, stressed live cells, intoxicated mouse liver tissue, and human hepatocellular carcinoma tissue. By tuning the number of hydroxyl groups, we optimize the selectivity of fluorescent protein chromophores toward aggregated proteins in these biological samples. In recombinant protein applications, the most hydrophobic P0 (cLogP = 5.28) offers the highest fold change (FC = 31.6), sensitivity (LLOD = 0.1 µM), and brightness (Φ = 0.20) upon binding to aggregated proteins. In contrast, P4 of balanced amphiphilicity (cLogP = 2.32) is required for selective detection of proteome stresses in live cells. In mouse and human liver histology tissues, hydrophobic P1 exhibits the best performance in staining the aggregated proteome. Overall, the amphiphilicity of fluorescent chromophores governs the sensor's performance by matching the diverse nature of different biological samples. Together with common extracellular amyloid sensors (e.g., Thioflavin T), these sensors developed herein for intracellular amorphous aggregation complement the toolbox to study protein aggregation.

Effect of the Competing Ligand on Ni-NTA/Histag Strength Revealed by Click Chemistry-Based Force Spectroscopy.

The specific interaction between Ni-nitrilotriacetic acid and the six-histidine tag may be one of the most important coordination bonds utilized in biological research because of its wide application for recombinant protein purification. The complex stability is critical for target protein binding. Thus, measurement of the mechanical stability of the system was attempted soon after the invention of atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) two decades ago. Moreover, the competing ligand imidazole and protons are the two critical factors for target protein elution. However, the mechanochemistry between the system and the imidazole/proton has not been determined. Here, an AFM-SMFS system using strain-promoted alkyne-azide cycloaddition and Cu-free click chemistry was used to characterize the system. Consequently, the destabilizing effect of the imidazole and proton on the interaction was revealed quantitatively, leading to a 3-fold increase in the bond dissociation rate.

Therapeutic blocking of VEGF binding to neuropilin-2 diminishes PD-L1 expression to activate antitumor immunity in prostate cancer.

Prostate cancers are largely unresponsive to immune checkpoint inhibitors (ICIs), and there is strong evidence that programmed death-ligand 1 (PD-L1) expression itself must be inhibited to activate antitumor immunity. Here, we report that neuropilin-2 (NRP2), which functions as a vascular endothelial growth factor (VEGF) receptor on tumor cells, is an attractive target to activate antitumor immunity in prostate cancer because VEGF-NRP2 signaling sustains PD-L1 expression. NRP2 depletion increased T cell activation in vitro. In a syngeneic model of prostate cancer that is resistant to ICI, inhibition of the binding of VEGF to NRP2 using a mouse-specific anti-NRP2 monoclonal antibody (mAb) resulted in necrosis and tumor regression compared with both an anti-PD-L1 mAb and control immunoglobulin G. This therapy also decreased tumor PD-L1 expression and increased immune cell infiltration. We observed that the NRP2, VEGFA, and VEGFC genes are amplified in metastatic castration-resistant and neuroendocrine prostate cancer. We also found that individuals with NRP2High PD-L1High metastatic tumors had lower androgen receptor expression and higher neuroendocrine prostate cancer scores than other individuals with prostate cancer. In organoids derived from patients with neuroendocrine prostate cancer, therapeutic inhibition of VEGF binding to NRP2 using a high-affinity humanized mAb suitable for clinical use also diminished PD-L1 expression and caused a substantial increase in immune-mediated tumor cell killing, consistent with the animal studies. These findings provide justification for the initiation of clinical trials using this function-blocking NRP2 mAb in prostate cancer, especially for patients with aggressive disease.