Position Matters: Pyridine Regioisomers Influence Secondary Structure and Micelle Morphology in Polymer-Homopolypeptide Micelles.

Polymer-homopolypeptide block copolymers are a class of bioinspired materials that combine the processability and stability of synthetic polymers with the biocompatibility and unique secondary structures of peptides, such as α-helices and ß-sheets. These properties make them ideal candidates for a wide variety of applications, for example, in the pharmaceutical field, where they are frequently explored as building blocks for polymeric micelle drug delivery systems. While homopolypeptide side chains can be furnished with an array of different moieties to impart the copolymers with desirable properties, such as stimulus responsivity, pyridine derivatives represent an underutilized functional group for this purpose. Additionally, the interplay between polypeptide side chain structure, secondary conformation, and micelle morphology is not yet well understood, particularly in the case of structural regioisomers. Therefore, in this work, a series of polymer-homopolypeptide copolymers were prepared from a poly(ethylene glycol)-b-poly(glutamic acid) (PEG-b-PGA) backbone, where the pendant carboxylic acid groups were covalently conjugated to a series of pyridine regioisomers by carbodiimide coupling. These pyridine regioisomers differed only in the position of the nitrogen heteroatom, ortho, meta or para, relative to the linking group, generating a series of PEG-b-poly(pyridinylmethyl glutamate) (PEG-b-PMG) copolymers. Following self-assembly of the copolymers in aqueous solutions, dynamic light scattering (DLS) revealed differences in micelle hydrodynamic diameter (Dh) (ranging from ∼60 to 120 nm), while transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS) revealed distinctive morphologies ranging from ellipsoidal, to cylindrical, and disc-like, suggesting that subtle changes in positional isomers in the polypeptide block may influence the micelle structure. Analysis of the PEG-b-PMG copolymer micelles by circular dichroism (CD) and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy revealed that differences in the morphology were associated with changes in polypeptide secondary structure, which in turn was influenced by the position of the pyridine heteroatom. Overall, these findings contribute to the broader understanding of the relationship between polypeptide structure and micelle morphology and serve as useful insight for the rational design of polymer-polypeptide nanoparticles.

Prevalence of obesity and associated health risks in soldiers of the German Armed Forces.

BACKGROUND: Obesity rates are rising in the armed forces of Western democratic countries, impacting military readiness and health. This highlights the need for preventive health risk assessments and countermeasures. METHODS: Using mandatory health examination data from 2018 to 2022, we analyzed the prevalence of obesity, health risks, and associated specific military risk factors (rank and unit) in 43,214 soldiers of the German Armed Forces. Statistical methods included χ2 contingencies and binary logistic regressions. RESULTS: The prevalence of obesity (BMI ≥ 30) was 18.0%. Male soldiers (OR = 3.776) and those with an officer's rank (OR = 1.244) had an increased chance for obesity. Serving in a combat unit reduced the chance of being obese (OR = .886). Considering BMI and waist circumference, 2.4% of the total sample faced extremely high cardiovascular and metabolic health risks, while 11.0% and 11.6% had very high or high health risks, respectively. CONCLUSIONS: Our data underscore the importance of targeting obesity-related health risk factors in soldiers to ensure their well-being and deployment readiness.

A systematic review and in silico analysis of studies investigating the ischaemic penumbra proteome in animal models of experimental stroke.

Ischaemic stroke results in the formation of a cerebral infarction bordered by an ischaemic penumbra. Characterising the proteins within the ischaemic penumbra may identify neuro-protective targets and novel circulating markers to improve patient care. This review assessed data from studies using proteomic platforms to compare ischaemic penumbra tissues to controls following experimental stroke in animal models. Proteins reported to differ significantly between penumbra and control tissues were analysed in silico to identify protein-protein interactions and over-represented pathways. Sixteen studies using rat (n = 12), mouse (n = 2) or primate (n = 2) models were included. Heterogeneity in the design of the studies and definition of the penumbra were observed. Analyses showed high abundance of p53 in the penumbra within 24 hours of permanent ischaemic stroke and was implicated in driving apoptosis, cell cycle progression, and ATM- MAPK- and p53- signalling. Between 1 and 7 days after stroke there were changes in the abundance of proteins involved in the complement and coagulation pathways. Favourable recovery 1 month after stroke was associated with an increase in the abundance of proteins involved in wound healing. Poor recovery was associated with increases in prostaglandin signalling. Findings suggest that p53 may be a target for novel therapeutics for ischaemic stroke.

Mass spectrometry imaging protocol for spatial mapping of lipids, N-glycans and peptides in murine lung tissue.

RATIONALE: The application of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to murine lungs is challenging due to the spongy nature of the tissue. Lungs consist of interconnected air sacs (alveoli) lined by a single layer of flattened epithelial cells, which requires inflation to maintain its natural structure. Therefore, a protocol that is compatible with both lung instillation and high spatial resolution is essential to enable multi-omic studies on murine lung disease models using MALDI-MSI. METHODS AND RESULTS: To maintain the structural integrity of the tissue, murine lungs were inflated with 8% (w/v) gelatin for lipid MSI of fresh frozen tissues or 4% (v/v) paraformaldehyde neutral buffer for N-glycan and peptide MSI of FFPE tissues. Tissues were sectioned and prepared for enzymatic digestion and/or matrix deposition. Glycerol-free PNGase F was applied for N-glycan MSI, while Trypsin Gold was applied for peptide MSI using the iMatrixSpray and ImagePrep Station, respectively. For lipid, N-glycan and peptide MSI, α-cyano-4-hydroxycinnamic acid matrix was deposited using the iMatrixSpray. MS data were acquired with 20 µm spatial resolution using a timsTOF fleX MS instrument followed by MS fragmentation of lipids, N-glycans and peptides. For lipid MSI, trapped ion mobility spectrometry was used to separate isomeric/isobaric lipid species. SCiLS™ Lab was used to visualize all MSI data. For analyte identification, MetaboScape®, GlycoMod and Mascot were used to annotate MS fragmentation spectra of lipids, N-glycans and tryptic peptides, respectively. CONCLUSIONS: Our protocol provides instructions on sample preparation for high spatial resolution MALDI-MSI, MS/MS data acquisition and lipid, N-glycan and peptide annotation and identification from murine lungs. This protocol will allow non-biased analyses of diseased lungs from preclinical murine models and provide further insight into disease models.

Use of tryptic peptide MALDI mass spectrometry imaging to identify the spatial proteomic landscape of colorectal cancer liver metastases.

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide. CRC liver metastases (CRLM) are often resistant to conventional treatments, with high rates of recurrence. Therefore, it is crucial to identify biomarkers for CRLM patients that predict cancer progression. This study utilised matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to spatially map the CRLM tumour proteome. CRLM tissue microarrays (TMAs) of 84 patients were analysed using tryptic peptide MALDI-MSI to spatially monitor peptide abundances across CRLM tissues. Abundance of peptides was compared between tumour vs stroma, male vs female and across three groups of patients based on overall survival (0-3 years, 4-6 years, and 7+ years). Peptides were then characterised and matched using LC-MS/MS. A total of 471 potential peptides were identified by MALDI-MSI. Our results show that two unidentified m/z values (1589.876 and 1092.727) had significantly higher intensities in tumours compared to stroma. Ten m/z values were identified to have correlation with biological sex. Survival analysis identified three peptides (Histone H4, Haemoglobin subunit alpha, and Inosine-5'-monophosphate dehydrogenase 2) and two unidentified m/z values (1305.840 and 1661.060) that were significantly higher in patients with shorter survival (0-3 years relative to 4-6 years and 7+ years). This is the first study using MALDI-MSI, combined with LC-MS/MS, on a large cohort of CRLM patients to identify the spatial proteome in this malignancy. Further, we identify several protein candidates that may be suitable for drug targeting or for future prognostic biomarker development.

Pretest PSA and Restaging PSMA PET/CT Predict Survival in Biochemically Recurrent Prostate Cancer.

BACKGROUND: A biochemical recurrence (BCR) risk model was created based on pretest prostate specific antigen (PSA) and groupings by restaging prostate specific membrane antigen (PSMA) PET/CT. METHODS: A cohort of 1216 BCR patients were analyzed for overall survival (OS) according to the PSA threshold and restaging PSMA PET/CT. A Cox regression analysis of OS was carried out to detect significant clinical characteristics. RESULTS: In the cohort, 271 patients had a pretest PSA of <0.5 ng/mL and 945 patients had higher PSA values. The restaging PSMA PET/CT was positive for 834 patients and negative for 369. Of 1203 patients, 133 (11%) died, including 19 of the 369 (5%) patients without positive sites on the restaging PSMA PET/CT, 82 of the 711 (12%) with 1-5 positive sites, and 32 of the 123 (26%) with >5 positive sites. In the Cox regression analysis, four variables significantly predicted OS: treatment center, International Society of Urologic Pathology (ISUP) grade, pretest PSA threshold, and the grouping of positive sites on the restaging PSMA PET/CT. CONCLUSIONS: The pretest PSA and PSMA PET/CT were important for the OS of the BCR patients. The findings argue for the new BCR risk model and serve as framework for ongoing trials.

The Influence of Hypothyroid Metabolic Status on Blood Coagulation and the Acquired von Willebrand Syndrome.

The intent of this prospective study aimed to identify the influence of hypothyroid metabolic status on the coagulation and fibrinolytic system and association with the acquired von Willebrand syndrome (VWS-ac). We compared 54 patients without substitution therapy after radical thyroidectomy with 58 control subjects without pathological thyroid-stimulating-hormone (TSH)-values. Patients with TSH > 17.5 mU/L over a period of >4 weeks were included. The control-collective was selected based on age and sex to match the patient-collective. The data were collected using laboratory coagulation tests and patient questionnaires; a bleeding score was determined. There were significant differences in the measurement of activated-partial-thromboplastin-time (aPTT/p = 0.009), coagulation-factor VIII (p < 0.001) and von-Willebrand-activity (VWF-ac/p = 0.004) between the patient and control groups. The patient cohort showed an increased aPTT and decreased factor VIII and VWF-ac. 29.7% of the patient-collective compared to 17.2% of the control subjects met the definition of VWS-Ac (p = 0.12). The bleeding score showed significantly more bleeding symptoms in patients with a laboratory constellation of VWS-ac (no family history; p = 0.04). Our results suggest hypocoagulability in hypothyroid patients. Hypothyroidism appears to have a higher incidence of VWS-ac. The increased risk of bleeding complications in hypothyroid patients may be of relevant importance for the outcome, especially in the context of invasive interventions.

Kinetic Effects of Transferrin-Conjugated Gold Nanoparticles on the Antioxidant Glutathione-Thioredoxin Pathway.

Nanoparticle-based therapeutics are being clinically translated for treating cancer. Even when thought to be biocompatible, nanoparticles are being increasingly identified as altering cell regulation and homeostasis. Antioxidant pathways are important for maintaining cell redox homeostasis and play important roles by maintaining ROS levels within tolerable ranges. Here, we sought to understand how a model of a relatively inert nanoparticle without any therapeutic agent itself could antagonize a cancer cell lines' antioxidant mechanism. A label-free protein expression approach was used to assess the glutathione-thioredoxin antioxidative pathway in a prostate cancer cell line (PC-3) after exposure to gold nanoparticles conjugated with a targeting moiety (transferrin). The impact of the nanoparticles was also corroborated through morphological analysis with TEM and classification of pro-apoptotic cells by way of the sub-G0/G1 population via the cell cycle and annexin V apoptosis assay. After a two-hour exposure to nanoparticles, major proteins associated with the glutathione-thioredoxin antioxidant pathway were downregulated. However, this response was acute, and in terms of protein expression, cells quickly recovered within 24 h once nanoparticle exposure ceased. The impact on PRDX-family proteins appears as the most influential factor in how these nanoparticles induced an oxidative stress response in the PC-3 cells. An apparent adaptive response was observed if exposure to nanoparticles continued. Acute exposure was observed to have a detrimental effect on cell viability compared to continuously exposed cells. Nanoparticle effects on cell regulation likely provide a compounding therapeutic advantage under some circumstances, in addition to the action of any cytotoxic agents; however, any therapeutic advantage offered by nanoparticles themselves with regard to vulnerabilities specific to the glutathione-thioredoxin antioxidative pathway is highly temporal.

Robotic Retroperitoneal Versus Transperitoneal Partial Nephrectomy: Outcomes and Learning Curve.

BACKGROUND/AIM: The robotic retroperitoneal approach for renal mass surgery was introduced in 2018 at the Department of Urology in the clinic of Leverkusen, Germany. Clinical criteria for the choice of the access site (trans- vs. retroperitoneal) are not clearly defined. The aim of this study was to explore the learning curve and the impact of the access site on clinical outcome, in order to elucidate which preoperative clinical criteria should be taken into account when choosing the renal approach site. PATIENTS AND METHODS: This retrospective study included 107 patients who underwent robotic tumor surgery between June 2018 and March 2022 at the Department of Urology in the Clinic of Leverkusen, Germany. Data from 86 patients with transperitoneal robotic surgery of the kidney and 21 patients with retroperitoneal access were available for analysis. We evaluated the data of patients in a trans- and a retroperitoneal access group. The preoperative clinical data included anthropomorphic data, the Body Mass Index (BMI) as well as the Preoperative Aspects and Dimensions Used for Anatomical Classification of Renal Masses (PADUA) - score. Intraoperative and postoperative data such as blood loss, clamping time, renal function and the learning curve of the surgeons was used to evaluate the outcomes of the two groups. RESULTS: Operation time in the retroperitoneal group was significantly shorter (p=0.015). Operation-specific variables showed no significant difference between the two groups. PADUA score and hilar clamping time showed no difference (p=0.345 and p=0.130, respectively). The learning curve in the retroperitoneal access group unveiled a noticeable difference in the experience and mastery of the involved surgeons. CONCLUSION: Mastery of the retroperitoneal approach is readily possible for surgeons with previous experience in robotic renal surgery without compromising the operative morbidity. The PADUA-score seems most suitable as a preoperative clinical criterion for choosing the renal approach site.

Label-Free Quantification Mass Spectrometry Identifies Protein Markers of Chemotherapy Response in High-Grade Serous Ovarian Cancer.

Eighty percent of ovarian cancer patients initially respond to chemotherapy, but the majority eventually experience a relapse and die from the disease with acquired chemoresistance. In addition, 20% of patients do not respond to treatment at all, as their disease is intrinsically chemotherapy resistant. Data-independent acquisition nano-flow liquid chromatography-mass spectrometry (DIA LC-MS) identified the three protein markers: gelsolin (GSN), calmodulin (CALM1), and thioredoxin (TXN), to be elevated in high-grade serous ovarian cancer (HGSOC) tissues from patients that responded to chemotherapy compared to those who did not; the differential expression of the three protein markers was confirmed by immunohistochemistry. Analysis of the online GENT2 database showed that mRNA levels of GSN, CALM1, and TXN were decreased in HGSOC compared to fallopian tube epithelium. Elevated levels of GSN and TXN mRNA expression correlated with increased overall and progression-free survival, respectively, in a Kaplan-Meier analysis of a large online repository of HGSOC patient data. Importantly, differential expression of the three protein markers was further confirmed when comparing parental OVCAR-5 cells to carboplatin-resistant OVCAR-5 cells using DIA LC-MS analysis. Our findings suggest that GSN, CALM1, and TXN may be useful biomarkers for predicting chemotherapy response and understanding the mechanisms of chemotherapy resistance. Proteomic data are available via ProteomeXchange with identifier PXD033785.

Assessment of yeast physiology during industrial-scale brewing practices using the redox-sensitive dye resazurin.

Beer refermentation in bottles is an industrial process utilized by breweries where yeast and fermentable extract are added to green beer. The beer is refermented for a minimum of 2 weeks before distribution, with the physiological state of the yeast a critical factor for successful refermentation. Ideally, fresh yeast that is propagated from a dedicated propagation plant should be used for refermentation in bottles. Here, we explored the applicability of the fluorescent and redox-sensitive dye, resazurin, to assess cellular metabolism in yeast and its ability to differentiate between growth stages. We applied this assay, with other markers of yeast physiology, to evaluate yeast quality during a full-scale industrial propagation. Resazurin allowed the discrimination between the different growth phases in yeast and afforded a more in-depth understanding of yeast metabolism during propagation. This assay can be used to optimize the yeast propagation process and cropping time to improve beer quality.

Detection Efficacy of 68Ga-PSMA-11 PET/CT in Biochemical Recurrence of Prostate Cancer with Very Low PSA Levels: A 7-Year, Two-Center "Real-World" Experience.

In biochemical recurrence of prostate cancer (BCR), prompt tumor localization guides early treatment, potentially improving patient outcomes. Gallium-68 prostate-specific membrane antigen-11 positron emission tomography/computed tomography (68Ga-PSMA-11 PET/CT) detection rates of lesions suspicious for prostate cancer are well known to rise along with prostate-specific antigen (PSA) concentration. However, published data are limited regarding very low values (≤0.2 ng/mL). We retrospectively analyzed ~7-year "real-world" experience in this setting in a large post-prostatectomy cohort (N = 115) from two academic clinics. Altogether 44 lesions were detected in 29/115 men (25.2%) (median [minimum-maximum] 1 [1-4]/positive scan). The apparent oligometastatic disease was found in nine patients (7.8%) at PSA as low as 0.03 ng/mL. Scan positivity rates were highest when PSA was >0.15 ng/mL, PSA doubling time was ≤12 months, or the Gleason score was ≥7b (in 83 and 107 patients, respectively, with available data); these findings were statistically significant (p ≤ 0.04), except regarding PSA level (p = 0.07). Given the benefits of promptly localizing recurrence, our observations suggest the potential value of 68Ga-PSMA-11 PET/CT in the very low PSA BCR setting, especially in cases with more rapid PSA doubling time or with high-risk histology.

Correction: von Eyben et al. A Risk Model for Patients with PSA-Only Recurrence (Biochemical Recurrence) Based on PSA and PSMA PET/CT: An Individual Patient Data Meta-Analysis. Cancers 2022, 14, 5461.

In the original publication [...].

Prediction of Significant Prostate Cancer in Equivocal Magnetic Resonance Imaging Lesions: A High-volume International Multicenter Study.

BACKGROUND: Decision of performing prostate biopsy in men with Prostate Imaging Reporting and Data System (PI-RADS) 3 findings in prostate magnetic resonance imaging (MRI) is challenging as they have a low but still relevant risk of harboring significant prostate cancer (sPC). OBJECTIVE: To identify clinical predictors of sPC in men with PI-RADS 3 lesions in prostate MRI and to analyze the hypothetical effect of incorporating prostate-specific antigen density (PSAD) into biopsy decision. DESIGN, SETTING, AND PARTICIPANTS: We analyzed a retrospective multinational cohort from ten academic centers comprising 1476 men who underwent a combined prostate biopsy (MRI targeted plus systematic biopsy) between February 2012 and April 2021 due to a PI-RADS 3 lesion in prostate MRI. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary outcome was the detection of sPC (ISUP ≥2) in a combined biopsy. Predictors were identified by a regression analysis. Descriptive statistics were applied to evaluate the hypothetical effect of involving PSAD into biopsy decision. RESULTS AND LIMITATIONS: Of all patients, 273/1476 (18.5%) were diagnosed with sPC. MRI-targeted biopsy diagnosed fewer sPC cases than combined strategy: 183/1476 (12.4%) versus 273/1476 (18.5%), p < 0.01. Age (odds ratio [OR] 1.10 [95% confidence interval {CI}: 1.05-1.15], p < 0.001), prior negative biopsy (OR 0.46 [0.24-0.89], p = 0.022), and PSAD (p < 0.001) were found to be independent predictors of sPC. Applying a PSAD cutoff of 0.15, 817/1398 (58.4%) biopsies would have been avoided at the cost of missing sPC in 91 (6.5%) men. Limitations were the retrospective design, heterogeneity of the study cohort due to the long inclusion period, and no central revision of MRI. CONCLUSIONS: Age, previous biopsy status, and PSAD were found to be independent predictors of sPC in men with equivocal prostate MRI. Implementation of PSAD into biopsy decision can avoid unnecessary biopsies. Clinical parameters such as PSAD need validation in a prospective setting. PATIENT SUMMARY: In this study, we looked for clinical predictors of significant prostate cancer in men with Prostate Imaging Reporting and Data System 3 lesions in prostate magnetic resonance imaging. We identified age, previous biopsy status, and especially prostate-specific antigen density as independent predictors.

Analgesic Use in Sports.

BACKGROUND: Consumption of medication to alleviate pain is widespread in Germany. Around 1.9 million men and women take analgesics every day; some 1.6 million persons are addicted to painkillers. Analgesic use is thought also to be common in sports, even in the absence of pain. The aim of this study was to assess the extent of painkiller use among athletes. METHODS: In line with the PRISMA criteria and the modified PICO(S) criteria, a systematic literature review was registered (Openscienceframework, https://doi. org/10.17605/OSF.IO/VQ94D) and carried out in PubMed and SURF. The publications identified (25 survey studies, 12 analyses of doping control forms, 18 reviews) were evaluated in standardized manner using the Newcastle Ottawa Scale (NOS) and AMSTAR (A MeaSurement Tool to Assess systematic Reviews). RESULTS: Analgesic use is widespread in elite sports. The prevalence varies between 2.8% (professional tennis) and 54.2% (professional soccer). Pain medication is also taken prophylactically in the absence of symptoms in some non-elite competitive sports. In the heterogeneous field of amateur sports the data are sparse and there is no reliable evidence of wide-reaching consumption of painkillers. Among endurance athletes, 2.1% of over 50 000 persons stated that they used analgesics at least once each month in connection with sports. CONCLUSION: Analgesic use has become a problem in many areas of professional/ competitive sports, while the consumption of pain medication apparently remains rare in amateur sports. In view of the increasing harmful use of or even addiction to painkillers in society as a whole, there is a need for better education and, above all, restrictions on advertising.

BMI Alterations and Prevalence of Overweight and Obesity Related to Service Duration at the German Armed Forces.

The aim of this study was to assess body mass index (BMI) and the prevalence of overweight and obesity at entry and release of service at the German Armed Forces and related associations to service duration. In a cohort study, 85,076 paired BMI data sets (entry and release of service) of German soldiers (5.4% females) between 2010 to 2022 were analyzed retrospectively to assess BMI alterations and the prevalence of overweight (BMI ≥ 25) and obesity (BMI ≥ 30) after service durations of ≤2 years, 2−5 years, or ≥5 years. Between 2010 and 2022, we observed a trend for BMI increases of about 0.5 kg/m2 (X2 = 27.104, p = 0.007). BMI increases differed significantly (X2 = 7622.858, p < 0.001) after ≤2 years (0.0 kg/m2), after 2−5 years (1.1 kg/m2), and after ≥5 years (2.4 kg/m2) and were correlated to service duration (r = 0.34, p < 0.001). The prevalence of overweight increased from 33.0% to 39.5%. Obesity prevalence increased from 3.7% to 6.3%. The switch to obesity was more pronounced for longer service durations. Although secular trends for BMI increases among soldiers were in line with the general population, service duration was related to BMI increases. Especially, the service time depending on pronounced prevalence of obesity should be a matter of debate leading to counteracting measures at the German Armed Forces.

[Postoperative outcomes and survival rates after aseptic revision total hip arthroplasty : What can patients expect from revision surgery?] / Postoperative Ergebnisse und Überlebensraten nach aseptischem Hüft-TEP-Wechsel : Was können Patient*innen von einem Revisionseingriff erwarten?

BACKGROUND: In 2020, more than 14,000 aseptic revision procedures for total hip arthroplasty (THA) were registered in Germany. Patient expectations of revision hip arthroplasty are not substantially different from expectations of primary hip replacement. OUTCOME: However, revision surgery is associated with increased complication rates and a higher proportion of dissatisfied patients. In particular, poorer postoperative function and mobility as well as increased pain levels following revision THA have been described compared to the outcome after primary THA. Quality of life and return-to-work can also be impaired. SURVIVAL RATE: Implant survival is influenced by age, BMI, and comorbidities of the patients, but also by the size and complexity of bone defects, the extent of periprosthetic soft tissue compromise and the choice of revision implant(s). In addition, the number of previous revision surgeries inversely correlates with the survival rates. Previous revisions have been shown to be associated with increased risks of aseptic loosening, instability and periprosthetic infection.

Proteomic analysis of holocarboxylase synthetase deficient-MDA-MB-231 breast cancer cells revealed the biochemical changes associated with cell death, impaired growth signaling, and metabolism.

We have previously shown that the holocarboxylase synthetase (HLCS) is overexpressed in breast cancer tissue of patients, and silencing of its expression in triple-negative cancer cell line inhibits growth and migration. Here we investigated the global biochemical changes associated with HLCS knockdown in MDA-MB-231 cells to discern the pathways that involve HLCS. Proteomic analysis of two independent HLCS knockdown cell lines identified 347 differentially expressed proteins (DEPs) whose expression change > 2-fold (p < 0.05) relative to the control cell line. GO enrichment analysis showed that these DEPs were mainly associated with the cellular process such as cellular metabolic process, cellular response to stimulus, and cellular component organization or biogenesis, metabolic process, biological regulation, response to stimuli, localization, and signaling. Among the 347 identified DEPs, 64 proteins were commonly found in both HLCS knockdown clones, confirming their authenticity. Validation of some of these DEPs by Western blot analysis showed that plasminogen activator inhibitor type 2 (SerpinB2) and interstitial collagenase (MMP1) were approximately 90% decreased in HLCS knockdown cells, consistent with a 50%-60% decrease in invasion ability of knockdown cells. Notably, argininosuccinate synthase 1 (ASS1), one of the enzymes in the urea cycle, showed approximately a 10-fold increase in the knockdown cells, suggesting the crucial role of HLCS in supporting the urea cycle in the triple-negative cancer cell line. Collectively, our proteomic data provide biochemical insights into how suppression of HLCS expression perturbs global changes in cellular processes and metabolic pathways, impairing cell growth and invasion.

A Risk Model for Patients with PSA-Only Recurrence (Biochemical Recurrence) Based on PSA and PSMA PET/CT: An Individual Patient Data Meta-Analysis.

An individual patient meta-analysis followed 1216 patients with PSA-only recurrence (biochemical recurrence, BCR) restaged with [68Ga]Ga-PSMA-11 PET/CT before the salvage treatment for median 3.5 years and analyzed the overall survival (OS). A new risk model included a good risk group with a prescan PSA < 0.5 ng/mL (26%), an intermediate risk group with a prescan PSA > 0.5 ng/mL and a PSMA PET/CT with 1 to 5 positive sites (65%), and a poor risk group with a prescan PSA > 0.5 ng/mL and a PSA PET/CT with > 5 positive sites (9%) (p < 0.0001, log rank test). The poor risk group had a five-year OS > 60%. Adding a BCR risk score by the European Association of Urology did not significantly improve the prediction of OS (p = 0.64). In conclusion, the restaging PSMA PET/CT markedly predicted the 5-year OS. The new risk model for patients with PSA-only relapse requires a restaging PSMA PET/CT for patients with a prescan PSA > 0.5 ng/mL and has a potential use in new trials aiming to improve the outcome for patients with PSA-only recurrence who have polysites prostate cancer detected on PSMA PET/CT.