For patients with high oxidant species at baseline, females showed a greater reduction in itching compared to males (−95% vs. −50%), while men had a higher amelioration in pain and morning stiffness . In conclusion, sulfureous crenotherapy can be a valuable strategy to improve cellular redox state in both sexes.Oxidative stress, a condition induced by an excessive amount of free radicals, such as reactive oxygen species (ROS), shows several gender-related differences in basal cellular redox state and antioxidant responses. Crenotherapy with sulfureous mineral water can improve the cellular redox state. In this retrospective observational study, gender-related differences in the efficacy of sulfureous crenotherapy in decreasing oxidant species were investigated. Seventy-eight patients, stratified by sex, with osteoarthritis or degenerative joint disease and Vulgar psoriasis who have received a cycle of sulfureous mud-bath therapy + sulfureous hydropinotherapy were enrolled. Plasma concentration of oxidant species and clinical outcomes were measured at baseline and at the end of treatment. After 2 weeks of sulfureous crenotherapy, a significant amelioration of clinical outcomes and a significant reduction of oxidant species were observed in both sexes, more marked in females than in males ( Oxidative stress is a condition induced by the presence of excessive levels of free radicals, such as Reactive Oxygen Species (ROS), resulting from increased production and/or reduced effectiveness of endogenous antioxidant systems to neutralize them ,2,3,4. RGender plays an important role in modulating basal cellular redox state and oxidative stress response, as females are more resistant to oxidative stress, such as chronic oxidative stress induced by iron accumulation in thalassemia ,9,10,11.Crenotherapy with sulfureous mineral water exerts beneficial effects in several diseases and also improves cellular redox state in both animal models and humans ,18,19,20Based on these considerations, in this retrospective observational study, we evaluated possible gender-related differences in the impact of sulfureous crenotherapy on plasma levels of ROMs and symptomatology in patients with rheumatic diseases and Vulgar psoriasis by stratifying for the first time by sex a real-life population treated with crenotherapy. We showed that sulfureous crenotherapy caused a more pronounced reduction of oxidant molecules in females with OA and psoriasis.p = 0.04). At baseline, 29% of females (N = 14) and males (N = 9) showed ROMs within normal values . Conversely, the majority of subjects (71% in both sexes) displayed increased circulating ROM levels at baseline, and females tended to have higher ROMs compared to males .The study population (N = 78) was stratified by sex and included 40% of males (N = 31) with a mean age of 59 ± 9.5 years  and a body mass index (BMI) of 27 ± 8.0, and 60% of females (N = 47) with a mean age of 57 ± 12.8 years  and a BMI of 26 ± 5.1 . No diffp = 0.19), and 285 ± 38 Carr.U. in males . Conversely, patients with high ROMs at baseline displayed a significant reduction after crenotherapy in both sexes , more marked in females with a 10-fold reduction  compared to males with a 7-fold decrease   .p < 0.05) (p = 0.004), as well as morning stiffness , while itch symptom was similar in both groups . After crenotherapy, pain, morning stiffness, and itch improved in both sexes. VAS-scores for morning stiffness were lower in males , while for itch in females   , includi < 0.05) . Convers= 0.030) .p = 0.020) and morning stiffness than males , while itch symptom was similar in both groups (p = 0.799). After crenotherapy, pain, morning stiffness, and itch improved in both sexes (p = 0.077), while men had a higher amelioration in pain and morning stiffness compared to females , with lower VAS-scores .Similarly, in subjects with higher ROM levels at baseline, females complained of more pain  complained of belching (on the third day) and fatigue (during the last two days), while 10% of females experienced pain exacerbation (on the third day) and fatigue (on the last two days). All enrolled subjects completed the treatment.salus per aquam has therapeutic effects on various disorders, especially in those triggered by oxidative stress, and in some cases, they represent an alternative approach to standard pharmacological treatments [Crenotherapy or spa eatments ,20,21,22eatments . Sulfureeatments ,24,25,26eatments ,28. Botheatments ,17,19,202S, that have a cell signaling function and plays important roles in combating ROS and other free radicals [In this retrospective observational real-life study, ROM plasma level changes were investigated after 2 weeks of sulfureous crenotherapy in a cohort of patients with OA or psoriasis stratified by sex. After crenotherapy, ROM plasma levels were significantly reduced in those subjects with increased ROMs at baseline, more marked in females than in males. This finding could be related to the antioxidant and anti-inflammatory effects of sulfureous mineral water used in crenotherapy (mud-bath therapy and hydropinotherapy). For example, sulfureous hydropinotherapy can provide the body’s supply of several minerals, like Hradicals ,29,30; mradicals ,31; and radicals ,33. In oradicals ,17,21,34radicals ,36,37. IThis study has some limitations: (i) the small sample size of each group and the absence of a control group, even though a reference ROM level range is already well-established; (ii) hydropinotherapy and mud-bath therapy were prescribed by different physicians, leading to treatment heterogeneity for similar conditions in our cohort; (iii) hormone levels or related signaling pathways were not investigated; and (iv) antioxidant status of subjects was not assessed to confirm the utility of sulfureous crenotherapy because of a small sample volume was available for each subject.A total of 78 Caucasian patients  who received a sulfureous crenotherapeutic cycle at the Telese Spa  were enrolled in this study after obtaining informed consent according to the Declaration of Helsinki and its amendments, and protocols approved by local Ethics Committee “Campania Sud” (no. 7 r.p.s.o./2020). Patients had a diagnosis of degenerative joint diseases  or Vulgar psoriasis (N = 27) according to international guidelines . InclusiCarratelli Units (1 Carr.U. = 0.08 mg/L of H2O2) and ranged between 250–300 Carr.U. [Plasma ROM concentrations were measured at baseline and after 2 weeks of treatment using a d-ROMs test , a spectrophotometric assay for ROM level assessment, mainly hydroperoxides (ROOH) ,33. Norm2 test. A p value < 0.05 was considered statistically significant.Data were analyzed using the STATA 16 statistics package. Continuous variables, expressed as mean ± standard deviation (SD), were analyzed with t Student’s tests for paired and unpaired normally distributed data, while categorical variables using χIn conclusion, spa therapy with mineral waters represents an alternative approach in musculoskeletal, skin, gastroenteric, and respiratory disorders because spa mineral waters influence several biological pathways via chemical stimuli, similar to common drugs. Therefore, sulfureous crenotherapy could be an effective alternative therapeutic strategy in OA and psoriasis treatment for both sexes, likely because of its antioxidant effects and in supplying the body with minerals important in muscle and bone homeostasis. Moreover, we showed for the first time that these crenotherapy-related effects could be more pronounced in females compared to males, probably because oxidative stress is also influenced by gender-related factors, like hormonal status. However, larger studies are needed to establish the type and extent of these beneficial effects from a gender-oriented perspective.


Non-coding RNAs (ncRNAs) are involved in the occurrence and development of PCa and the response to PCa treatment. ncRNAs are known to execute diverse regulatory roles in cellular processes, despite their inability to encode proteins. Among them, microRNAs, long non-coding RNAs, and circular RNAs play key roles in the regulation of the Wnt signaling pathway in PCa. Aberrant expression of these ncRNAs and dysregulation of the Wnt signaling pathway are one of the causes of cell proliferation, apoptosis, invasion, migration, and angiogenesis in PCa. Moreover, these ncRNAs affect the characteristics of PCa cells and hold promise as diagnostic and prognostic biomarkers. Herein, we summarize the role of ncRNAs in the regulation of the Wnt signaling pathway during the development of PCa. Additionally, we present an overview of the current progress in research on the correlation between these molecules and clinical features of the disease to provide novel insights and strategies for the treatment of PCa.Prostate cancer (PCa) is one of the most common cancers in males, exhibiting a wide spectrum of clinical manifestations that pose challenges in its diagnosis and treatment. The Wnt signaling pathway, a conserved and complex pathway, is crucial for embryonic development, tissue homeostasis, and various physiological processes. Apart from the classical Wnt/β-catenin signaling pathway, there exist multiple non-classical Wnt signaling pathways, including the Wnt/PCP and Wnt/Ca In males, prostate cancer (PCa) is one of the most common cancers and the fifth leading cause of cancer-related deaths . Accordi2+ signal transduction  have been recognized as critical regulatory factors in gene expression and signal transduction in both normal physiology and disease pathogenesis, particularly in PCa . ncRNAs In conclusion, our review presents a comprehensive elucidation of the intricate interplay between ncRNAs and the Wnt signaling pathway in the context of PCa occurrence, progression, and therapeutic approaches. Significantly, we underscore the profound implications of ncRNAs as promising diagnostic and prognostic biomarkers, accentuating their pivotal role in modulating PCa aggressiveness and therapeutic response. Moreover, we delve deep into the immense potential of targeting ncRNAs as therapeutic interventions for PCa, exploring a plethora of strategic avenues. Finally, we address the contemporary challenges encountered in this ever-evolving field.2+ signaling pathways  signaling pathway, is gaining recognition as a key factor in the pathogenesis of PCa . This prThe dysregulation of ncRNAs has been associated with the occurrence and progression of PCa . Recent Despite the aforementioned findings, the molecular mechanism of the regulation of the Wnt signaling pathway by ncRNAs in PCa is not entirely understood, and additional investigations are required to elucidate the complex interplay between various types of ncRNAs and other regulatory factors that activate or inhibit the Wnt signaling pathway in PCa. Furthermore, the investigation of ncRNAs poses technical challenges, owing to their low abundance and high sequence variability . AdvanceThe Wnt signaling pathway plays a key role in the progression of PCa, with ncRNAs as crucial regulatory factors of this pathway . Recent miRNAs are RNA molecules that are 22 nucleotides in length and regulate gene expression by binding to complementary target messenger RNAs (mRNAs) . The tisLncRNAs are RNA molecules that are 200 nucleotides in length and do not encode proteins . AlthougCircRNAs were first discovered in plant viruses and the Sendai virus through electron microscopy in 1976 . HoweverMetastasis is the primary cause of most cancer-related deaths . InvasioLi et al. demonstrated that miR-1297 directly targets the 3′-untranslated region of AEG-1 and regulates its mRNA and protein expression levels . In addi+ cells during IL-6-induced PCa cell progression, thereby suppressing cell proliferation, cell cycle progression, and invasion  in diameter and originate from endosomes . Many stEarly detection of PCa is crucial for effective treatment and improved survival rates . For exaEarly screening and diagnosis of cancer are crucial for patient survival . The idePatient prognostic information is essential in the process of making informed treatment decisions . MountinCancer treatment has long been regarded as one of the most formidable challenges worldwide, despite the progress made in treatment modalities . TargeteTargeting ncRNAs has been considered an attractive strategy for cancer treatment . In addiChemoresistance is a notable concern in cancer treatment, and enhancing chemosensitivity in PCa through ncRNA intervention has become a strategy that is increasingly being recognized and investigated . Targetiin vivo has hindered their potential clinical application, necessitating the exploration of more reliable strategies to target ncRNAs.This review provided a comprehensive overview of the role of the Wnt signaling pathway and related ncRNAs  in PCa. MiRNAs affect the expression of target genes associated with the Wnt signaling pathway. Additionally, the Wnt signaling pathway establishes feedback mechanisms and functions as an upstream mediator of miRNAs. In most cases, lncRNAs regulate the expression of proteins in the Wnt signaling pathway by serving as sponges for miRNAs. CircRNAs also regulate the expression of the Wnt signaling pathway; however, similar to lncRNAs, they primarily regulate the expression of the Wnt signaling pathway by targeting miRNAs. We also discussed the novel avenues for the development of siRNAs and shRNAs that target the Wnt signaling pathway and their potential clinical applications. However, the limited efficacy of siRNAs and shRNAs Although the regulation of the Wnt signaling pathway through ncRNA intervention is a promising avenue for the treatment of PCa, certain issues need to be addressed. First, the Wnt signaling pathway is highly intricate, consisting of 19 distinct types of Wnt-secreted glycoproteins and over 15 types of Wnt receptors in humans, which activate various downstream pathways. Second, the differences and balance between the classical and non-classical Wnt signals are difficult to capture, making targeting the Wnt signaling pathway even more challenging. Third, the Wnt signaling pathway plays a fundamental role in the dynamic balance of systems, such as the digestive and hematopoietic systems; therefore, blocking the Wnt signaling pathway may lead to systemic toxicity. Hence, modulation of the Wnt signaling pathway as a therapeutic strategy for PCa is both an opportunity and a challenge, warranting further research. In the future, it will be necessary to conduct additional investigations of the interplay between the Wnt signaling pathway and ncRNAs and develop more reliable methods for targeting ncRNAs for their clinical application. Simultaneously, alternative therapeutic strategies for PCa should also be explored to improve the efficacy of treatment and the quality of life of patients.


Persistent knee joint instability has been recognized as a potential postoperative consequence after Tibial Tuberosity Advancement (TTA) causing chronic lameness. A six-year-old male Labrador retriever (38 kg) with persistent lameness lasting six months after TTA Rapid surgery has been examined. During orthopedic examination, the lameness was subjectively graded 3/5 and the positive drawer and tibial compression tests were performed. Radiographical measurements were performed; the preoperative tibial plateau angle was 27° and the patella ligament angle relative to the tibial plateau was 102°. The decision was made to completely remove the TTA Rapid cage and all screws to have enough room to perform a TPLO and apply a 3.5 LCP plate. Long-term follow-up showed radiologically excellent healing with fusion of the gap and disappearance of the osteotomy line. Six months postoperatively, no lameness was detected at a walk and trot. The owner was completely satisfied and reported the dog being free of lameness even after long walks.The purpose of this case report is to describe the functional and clinical outcome of a tibial plateau leveling osteotomy (TPLO) in a dog with joint instability and persistent lameness following a Tibial Tuberosity Advancement surgery (TTA) Rapid. A six-year-old male Labrador retriever (38 kg) with a tibial plateau angle of 27° and a patella ligament to tibial plateau angle of 102° and persistent lameness lasting six months after TTA Rapid surgery has been examined. During orthopedic examination, the lameness was subjectively graded 3/5 and the positive drawer and tibial compression tests were performed. The TTA Rapid cage and all screws were completely removed from the tibia to have enough room to perform a TPLO radial cut. A lameness score evaluation, client satisfaction and radiographic follow-up were performed at 4 weeks, 8 weeks and 6 months postoperatively. Long-term follow-up showed radiologically excellent healing with fusion of the gap and disappearance of the osteotomy line. Six months postoperatively, no lameness was detected at a walk and trot. The owner was completely satisfied and reported the dog being free from lameness even after long walks. No complications related to the TPLO surgery occurred. Cranial cruciate ligament (CCL) rupture is the most common orthopedic condition affecting the hind limb in dogs . SeveralThe surgical procedures are associated with a wide range of postoperative complications, ranging in intensity from mild to catastrophic ,14. SomeProlonged knee joint instability has been recognized as a possible postoperative consequence after TTA and TPLO ,16. It iA six-year-old male Labrador retriever (38 kg) with persistent lameness after TTA Rapid was referred for assessment of right pelvic limb lameness. The dog underwent the initial TTA surgery six months prior to referral due to cranial cruciate rupture. According to the owner, the dog had never regained complete limb function following the initial TTA procedure. During orthopedic examination, the lameness was subjectively graded as 3/5 and the positive drawer and tibial compression tests were performed.Preoperative craniocaudal and mediolateral radiographs of the stifle joint were obtained under sedation and surgical planning for a TPLO radial cut was undertaken using vPOP-PRO software (ver. 2.9.2). Preoperative radiographs showed complete osteointegration of TTA Rapid cage to the tibia, signs of stifle joint effusion and the presence of osteophytes consistent with osteoarthritis (OA) associated with cranial cruciate disease. The measurements were performed based on previously described methods; the preoperative tibial plateau angle (TPA) was 27° and the patella ligament angle relative to the tibial plateau (PLA) was 102°  Figure A. A deciPrior to surgery, the patient received dexmedetomidine  and butorphanol  as premedication. General anesthesia was induced with propofol (2–4 mg/kg IV) and maintained with isoflurane in 100% oxygen. Cefazolin (22 mg/kg IV) was administered 30 min prior to surgery and then after 90 min.® Acculan 4 TPLO Saw, B Braun, Melsungen, Germany). The tibial plateau segment was rotated to 8 mm and temporarily fixed in a position with a 1.6 mm Kirschner pin. The 3.5 mm anatomically pre-countered TPLO plate  was positioned on the caudal part of the proximal tibia in a way that best fit the bone surface and osteotomy. The TPLO plate was attached to the tibia using two 3.5 mm standard cortical screws for dynamic compression and five 3.5 mm locking screws . A 1.6 mm Kirschner pin in the cranio-caudal direction was inserted for additional stabilization. The incision was closed in three layers. Two orthogonal postoperative radiographs were performed to evaluate the implants’ position, apposition of the bone fragments and postoperative TPA. Postoperative radiographic TPA was 10°.The surgical approach was started from the medial parapatellar skin incision. The subcutaneous tissues were dissected blindly and a medial mini parapatellar arthrotomy was performed for initial inspection of the stifle, to examine any meniscal lesions by visualization and probing. The menisci were intact and then not released. A medial approach to the proximal tibia was performed and TTA screws were gently removed. By using a mallet, a small osteotome was advanced in a mediolateral direction against and parallel to the external sides of the TTA Rapid cage to a depth approximately equal to the length of the cage. After the rim of the cage appeared to be separated from the tibia, the cage was rocked back and forth in a proximal-distal direction by using a periosteal elevator until it was evidently loose. Cranio-caudal manipulations were clearly avoided to minimize fracture of the tibial tuberosity. Gentle steady traction was applied to complete the TTA Rapid cage removal. A 24 mm crescentic TPLO saw blade was used. The location of the tibial osteotomy was determined by preoperative planning from a mediolateral radiograph. Due to not having enough room for the TPLO plate and jig, the procedure was performed without a jig. The osteotomy was performed with an oscillating saw . All received images were evaluated by the Authors. Radiographs were critically assessed for implant position, alignment and bone healing of the osteotomy and TTA gap site. No postoperative complications related to the TPLO procedure or surgical site infection (SSI) occurred.At the 4-week follow-up, the dog significantly improved after surgery with a lower subjective lameness score of 1/5 at a walk and at a trot. Kirschner pins and TPLO plate were aligned and positioned without displacement. Radiographically, the disappearance of the TTA gap and osteotomy gap and the presence of bridging callus were noted C. The owTo the best of our knowledge, this is the only case that shows the viability of applying a TPLO radial cut to address chronic lameness and instability after TTA Rapid cage removal.Prolonged knee joint instability has been recognized as a potential consequence following proximal tibial osteotomies. In comparison to TPLO procedures, the reported score of chronic stifle instability appears to be greater following TTA procedures ,16. The The reason for persistent instability after TTA surgery is still unknown; however, there are two proposed theories including faults in surgical planning and execution of the procedure or in the biomechanical concept . The TTAOne of the causes of lameness after stifle surgery could be late meniscal injury. Some clinical studies reported that the severity and frequency of eventual meniscal pathology is higher after TTA compared to TPLO at short-term and long-term evaluations . In an iMobility of bone fragments at the osteotomy line, even without significant loss of reduction, has also been implicated in increased rates of complications, especially in large- and giant-breed dogs . SeveralThe preoperative planning of TPLO does not require a stifle flexion angle of 135° . The tibThis report has several limitations including that it is a case report. A kinematic study of the dogs prior to and following surgery might provide a more reliable pre- and post-operative assessment of the lameness. To confirm the suitability of the TPLO procedure after TTA Rapid cage removal, a similar procedure must be performed on a larger number of patients.In conclusion, TPLO surgery was successfully performed after TTA Rapid cage removal to treat persistent stifle joint instability and chronic lameness in a six-year-old Labrador retriever. TPLO following initial TTA yielded good to excellent clinical outcomes and improved clinical function. In our case report, TPLO was performed after the TTA implant was removed in order to improve stifle stability and function.


The in vivo biodistribution of Bi-212-MAA was investigated in Balb/c and C57BL/6 mice with 4T1 and EO771 orthotopic breast tumors, respectively. The same orthotopic breast cancer models were used to evaluate the treatment efficacy of Bi-212-MAA. Our results showed that macroaggregated albumin can be stably radiolabeled with Bi-212 and that Bi-212-MAA can deliver significant radiation therapy to reduce the growth and clonogenic potential of 4T1 and EO771 cells in vitro. Additionally, Bi-212-MAA treatment upregulated γH2AX and cleaved Caspase-3 expression in 4T1 cells. Biodistribution analyses showed 87–93% of the Bi-212-MAA remained in 4T1 and EO771 tumors 2 and 4 h after injection. Following single-tumor treatments with Bi-212-MAA there was a significant reduction in the growth of both 4T1 and EO771 breast tumors over the 18-day monitoring period. Overall, these findings showed that Bi-212-MAA was stably radiolabeled and inhibited breast cancer growth. Bi-212-MAA is an exciting platform to study α-particle therapy and will be easily translatable to larger animal models and human clinical trials.Intravascularly administered radiation therapy using beta (β-)-emitting radioisotopes has relied on either intravenously injected radiolabeled peptides that target cancer or radiolabeled microspheres that are trapped in the tumor following intra-arterial delivery. More recently, targeted intravenous radiopeptide therapies have explored the use of alpha (α)-particle emitting radioisotopes, but microspheres radiolabeled with α-particle emitters have not yet been studied. Here, FDA-approved macroaggregated albumin (MAA) particles were radiolabeled with Bismuth-212 (Bi-212-MAA) and evaluated using clonogenic and survival assays  Radiation therapy has been essential for cancer management since its inception . Many fohttps://www.niowaveinc.com/index.php/alpha-emitters/)), Ac-225 ; and future availability of At-211 ).Alpha (α)-particle emitting radionuclides are an exciting alternative to β-emitting radionuclides due to their high linear energy transfer (LET) and shorter range in tissue . Alpha . ImportaInterventional oncology is a field of medicine that utilizes image guidance to deliver cancer therapies directly into tumors . The priin vitro and in vivo.FDA-approved macroaggregated albumin (MAA) are particles that range in size from 10–70 μm  and can be labeled with Tc-99m for lung perfusion studies in nuclear medicine or for SIRT pre-treatment planning . MAA is 3 and evaporated to dryness 3 times. The dried vials containing the Bi-212 were reconstituted with 100 μL of 0.1 M HNO3 for transfer to vials containing 10 μL of 1 M NaOH for neutralization. To confirm the purity of the Bi-212 solution, a small sample from each elution was evaluated with a gamma counter  for the energy peak corresponding to Bi-212 only (600 keV). Bi-212 purity was also confirmed by repeatedly measuring Bi-212 samples over time with a dose calibrator  to measure half-life.The Ra-224/Pb-212 generators (5 mCi) were provided by Oak Ridge National Lab . Elution and preparation of the isotope was done similarly to as previously described . The geng for 5 min with the pellet containing the Bi-212 bound MAA and the supernatant containing unbound Bi-212 that was easily removed. The percentage of Bi-212 bound to MAA was determined with instant thin layer chromatography (iTLC) on paper silica gel impregnated strips using 10 mM EDTA in 0.15 M NH4OAc as the mobile phase.FDA-approved MAA kits (Pulmotech) were purchased from Cardinal Health . For radiolabeling MAA with Bi-212, 3 mg of the MAA kit (0.33 mg MAA) was resuspended in 500 μL 1X PBS (pH 7.0) and added to the neutralized Bi-212. The Bi-212-MAA solution was incubated for 10 min at 70 °C with 500 RPM shaking. Bi-212-bound MAA was purified by centrifugation at 1000 2. Cells were subcultured upon reaching 90% confluency and reseeded at 20% confluency.4T1 and EO771 cells were purchased from ATCC. Cells were cultured in complete media , respectively) and incubated at 37°C with 5% COFor generation of stably luciferase expressing cell tines, both 4T1 and EO771 cells were transduced by PLV-10170-pLV-CAG-Firefly luciferase-PGK-Puro plasmid using lentivirus (Cellomics Technology) and luciferase positive clones were selected using puromycin (Thermo Fisher Scientific).3 4T1 and EO771 Luc + cells were seeded into 6-well plates 12 h prior to treatment. Cells were treated in triplicate with 0, 5, 10 and 20 μCi Bi-212-MAA and incubated for 1 week. Bland, unlabeled MAA particles were used as 0 uCi condition (control group) to match the number of particles used for the highest treated condition (20 μCi). For IVIS imaging and quantification, cells had media removed and replaced with 150 μg/mL luciferin diluted in complete media. Cells were imaged using IVIS Lumina bioluminescence imaging (BLI) with autoexposure setting. Data were analyzed using ROI and radiance (photon/second). After imaging, colonies were stained with crystal violet and manually counted for the total number of colonies per well. Colonies were considered for clusters with greater than 50 cells.The clonogenic assay was performed similarly to as previously described . In this4 4T1 and EO771 Luc + cells were seeded into a 24-well plate. 12 h later, cells were treated with 0, 2.5, 5, 10, or 20 μCi of Bi-212-MAA and incubated for 48 h. Bland, unlabeled MAA was again used as the 0 μCi condition (Control group), with the number of particles matching those used in the 20 μCi condition. IVIS imaging and quantification were done identically to the clonogenic assay as described.For the survival assay, 5 × 10Total protein was extracted by lysing the 4T1 cells using RIPA buffer (Thermo Fisher Scientific) with 1X proteinase inhibitor cocktail added (Abcam) and incubated at 4°C for 30 min and vortexed 45 s every 10 min. Cell lysates were centrifuged at 30,000 g for 30 min at 4°C, and the supernatants were collected and stored at −80°C for further use. Protein concentration was determined using the Bradford protein assay method. Protein samples were denatured by adding 4 x Laemmli sample buffer (BioRad) and heating 95°C for 5 min. Further, 40 μg of proteins were run on 4–15% SDS-PAGE gels. Proteins were transferred onto the nitrocellulose membrane, followed by blocking with a blocking buffer for 1 h at room temperature. Next, membranes were incubated with diluted primary antibody 1:1000;  in a bloAll animals were purchased from Charles River Laboratory. All animals used in the experiments were of Balb/c and C57BL/6 background  and 8 weeks of age. The animal studies were approved by the Institutional Animal Care and Use Committee (IACUC), and all animal experiments were conducted according to IACUC guidelines.5 4T1 and EO771 cells (respectively) in the left 4th mammary gland using a 50:50 mixture of Matrigel (Thermo Fisher Scientific) and PBS in a total volume of 50 uL. After 10 and 11 days , 4 mice with breast tumors were injected intratumorally (IT) with either 11 μCi (4T1) or 7 μCi (EO771) of Bi-212-MAA in 20 μL of 0.9% sterile saline using 25-gauge integrated needle syringes with zero dead volume. The 4 mice plus 1 additional untreated mouse with a breast tumor were then imaged 2 h later using Cherenkov luminescence imaging (CLI) on IVIS Spectrum for radiation localization. After imaging, all 5 mice were injected with luciferin (3 mg) intraperitoneally and imaged 5 min later using BLI for tumor localization. Mice were sacrificed after the imaging session and had organs removed for gamma counting. Injection data and gamma counted data were all decay corrected to the counting start time. Mice in the 4-h group underwent biodistribution without an imaging session and were counted in the same fashion as the 2-h group with proper decay correction.Balb/c and C57BL/6 mice (8 weeks old) were implanted with 1 × 105 4T1 and EO771 Luc + cells (respectively) in the left 4th mammary gland. After 7- and 8-days post-implantation, 4T1 and EO771 tumors, respectively, were injected IT with Bi-212-MAA or vehicle control . 4T1 mice received either 25 or 50 uCi of Bi-212-MAA or control, while EO771 mice received either 50 or 100 uCi Bi-212-MAA or control suspended in 20 μL of 0.9% sterile saline using 25-gauge integrated needle syringes with zero dead volume. Mice were then tracked for tumor growth using digital caliper measurement. All groups were euthanized once the tumor size reached 2 cm in length in any group. Tumor volume was calculated using the equation (longest diameter x ((shortest diameter/2)^2)).Balb/c and C57BL/6 mice (8 weeks) were implanted with 1 × 10p-values <0.05 were considered statistically significant.Statistical analysis was performed using GraphPad. One-way analysis of variance (ANOVA) with Dunnett’s test was used to compare the experimental groups to the control group. Data are reported as mean ± standard error of the mean (SEM). in vitro and in vivo studies.Bi-212 was successfully eluted from the Ra-224 column (Pb-212 generator) and used for all assays. The decay scheme for Pb-212 is shown in 3 4T1 and EO771 breast cancer cells in a 6-well plate format to assess radiation therapy effectiveness on cancer cell clonogenicity. Increased levels of Bi-212-MAA led to significantly less total colony formation in both 4T1 and EO771 cells  is a well-known marker of an early cellular response to DNA double-strand breaks, and Caspase 3 is a key molecule involved in cell death . In thisNext, the expression of cell cycle checkpoint markers was studied to elucidate if 4T1 cells could effectively initiate DNA damage repair response . The resMice are too small to use IA delivery of drugs into tumor vasculature, so direct IT injection was needed for delivery of radioembolics into tumor tissue. It is well known that large particles can be retained in solid tumors after injection . Mice wiAt 2- and 4-h post-injection, greater than 90% of the total injected dose remained within the tumor for both 4T1 and EO771 . A full in vivo, 4T1 and EO771 tumors in Balb/c and C57BL/6 mice, respectively, were treated with Bi-212-MAA by injecting IT. The Bi-212-MAA caused a significant reduction in total growth compared to bland MAA-treated controls in both 4T1 and EO771 tumors (Further, to evaluate the effectiveness of Bi-212-MAA in killing breast cancer cells 1 tumors . These r1 tumors .A standard, commercially available MAA kit was rapidly labeled with Bi-212 and easily purified. The purification method was easier compared to other methods, that can take additional time, and the product can be lost during the process. The decay scheme of Pb-212 and pure elution of Bi-212 allowed for simple labeling and quantification of Bi-212-MAA. The process can be improved even further by using clinical grade generators when they become available.in vitro in both the clonogenic and survival assays. Importantly, we can perform further in vitro studies to study the molecular effects of α-particle radiation on cancer cells.Due to its size and density, MAA sank in solution and thus contacted cells in monolayer cell culture when added. With the short range of α-particles, it was unknown whether the MAA would bring the Bi-212 close enough to cells to deliver an effective therapy. However, the results clearly indicate that the Bi-212-MAA was an effective therapeutic against mouse breast cancer cells It was found that classic markers of apoptosis and general DNA-damage response markers were elevated with increasing levels of Bi-212-MAA. Further, no change was seen in markers of radioresistance, specifically proteins involved in cell cycle checkpoint control. It is known that increased radiation dose can lead to cell cycle arrest and cell survival, making it critical to find a sensitive dose . Alpha (The large size of MAA allows it to remain in tumor when delivered IT as seen with our biodistribution studies. The 2- and 4-h time points both showed high levels of MAA retention. These time points represented 75% and 94% of the total decay of Bi-212, respectively. The low dose present in healthy tissues shows that MAA stayed lodged within the tumor and that the radiopharmaceutical was stable and not releasing Bi-212. Further time points were not needed as greater than 90% of the dose was still present in the tumor at the 94% decay point. The biodistribution data were corroborated using CLI and BLI.Both 4T1 and EO771 tumors showed a response to IT delivery of Bi-212-MAA. This is an impressive result since the MAA is likely not distributed uniformly throughout the tumor and is therefore not leading to a complete tumor tissue dose. Although the CLI data shows retention of Bi-212-MAA in tumors, it does not cover the margins of the BLI data, indicating a smaller area of localization at the injection site. In an IA delivery model, the distribution would be much more uniform. Additionally, the expected range of α-particles from Bi-212 decay would span multiple cell diameters, allowing for a local crossfire effect in the tumor without causing toxicity to surround healthy parenchyma. Mice vessels were too small to utilize image guidance; however, there was precedence for direct injection of drugs into tumors to study therapy. With the short half-life of Bi-212, Bi-212-MAA only needed to remain within the tumor for a few hours to deliver a large radiation dose to tumors, which explains the success of our therapy studies.in vitro because of its agnostic targeting, similar to external beam radiation. Further, the short half-life of Bi-212 allows for a similar fractionated therapy schedule compared to the external beam, while long-lived isotopes such as Y-90 will continually dose the tumor once delivered (Due to Bi-212-MAA being agnostic to tumor receptor presence or density, it can be used to explore the effects of α-particles on any solid tumor model. Critically, it was recently found that fractionated external beam radiation therapy combined with immunotherapy led to an abscopal response in mice . Systemielivered .Bi-212-MAA also represents an easily translatable α-particle emitting radiopharmaceutical. As MAA and Ra-224/Pb-212 generators are already used in clinical trials, Bi-212-MAA has great promise for translation to human studies as an IA delivered drug. SIRT is a widely used treatment option for reducing hepatic tumor size and bridging patients to either surgical or transplantation options. The recent success of SIRT in the prostate highlights the potential for radioembolization in tissues outside of the liver . The breIn this study, we were able to effectively radiolabel MAA with the short-lived α-particle emitter Bi-212. Additionally, Bi-212-MAA was successfully used to inhibit mouse breast cancer growth in monolayer cell culture and in orthotopic tumor models. Bi-212-MAA represents a widely applicable platform for studying the effects of α-particle therapy on cancer cells, including molecular mechanisms of radioresistance and immunogenicity. Further, Bi-212-MAA is uniquely poised to be translated into clinical trials due to all reagents and its required delivery method being FDA approved.


The benefits of breastfeeding are extensive and well known. However, in certain medical conditions it is contraindicated, such as in women living with a human T-cell lymphotropic virus type 1 (HTLV-1) infection.),,, and mother-to-child transmission, mainly through breastfeeding. There is no vaccine or curative treatment for HTLV-1.),6,(7),, is also a strategy recommended in several countries, such as Japan, Chile, Colombia, Uruguay, Santa Lucia and Canada, as well as agencies, such as the National Institute of Health in the United States),10,, stands out. Information Note No. 4/2021-CGIST/DCCI/SVS/MS addresses the recommendation for the use of cabergoline in lactation inhibition and infant formula in the prevention of mother-to-child transmission of HIV and HTLV.)- intensification of capacity building for healthcare professionals and public policymakers; (ii) expanding HTLV-1 testing coverage in antenatal care; (iii) ensuring access to infant formula; (iv) establishing and strengthening multidisciplinary care networks for the comprehensive support and clinical follow-up of pregnant women living with HTLV-1 and HTLV-1 exposed children; and (v) implementing a surveillance system for HTLV-1 infection in pregnant women and exposed children .The sensitization of managers is of fundamental importance for the inclusion of HTLV-1 in the public health agenda, regarding capacity building for professionals, especially in primary healthcare. This is a crucial action to ensure that mothers have access to adequate information about HTLV-1, modes of transmission, associated diseases, and methods for reducing transmission risks. The Ministry of Health has included HTLV-1 in a training course on STIs and regularly conducts awareness campaigns.The identification of mothers living with HTLV-1 is essential for the success of any strategy aimed at reducing mother-to-child transmission of the virus. However, despite its cost-effectiveness, testing for HTLV-1 during antenatal care),,, intervention. Although there is limited data, the literature demonstrates that prenatal testing and intervention have high acceptance rates: over 90% of mothers choose to follow medical recommendations, a finding confirmed by representatives of people living with HTLV-1, both in Brazil and in other countries.),16,, HTLV-1 testing during antenatal care and (ii) an AFASS intervention. Access to free infant formula plays an important role in promoting adherence; however, many mothers living with the human T-cell lymphotropic virus type 1 - HTLV-1 are unaware of this right. Despite the advancements achieved, it is necessary to expand and strengthen public policies aimed at controlling this important, yet neglected disease.


Vibrio fluvialis QY27. Here, we investigated the molecular mechanism of HHP-responsive regulation of TMAO reductase TorA. By constructing torR and torS deletion mutants, we demonstrated that the two-component regulator TorR and sensor TorS are responsible for the HHP-responsive regulation of torA. Unlike known HHP-responsive regulatory system, the abundance of torR and torS was not affected by HHP. Complementation of the ΔtorS mutant with TorS altered at conserved phosphorylation sites revealed that the three sites were indispensable for substrate-induced regulation, but only the histidine located in the alternative transmitter domain was involved in pressure-responsive regulation. Taken together, we demonstrated that the induction of TMAO reductase by HHP is mediated through the TorRS system and proposed a bifurcation of signal transduction in pressure-responsive regulation from the substrate-induction. This work provides novel knowledge of the pressure regulated gene expression and will promote the understanding of the microbial adaptation to the deep-sea HHP environment.High hydrostatic pressure (HHP) regulated gene expression is one of the most commonly adopted strategies for microbial adaptation to the deep-sea environments. Previously we showed that the HHP-inducible trimethylamine N-oxide (TMAO) reductase improves the pressure tolerance of deep-sea strain  MicroorShewanella violacea DSS12 discovered a HHP-inducible glutamine synthetase (encoded by glnA), whose expression was under the control of sigma54 factor and two-component system NtrBC , inner membrane c-type cytochrome (torC) and a chaperone protein (torD) form the torCAD cluster, whose expression was induced by the presence of substrate through the periplasmic substrate-binding protein TorT and two-component regulatory system TorRS  is a tertiary amine oxide widespread in sea water. As an osmolyte and a piezolyte, TMAO favors for the adaptation of deep-sea organism to the low-temperature and high-pressure environment . It alsoem TorRS . Bindingem TorRS . The coneductase .P. profundum SS9. Strain SS9 encodes three sets of TMAO reductase, one of them (PBPRA1467-1468) exhibited higher expression level at 28 MPa compared to that at atmospheric pressure  significantly increased by HHP , and the expression of torAC is significantly induced by both substrate and elevated pressure, while the other set had a constitutive expression at a low level. By constructing deletion mutant, we further demonstrated that the HHP-inducible torA led to higher TMAO reduction efficiency under HHP condition, and improved pressure tolerance of strain QY27 in the presence of TMAO  auxotrophic E. coli strain WM3064 was cultured with the supplementation of 0.3 M of DAP. The E. coli strains carrying pBBR1MCS2 derived and pRE112 derived plasmids were maintained with 30 μg/mL kanamycin and 30 μg/mL chloramphenicol, respectively.d before . The culd before . TMAO wa2.2.torR contained cas9 gene under the control of Ptac, the sgRNA targeting at torR and the 1 kb fragments flanking the torR gene as the template sequence for genome editing. The gRNA sequences for torR was 5’-AGTTGATGTCCAGCATCACC-3′. The primers used to amplify upstream and downstream fragments flanking torR were as follows: primer 1, 5’-TCCCCCCGGGGTTTGCGGCCAATATAGTAGTC-3′; primer 2, 5’-CGGGATCCACACCACTTTATGCTCATTC-3′; primer 3, 5’-CGGGATCCGCGTTGACGCAGAAAG-3′; and primer 4, 5’-TCCCCGCGGATTCTGTCATAGGAATGGC-3′. The plasmid pBBR-tac-Cas9-torR was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. The plasmid was first transferred into the nutritional deficiency strain E. coli MW3064 by electroporation , and then introduced into strain QY27 by conjugation. The transformants were cultivated for 24 h with the presence of 0.2 mM isopropyl β-D-thiogalactoside (IPTG) that induced the expression of Cas9 protein for DNA cleavage.TorR deletion mutant was constructed using CRISPR-Cas9 system as previously described . The knotorS was constructed by means of homologous recombination double exchange. To construct plasmid pRE112-torS, DNA fragments flanking torS were amplified from the genomic DNA of QY27 with primer 1, 5′- CATGCCATGGTACCCGGGATGCCTTCGTATTG-3′; primer 2, 5′- GAGCAGGCGAGGATCCAGGCTCAGC-3′; primer 3, 5′- CTGCTGAGCCTGGATCCTCGCCTGC-3′; and primer 4, 5′- CGATCCACTAGTTCTAGAGGGTGAGTTTGGCG-3′. The gentamycin gene cassette was acquired from the vector pUCGm. The three fragments were assembled by fusion PCR and introduced into pRE112. The resulting plasmid was transferred into the nutritional deficiency strain E. coli MW3064 by electroporation , and then introduced into strain QY27 by conjugation. The double crossover transformants were screened by a two-step strategy, first culturing on the plates of YPG with chloramphenicol and then on plates of YPG supplemented with 15% (w/v) sucrose.The deletion mutant of torS genes were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and introduced into pBBR1MCS2. The plamids carrying mutated torS were introduced into deletion mutant of torS by conjugation and screened by growth on YPG plates with kanamycin.The point mutated torR or torS gene and the complementation of torS from the plasmid were confirmed by PCR amplification and sequencing .To carry out bi-parental conjugation, the donor strain of WM3064 cell carrying desired plasmid was cultivated at 37°C in LB medium to exponential phase. One milliliter of WM3064 culture was washed twice with LB medium before mixed with 1 mL of culture of QY27 strain. The mixture was suspended with 30 μL of YPG medium supplemented with DAP and placed on a filter on a plate of YPG medium with DAP overnight for conjugation. The mixture was transferred into an EP tube with YPG medium and incubated at 25°C for an hour before spread on plates for the selection of transformants. The full-length deletion of 2.3.7 cells were collected and treated with trizol and chloroform. The nucleic acids were precipitated and washed with isopropanol and ethanol, and then dissolved in RNase-free water. Residual DNA was digested with DNase I and the reverse transcription was performed with a PrimeScript™II 1st strand cDNA synthesis kit . RT-PCR was conducted with StepOne Software (ABI). The relative expression of the target gene was normalized to the reference gene of rpoD. Three replications were set for each assay for the calculation of the mean value and the standard deviation.Total RNA of QY27 cells was extracted as previously described . Briefly2.4.−1 to 2000 cm−1, using a portable fiber Raman system composed of a fiber optic Raman sensor RL-RP-785-(F/S)S  and a spectrometer . The peak at 751 cm−1 represents TMAO (PTMAO) and the peak at 2500 cm−1 represents H2O (PH2O). The peak area ratio was calculated by dividing the area value of PTMAO with that of PH2O. To quantify concentration of TMAO, a standard curve was first established. The Raman spectra of YPG medium supplemented with different concentrations of TMAO  were collected and the area of PTMAO and PH2O peaks were measured and the ratio was calculated. For each sample, five spectra were collected, and the ratios of PTMAO/PH2O were calculated for each spectrum and the average and standard deviation were calculated. The spectrum data were processed by NGSLabSpec and Origin softwares as previously described  for the production of his-tagged TorR. TorR was expressed in E. coli BL21  and purified with Ni- affinity chromatography. Briefly, IPTG was added to a final concentration of 0.8 mM for induction. After 4 h’ induction at 28°C, cells were collected by centrifuge and washed twice with 20 mM Tris–HCl pH 8.0. The cell pellet was suspended in lysis buffer  supplemented with Pierce™ protease inhibitor (Thermofisher) and lysed by sonication. The cellular lysis was centrifuged at 5000 rpm for 30 min at 4°C, and the supernatant was then loaded to Ni-NTA Superflow . The TorR was eluted with elution buffer containing 100 mM imidazole. The eluted fractions were analyzed by SDS-PAGE.The E. coli cells. For short, E. coli BL21 cells containing pET-28(a) plasmid were collected and washed with Tris–HCl buffer before incubated in pre-cooled acetone. The treated E. coli cells were air-dried into powder and incubated with the antiserum for the purification of TorR antibody.The antibody against TorR was prepared by ChinaPeptides (QYAOBIO)  and then purified with For western-blot analysis, cells grown to exponential phase were collected and suspended in 40 mM Trhis-HCl (pH 7.4) containing Pierce™ protease inhibitor (Thermofisher) and lysed by sonication. The crude extract was collected by centrifuge and the protein concentration was determined by Pierce™ BCA Protein Assay Kits (Thermofisher). The crude extract was separated by SDS-PAGE and detected with anti-TorR as first antibody, HRP conjugated goat anti rabbit IgG as second antibody and developed with DAB horseradish peroxidase color development kit (Beyotime).3.3.1.torA at different growth phases. Strain QY27 was usually cultivated in half filled syringe (2.5 mL syringe filled with 1 mL medium and 1.5 mL air), to provide oxygen for respiration. However, recent studies showed that unlike most anaerobic respiratory systems, TMAO reductase is expressed under aerobic conditions, but with strong cell-to-cell fluctuation  while HHP-dependent induction was observed in cells collected at both exponential phase (around 4-fold) and stationary phase (around 20-fold)  . Collect3.2.torCA and torYZ, respectively, the third consisted of torS and torT, and the fourth of torR and torD. It was noted that, although torS and torR are adjacent to torT and torD, respectively, and they transcribe in opposite directions. The torS and torR locate on the reverse strand, upstream of torT and torD that are both on the forward strand. Sequence analysis showed that TorS and TorR from strain QY27 shared merely 33 and 53% identity in amino acid sequences to their homologs in E. coli, respectively.Examination of QY27 genome identified eight components involved in the synthesis of TMAO reductase: two sets of TMAO reductase catalytic subunit (TorA and TorZ) and corresponding cytochrome c (TorC and TorY), a TMAO reductase maturation chaperon TorD, a periplasmic substrate TMAO binding protein TorT, and the substrate-responsive two-component regulatory system composed of a histidine kinase TorS and a response regulator TorR . The gentorA in strain QY27, we constructed deletion mutants of torR and torS, respectively, and characterized their growth and expression of torA. The two mutants exhibited same growth curve as the wild-type strain when cultivated in plain medium, but reacted differently to the supplementation of TMAO, as the addition of TMAO slowly stimulated the growth of ΔtorR but not ΔtorS   . In contdeletion .torA was observed in the two mutants. As expected, torA was no longer induced by the presence of TMAO. Meanwhile, its expression was weakly up-regulated (barely 2-fold) by elevated pressure, and the induction appeared only at stationary phase  by the function of pressure . To examd 30 MPa . We circ of torR. Taken t3.4.E. coli. They are the two histidines in the classical transmitter domain (H443) and the alternative transmitter domain (H850), and an aspartic acid in the sensor receiver domain (D723)   , 1997. Snd H902) .H479Q and TorSH902Q), and the aspartic acid with alanine (TorSD762A), respectively, and introduce the mutated TorS proteins into the ΔtorS mutant  did not change the growth profile of the ΔtorS, thus failed to utilize TMAO  of TorS partially impaired the substrate-dependent regulation but had no influence on the HHP-dependent regulation. The mutation of the aspartic acid in the sensor receiver domain (D762A) preserved only the function of HHP-responsive regulation, while substitution of the histidine in the alternative transmitter domain (H902Q) abolished the capacity to induce torA in response to both stimuli. Apparently, the TorRS regulatory system from deep-sea strain QY27 adopted different signaling pathways during substrate- and HHP- dependent regulation. Substrate triggered phosphoryl-transfer was same as the classical TorRS system from E. coli, with all the three conserved phosphorylation sites are involved. However, the phosphorylation site located in the alternative transmitter domain alone is essential for the HHP-dependent regulation  are known responsible for HHP-dependent regulation. In both cases, the abundance of the regulator changes along with elevated pressure, and is thus believed a common strategy of pressure sensing  failed to regulate expression of torA in response to HHP (data not shown). It suggested the possibility that membrane localization is required for TorS to sense the stimulus of HHP. Mutation studies of residues within or close to the transmembrane segments may provide more biochemical evidence to illustrate the mechanism of HHP sensation by the TorRS regulatory system.Microorganisms receive diverse external stimuli, such as temperature and viscosity, by membrane and converts them into chemical signals through membrane embedded or associated protein complex. For example, inner membrane histidine kinase DesK detects low temperature through the increased thickness of membrane and activates the synthesis of unsaturated fatty acids which helps to maintain the fluidity of cellular membrane . HHP has902) alone participated in the HHP-dependent regulation. It is generally believed that autophosphorylation of the histidine in the classical transmitter domain initiates signal transduction of two-component regulatory systems. Its absence in the HHP-responsive signaling pathway brings up a series of questions. For example, does the histidine located in the C-terminal alternative transmitter domain (H902) receive the phosphoryl group directly from ATP (autophosphorylation), or from other residues. It has been shown that the alternative transmitter domain of TorS from E. coli could be phosphorylated through a phosphotransfer involving a protein other than TorS (902 of TorS in deep-sea strain QY27, and its downstream receiver remain unknown for now.By constructing mutated TorS, we demonstrated that the HHP-responsive regulation of TorRS system was carried out through an alternative signaling pathway different from the well-known substrate-dependent regulation. All the three conserved phosphorylation sites in TorS were involved in the substrate-dependent regulation in deep-sea strain QY27, while the histidine located in the C-terminal alternative transmitter domain (Hhan TorS . But theE. coli showed that TorRS system also regulates genes involved in alkaline-resistance, such as tnaA, gadA and hdeABD, facilitating the cells to deal with the increase of pH resulted from TMAO reduction (In addition, we showed that the TorA could be induced by HHP under both aerobic and microaerobic conditions, but the induction was more significant during exponential phase under aerobic condition, and during stationary phase under microaerobic condition. Since the pressure remained stable throughout the growth experiment, this difference suggested that the HHP-responsive regulation of TorA in deep-sea strain QY27 might be correlated with other physicochemical or physiological factors. Increasing evidence showed that several extreme environmental conditions, e.g., pressure, temperature, salinity, and pH, had similar effect on microbial cells and will interfere with the redox homeostasis. Accordingly, a common adaptation mechanism is adopted to cope with different types of stresses . Studieseduction . It woultorA in both ΔtorR and ΔtorS mutants, the former was still capable of TMAO reduction slowly, but not the latter. It suggested the existence of another TMAO reductase whose function required the presence of TorS but not TorR. Previously, we have showed that, between the two TMAO reductase systems harbored by strain QY27, the torCA played a dominant role and was inducible by HHP, while the torZY expressed at a much lower level and its function and regulation were still mystery (ΔtorR was catalyzed by TorZY. Further investigation will be necessary to decipher the function of TorS and if there is another regulator that replaces TorR in the regulation of TorZY. Collectively, we identified that TorRS two-component system is responsible for the HHP-responsive regulation of TMAO reductase and discovered that HHP triggered a novel signal transduction pathway of TorRS system that was different from the classical substrate-dependent regulation. These findings provide novel understanding of the regulation of energy metabolism in response to HHP and the microbial adaptation to the deep-sea environment.In this study, we noticed that despite a constitutive expression of  mystery . It is pThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.NL: Conceptualization, Formal analysis, Investigation, Methodology, Writing – review & editing. TJ: Conceptualization, Formal analysis, Investigation, Methodology, Writing – review & editing. W-PC: Methodology, Writing – review & editing. X-QQ: Investigation, Project administration, Writing – review & editing. X-GL: Formal analysis, Supervision, Writing – review & editing. YL: Methodology, Supervision, Writing – review & editing. L-FW: Conceptualization, Funding acquisition, Supervision, Writing – review & editing, Writing – original draft. W-JZ: Conceptualization, Funding acquisition, Supervision, Writing – original draft, Writing – review & editing.


The relationship between cognitive ability (CA) and childbearing remains unsettled. Using Norwegian administrative registers with population coverage, we study how male lifetime fertility patterns differ across cognitive score groups, and how these changed across the 1950–1981 birth cohorts, covering a period characterized by rapid social and economic change. The analyses reveal systematic differences in fertility and fertility timing across CA groups, with high scoring males having delayed but ultimately higher fertility than lower scoring males. This pattern remains stable over time despite strong trends towards delayed and reduced fertility. The overall positive relationship between CA and fertility is primarily driven by high rates of childlessness in the lowest scoring group, with low-scoring males showing higher rates of parity progression at higher parities. Indeed, a recent systematic review concluded that a negative association between cognitive ability (CA) and fertility in modern Western societies strengthened over the course of the twentieth century, attributing the development to social and economic progress that disproportionately raised the reproduction rates in the lower part of the ability distribution . The lonThe hypothesis that social welfare policies raise the relative fertility of low-ability parents, contrasts with the results of a recent study from Sweden. The study used administrative data with full population coverage to assess the CA–fertility gradient for the 1951–1967 male birth cohorts . AlthougIn this paper, we examine the case of Norway. Using administrative register data, we extend the analysis to cover more than 30 birth cohorts. This enables us to examine in detail how the association between male CA and fertility changed across a period of extensive societal change: significant expansions of the educational system, rising female participation in the labour force, and the increasing availability of contraception and legal abortion. The period also saw rapid and substantial shifts in overall fertility patterns, with overall male fertility (measured at age 40) declining from 1.9 to 1.5 as the average age of fathers increased by 3 years . The causes of this rapid decline remain unclear [Our analysis covers 953 692 Norwegian-born males across the 1950–1981 birth cohorts, using stanine scores from military conscription tests and fertility data from national administrative registers with full population coverage. Conscription was voluntary and rare for women, precluding further analysis. The registers cover all births from 1960 through 2021, totaling 1 706 438 observed births registered to our paternal cohorts. The conscription test data cover the majority of males in every birth cohort, with coverage increasing from 72% to 86% across the 1950–1960 cohorts and exceeding 90% for all but two cohorts born after 1961 .We employ two statistical models to describe nuanced fertility patterns within these data. The first model examines average fertility at different ages for men with different CA stanine scores born in different years, using a hierarchical structure with shrinkage to smooth patterns. The second model assesses individual fertility histories (up to age 50) across cells defined by the father's birth cohort and stanine score, exploring differences and trends in childlessness rates and parity progression probabilities. Finally, we reproduce key patterns uncovered by these analyses using the ‘raw’ register data to ensure that results are not the artefacts of statistical modelling.. 21The data for this study were obtained from Norwegian administrative registers, provided by Statistics Norway . IndividDescriptive statistical models are used to smooth random variation in the data. Model 1 estimates the average probability of childbirths within cells defined by the father's birth cohort, age and stanine score using a logit specification. The model parameters are divided into two blocks, with the first block containing coefficients for first-order indices , and the second block containing second-order or interaction indices representing all combinations of the stanine score levels with each of the first-order indices. Both blocks of coefficients are assigned zero-centred normal distribution priors, with hyperparameters controlling the scales of the priors. See electronic supplementary material for further details.In brief, the model assumes that there are underlying patterns of similarities across cells (e.g. fertility being higher at certain ages), which can be exploited to better predict fertility propensity within each individual cell. The hierarchical structure reduces overfitting to noise by inducing shrinkage in the estimates.Model 2 estimates the conditional probability of having additional children before the age of 50, based on the number of previous children . This is treated as analogous to a duration model, where all males in a birth cohort are at risk of having a child, and only those who have a child are at risk of having a second child, and so on. Children of parity >5 are excluded from the parity progression analysis.The probabilities are specified using a logit model, with parameters again divided into two blocks. The first block (first-order) consists of indices for birth cohort, stanine score and past-parity, while the second block (second-order or interactions) includes indices for all two-way interactions between the first-order indices . The hyperparameters for the second-order block are set more conservatively than those for the first-order block to reduce volatility and increase smoothing. For further details and model code, see the electronic supplementary material (section D).Both models are specified and estimated using the Stan programming language for probabilistic modelling . See ele. 32 This difference indicates the generational shift in IQ scores if each child were to inherit their father's stanine score. For 22 cohorts observed to age 50 (1950–1971), comprising 572 029 scored potential fathers with 1 104 594 children, the child-weighted and unweighted stanine means are 5.045 and 4.982, respectively. The difference of 0.064 stanines, equivalent to IQ point increases of approximately 0.5 over one generation or about 0.17 IQ point per decade, implies that the observed fertility across the full sample was weighted towards those with above-average CA scores. Note that this is only intended as a summary indicator of the cognitive fertility gradient for men. It should not be viewed as an estimate of generational IQ change, which will also be affected by the cognitive fertility gradient for women as well as the heritability of CA.A summary measure of the CA–fertility association is the difference between the child-weighted and the unweighted mean CA score of the parent generation.a). This indicates a stable, overall positive association between CA and male fertility. Although fertility is declining monotonically over birth cohorts, the decline is of similar magnitude across stanine groups (b). Notably, the lowest-scoring males consistently have markedly lower fertility within each cohort.Estimated at the birth cohort level, this summary measure of the CA–fertility association shows no long-term trend, varying around the overall sample mean a. This ie groups b. Notablc). There has been a general shift towards delayed and reduced fertility for all CA groups across the studied cohorts.Distinct CA-specific age profiles of fertility emerge from the data. Low-scoring males persistently have the highest fertility at young ages, while higher-scoring males begin later but ultimately end up with higher fertility c. There a, by excluding unscored males, likely yields conservative estimates, as unscored males tend to have scores in the lower part of the CA distribution [figure 1b). Their inclusion would consequently further increase the gap between the child-weighted and unweighted means.Note that figure 1ribution  with fera). This reveals a stark decline in fertility at ages below 30 across all CA groups, partly offset in the higher CA groups by an increase in fertility after the age of 30 (b).3 Repeating the analysis to assess cohort shifts across shorter time spans finds large shifts between 1950–1960, smaller shifts in the same direction 1960–1970, and a new pattern 1970–1980 where fertility grew increasingly concentrated around the early thirties with reduced fertility at both lower and higher ages .To assess how fertility age profiles have shifted over time for different score groups, we compare the fertility–age profiles of the 1950 and 1980 birth cohorts for three different score groups a. This rge of 30 b.3 Repeaa). Conditional on having a child (i.e. of parity 1), the probability of having an additional child increases with stanine score . For higher parity progressions, the gradient is reversed, with low-scoring males more likely to have additional children. Comparing trends across all cohorts finds childlessness rates almost doubling, with larger absolute changes for low-scoring males. Changes at later parity progressions are more moderate .Using model 2 to estimate parity progression rates as a function of birth year and stanine score, we find that the main driver of the CA–fertility gradient is a higher rate of childlessness among males in the lowest-scoring groups . The changes were large (e.g. a decline of seven percentage points in the probability of having a child at age 26) but occurred across stanine groups , with stronger fertility reductions in low-scoring groups . Third, while the shift towards delayed fertility seems to have stopped, fertility patterns have not reversed: births at ages below 30 and above 35 have declined in recent cohorts, concentrating fertility within a narrower age range . Lastly, the family size distribution for low-scoring males is more polarized, exhibiting both heightened childlessness rates and raised parity progression rates at higher parities .male fertility in these parts of the Scandinavian peninsula during this period.These patterns are systematic, sizeable and precisely inferred by conservative hierarchical models with parameter shrinkage. They are also corroborated by raw data visualizations . Although our data sample only covers Norway, similar patterns have been observed in neighbouring Sweden , indicatTwo important limitations should be stressed. First, military conscription historically only covered males, so the analyses cannot assess the female CA–fertility gradient. Other studies report stronger negative CA–fertility gradients for women . This al


We constructed a high-performance biosensor for detecting uric acid by immobilizing an engineered urate oxidase on gold nanoparticles deposited on a carbon-glass electrode. This biosensor showed a low limit-of-detection (9.16 nM), a high sensitivity (14 μA/μM), a wide range of linearity (50 nM–1 mM), and more than 28 days lifetime. We constructed a high-performance biosensor for detecting uric acid by immobilizing an engineered urate oxidase on gold nanoparticles deposited on a carbon-glass electrode. The most frequently employed strategy in the field of biosensing consists in the encapsulation of the enzymes into porous network materials or the immobilization on nanofibers.6,7 The confinement of the enzymes can physically protect them from the outside hyperthermia and reduce their exposure to the potentially harsh environment. Furthermore, the structural stability of enzymes can be enhanced through coordination and intermolecular interactions inside the cage.8 However, the protection conferred by these materials has the disadvantage to decrease the actual catalytical activity of enzymes due to the reduced accessibility of analytes and the possible occupation of the active sites by the inner materials.9 Consequently, this methodology sacrifices catalytic performance to improve thermal stability, which is not ideal for the development of more efficient biosensing platforms. In view of this, a rational strategy would be expected that can endow enzymes with both high thermal stability and outstanding catalytical activity.Biosensors based on oxidoreductase enzymes have been intensively studied during the last decade due to the requirement for more rapid and precise diagnosis protocols.e.g., thermal, pH, additives) while retaining of even enhancing the catalytical efficiency.10–14 The specificity of the enzyme toward a substrate or a cofactor can also be optimized along with its regioselectivity, and stereoselectivity in some catalytic reactions.15,16 Thermal stabilization can also be enhanced by introducing salt bridges and/or disulfide bonds.17 Thus, protein engineering can improve the catalytic efficiency of enzymes for existing as well as new electrochemical biosensors.Structural-guided enzyme engineering with the aid of computational methods or the directed evolution of an enzyme can provide a solution to this dilemma by increasing the enzyme stability  onto electrodes through the formation of the disulfide bond between BSA and glutathione  was selected as the model analyte and we used urate oxidase (UOx) as the model enzyme in this study.GSH, see .21Bacillus sp. TB-90  was recombinantly produced in E. coli and purified by Ni2+-affinity chromatography in our group  and GSH were incubated in presence of H2O2, a molecular weight increase was observed by size-exclusion chromatography-mass spectrometry  image clearly showed the deposited AuNPs on the surface of the electrode with a uniform distribution  while thncreased . This shThe characterization of the stepwise procedure for the fabrication of the UA biosensor was also performed electrochemically by cyclic voltammetry CV, . CompareTo further characterize the construction procedure, we conducted electrochemical impedance spectroscopy (EIS) to monitor the impedance of the biosensing interface . The nonWe then studied the influence of different parameters on the performance of the biosensor, comprising concentration of thioglycolic acid and glutathione, incubation time for them, and the catalytic reaction time  and the logarithm of the UA concentrations in the range of 0.05–1000 μM  produced a great decrease of the current value (ca. 60 μA). Moreover, a sample obtained by mixing all the interferents  with UA (100 μM) produced the same current as for a sample with UA alone (100 μM). These data demonstrates the high selectivity over competition for our UA biosensor, in agreement with the literature.30 Finally, the reliability of our biosensor was evidenced by measuring standard samples, since a regression coefficient of 0.9998 was calculated , linear detection range, selectivity, and reliability. The current in square wave voltametric test (SWV) decreased at increasing concentration of UA . I current value decreased of 15.44 μA for commercial UOx-based biosensor and of 63.80 μA for engUOx-based biosensor  that supports the different catalytic behavior  was almost two-fold higher than the one with commercial UOx (8.38 μA[logCUA]−1), which allows for a more precise quantification of UA with the former . Notably, the LOD was thousand-fold lower and the linear range was hundred-fold higher for the biosensor with engUOx compared with the same with commercial UOx. The sensitivity of UA biosensor with engUOx  for PhD scholarship funding. F. G. M. thanks the NWO Sector Plan for Physics and Chemistry for funding.There are no conflicts to declare.CC-059-D3CC01869E-s001


This paper uses a SWOT  analysis to overview the option of fertility preservation in women with genetic diseases, who would later use preimplantation genetic testing for monogenic disorders, in order to not transmit their condition. Strengths associated with elective oocyte freezing are ethical considerations, overall maternal and fetal safety, and effectiveness, if performed at <35 years of age. Weaknesses are related to costs and rare but present (<1–3%) risks of maternal complications. Counselling on fertility management aimed at preventing infertility offers a valuable opportunity, the same as it has been in oncological patients’ care. The potentially high percentage of women with genetic conditions who would return to use their frozen oocytes also represents an opportunity together with the minimization of the need for egg donation, which has higher obstetrical risks compared to the use of autologous oocytes. Finally, a threat is represented by the potential psychological distress to young women who could never attempt to become pregnant through preimplantation genetic testing, or do it before any decline in their fertility. Potential unknown future long-term health risks for children conceived after egg vitrification/thawing are also a threat, but current knowledge is reassuring. Altogether, early counselling on the option of fertility preservation should thus be incorporated into standard care of all patients with any genetic condition. The purpose of fertility preservation is to safeguard the reproductive potential of patients whose future fertility is endangered by various medical conditions, such as cancer or other diseases, or by aging. It is well known that female fertility declines with the passage of time and, in this sense, fertility preservation allows women to protect their parental project for possible future realization . A growiOocyte cryopreservation practice is a feasible strategy to preserve female fertility potential . Oocyte Other patients who could benefit from egg freezing include carriers or patients with genetic diseases. In particular, reproductive consequences of Turner syndrome include primary amenorrhea, premature ovarian insufficiency, and infertility. Very recently, the first live birth achieved using cryopreserved oocytes in a woman diagnosed with mosaic Turner syndrome has been reported, paving an optimistic path for the use of this technique in the future . Other gOocyte collection and in vitro fertilization, however, also allows preimplantation genetic testing for monogenic diseases (PGT-M), which identifies embryos that will be affected by the disease in order to transfer only those without the mutation. Currently, PGT-M can be used to detect more than 600 genetic conditions, and its use is rapidly increasing .Interest in PGT-M for affected individuals/carriers of single-gene disorders is explained by two main crucial domains, namely (i) avoidance of the suffering of an affected child and of the termination of an affected pregnancy  and (ii)While this evidence supports the employment of PGT-M in patients with genetic diseases, awareness about their reproductive prognosis in assisted fertilization cycles is still scarce. Van Der Keler and colleagues have recently published data from a study of 2265 PGT-M cycles involving couples with monogenic diseases not associated with reduced ovarian reserve or ovarian response. The authors showed that the study population has a lower reproductive prognosis in terms of cumulative live birth rate (CLBR) than infertile couples who do not perform PGT for intention-to-treat, despite a younger age. Importantly, this effect was observed in the absence of any known impact of the genetic disease on female ovarian function. While the overall prognosis of PGT-M remains good (CLBR 29.4% compared to 35.0% in non-PGT-M couples), , this imHowever, even with as few as two retrieved oocytes or a single embryo available for transfer, it is possible to predict a cumulative live birth rate (CLBR) of over 10% in PGT-M cycles . This suIn this context, the strategy of oocyte cryopreservation at a young age would prevent the detrimental effects of the reduced ovarian reserve and the age-related increase in aneuploidy and would allow the storage of a high number of oocytes and therefore blastocysts for PGT-M.On these bases, an analysis of strengths, weaknesses, opportunities, and threats  was conducted to overview oocyte cryopreservation for women who are at high risk of having offspring affected by genetic diseases .The Ethics Committee of the American Society for Reproductive Medicine found elective egg freezing to be ethically permissible based on the main arguments of enhancing reproductive autonomy and promoting social equality .Additionally, avoidance of suffering of an affected child and avoidance of termination of an affected pregnancy  seem to Safety issues such as potential harm to the embryos and short- and long-term health problems for the baby have been identified as the main concern for couples with monogenic diseases in relation to assisted reproduction . ImportaOocyte freezing could therefore raise the number of oocytes available for those women who, due to their risk of transmitting a genetic disorder to their children, intend to proceed with preimplantation diagnosis in the future to obtain a healthy baby. This strategy has already been applied to build larger cohorts of available oocytes ,25, and High rates of live births can be achieved when egg freezing is performed at a young age, approximately before the age of 38. Literature however shows a lack of agreement on the optimal time for cryopreservation of oocytes. Some studies show that the highest probability of achieving successful births from cryopreserved oocytes is before the age range of 36–38 years ,30. OtheThe cost of elective oocyte freezing can vary depending on several factors, including the geographical location, the clinic or fertility center chosen, and any additional services or medications required. In most countries, non-medical fertility preservation is not supported by public health systems and is expensive. Financial issues also play a strong role in patient decision-making. This means that this strategy is not available to all women, exacerbating existing inequalities and raising concerns about equity, as resources would be diverted from solving other fertility issues that are, by contrast, more evenly distributed across the population. These considerations highlight the complex nature of incorporating elective freezing into public reimbursement schemes. Healthcare systems must carefully evaluate the potential benefits and drawbacks, taking into account issues of equity, cost-effectiveness, and the broader context of fertility care in patients with genetic disorders.It is not always clear what constitutes as a medical reason for fertility preservation, and some argue that this represents an unnecessary medicalization of women, bearing in mind the minimal, but real, risks of ovarian stimulation and ovarian retrieval. One of the most frequent consequences of ovarian stimulation is ovarian hyperstimulation syndrome (OHSS) . Mild toWomen with a compromised ovarian reserve may need several controlled ovarian stimulations and ovarian retrievals to obtain a favorable number of oocytes. Multiple cycles of oocyte accumulation can be costly and can potentially have a physical and psychological impact. This might apply, for example, to patients with sickle cell disease , galactoThere is a need to improve fertility education for the general public. Many women report having received information about elective egg freezing mostly from friends and non-medical sources . When inOne of the main criticisms directed at elective egg freezing for non-medical reasons is a low cost–benefit ratio, and also due to a low usage rate of previously frozen oocytes. The largest published study reports that only 12.1% of women return to use their oocytes . This isEuropean statistics indicate a growing demand for donated oocytes in assisted reproductive technology. This highlights the escalating need for donated oocytes and the importance of addressing this demand to support couples undergoing fertility treatments in Europe . The useDiscussing the potential of elective egg freezing with women with genetic disorders, who have never thought about family issues due to their young age or lack of a partner, could encourage the patient to further postpone pregnancy, which increases the risk of maternal and neonatal complications . In someThe laws that regulate patients’ access to the procedure vary from country to country. For example, there are European countries where this procedure is forbidden, others where it is allowed in accordance with some medical conditions, and some others where it is also allowed without any clinical indication. Similarly, different states in the USA have different legislations regarding fertility preservation and relative insurance coverage . FurtherMany studies reported very good results in terms of survival and clinical outcomes after thawing vitrified oocytes, as we have already discussed above. Nevertheless, there are currently no data about the survival rate and clinical outcomes of oocytes affected by or carrying monogenic mutations. Moreover, these could also be very heterogeneous due to the heterogeneity that exists between the various monogenic disorders. Finally, although it appears reassuring that no major obstetrical and perinatal risks have been found in pregnancies carried out with frozen oocytes so far, these risks cannot be completely ruled out ,19,21.Fertility counseling should be an integral part of the management of monogenic disorders. Unfortunately, very often, the discussion of the genetic disorder’s impact takes place when the woman begins to contemplate her parental plan, thus more frequently at an advanced age and potentially too late to intervene effectively . MoreoveIn fact, family planning remains poorly discussed. Among pediatric and adult nephrologists involved in autosomal dominant polycystic kidney disease care, only 20–40% reported informing their patients about the available pre-implantation diagnostic options .Not surprisingly, the awareness and attitude regarding fertility preservation have never been investigated among professionals caring for patients affected by monogenic diseases.On the patients’ side, to the best of our knowledge, a survey regarding fertility preservation in non-oncologic genetic diseases is only available for female patients with cystic fibrosis , among wEven for genetic diseases leading to premature ovarian insufficiency, no specific recommendations concerning fertility preservation are available , and theAt the present stage, it seems therefore advisable for the professionals involved to refer patients to reproductive healthcare experts, in order to ensure adequate counseling and management through a multidisciplinary approach.As a matter of fact, as it is still very challenging to outline what qualifies as a medical reason to cryopreserve oocytes, women need to be informed about the pros and cons of elective egg freezing by a fertility preservation team, ideally consisting of a gynecologist, an embryologist, a psychologist or counselor, and a geneticist. This review aims at providing useful information to be shared with patients in this context.Non-invasive prenatal diagnosis (NIPD) of monogenic disorders has been developed thanks to the presence of cell-free fetal DNA that is detectable in the maternal circulation, and it can be offered to couples at known increased risk without the associated miscarriage risk that accompanies invasive prenatal testing .Detection of paternally inherited and de novo mutations has become quite uncomplicated, and several techniques have been improved to be used in the clinical setting, but a greater challenge is represented by the detection of maternally inherited mutations due to the abundant presence of maternal cell-free DNA .The spectrum of monogenic disorders that can be tested for by using NIPD includes cystic fibrosis, spinal muscular atrophy, and Duchenne and Becker muscular dystrophies. Nevertheless, many monogenic conditions are relatively rare, and therefore often a custom-made and disease-specific detection method is necessary for rare familial variants. In addition, these tests need to be worked up prior to pregnancy in order to be appropriately validated and therefore usable at the correct timing of pregnancy to aid in decision-making. The testing is technically challenging, making it less readily available for commercialization and diffusion in the clinical setting .Despite its undoubted usefulness, NIPD is still surrounded by some concerns in the case of rare monogenic diseases, such as the unawareness about critical issues regarding incomplete penetrance and variable expressivity, and the absence of large-scale validation and patient follow-up. This ultimately leads to patients making decisions about their pregnancy based on incomplete information.Moreover, expanding the procedure to low-risk pregnancies might increase detection of variants of uncertain or unknown significance, with no clear clinical interpretation, which challenges patient counseling .Most importantly, when a mutation or a gene involved in a specific disease is detected, couples must be informed about the risks and alternatives of either continuing or interrupting the pregnancy. Even if therapeutic interruption of pregnancy might have positive outcomes, especially for patients with strong distress caused by the diagnosis of fetal malformation, it undoubtedly has important consequences on the mental and reproductive health of women, as it carries a great traumatic burden and psychopathological effect .Monogenic causes of female infertility have increased dramatically over the last few decades. Hundreds of genes have been identified, and the clinical validation of just as many gene–disease relationships has been evaluated . In addiAdvancements in this field will imply moving towards personalized reproductive medicine, with an emphasis on molecular genetic diagnostics and, hence, prevention. In addition, the literature shows that even patients carrying a disease with an unknown association with female fertility  still have a lower prognosis in terms of cumulative live birth rate compared to non-PGT couples . In thisThe main limitation of our analysis is that it conveys information that might not be widely applicable to all clinical contexts. As a matter of fact, relevant technical difficulties exist for both oocyte cryopreservation and PGT-M, and a very specific counselling is required for both, as outlined by current guidelines ,54. Pati