Cinara cedri (Lachninae), Eriosoma lanigerum (Eriosomatinae), and Therioaphis trifolii . In this study, the morphology and distribution of the antennal sensilla of apterous adults were observed by scanning electron microscopy. Three morphological types were identified ; the first two were distributed on the antennal primary rhinaria. A pattern of primary rhinaria in C. cedri was found that differed from that of E. lanigerum and T. trifolii and consists of 1 large placoid sensillum (LP) on the 4th segment, 2 LPs on the 5th segment, and a group of sensilla on the 6th antennal segments. Later, we recorded and compared neuronal responses of the distinct placoid sensilla in the primary rhinaria of the three aphid species to 18 plant volatiles using a single sensillum recording (SSR) technique. The results indicated that the functional profiles based on the tested odorants of the primary rhinaria of the three investigated aphid species were clustered into three classes, and exhibited excitatory responses to certain types of odorants, especially terpenes. In C. cedri, the ORNs in LP6 exhibited the highest responses to (±)-citronellal across all tested chemicals, and showed greater sensitivity to (±)-citronellal than to (+)-limonene. ORNs in LP5 were partially responsive to α-pinene and (–)-β-pinene in a dose-dependent manner. Across different species, E. lanigerum showed significantly stronger neuronal responses of LP5 to several terpenes, such as (–)-linalool and α-terpineol, compared to other species. In T. trifolii, the neuronal activities in LP6 showed a greater response to methyl salicylate as compared to LP5. Overall, our results preliminarily illustrate the functional divergence of ORNs in the primary rhinaria of aphids from three subfamilies of Aphididae and provide a basis for better understanding the mechanism of olfactory recognition in aphids.Olfaction is important for mediating aphid behaviors and is involved in host location and mating. Antennal primary rhinaria play a key role in the chemoreception of aphids. The function of the peripheral olfactory system in the subfamily Aphidinae has been intensively studied, but little is known about other subfamilies of Aphididae. Therefore, three aphid species were selected to study the olfactory reception of plant volatiles:  Olfaction plays an important role in host location as well as mating in insects  -3-hexen-1-ol, is a main attractant at high concentrations  in A. pisum  cis-jasmone  technology. The neuronal response profile of three aphid species to 18 plant-derived volatiles was mapped. Additionally, the neuronal functions of placoid sensilla in the primary rhinaria were compared based on sensillum type and aphid species. Our results preliminarily illustrate the neuronal mechanisms of olfactory detection at the peripheral nervous level in three aphid species, beyond Aphidinae, to plant volatiles.Although previous studies have reported that the primary rhinaria were mainly responsible for semiochemical detection in several Aphidinae species, little is known about the odorant detection beyond the subfamily Aphidinae. To better understand the mechanism of olfactory recognition in different subfamilies of Aphididae, we selected three aphid species, C. cedri was collected from Cedrus deodara (Roxb.) in the Institute of Plant Protection, Chinese Academy of Agricultural Sciences (CAAS), Beijing, China . T. trifolii was collected from the alfalfa fields at the Langfang Experiment Station of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences (CAAS), Langfang, Hebei province, China , and reared in our laboratory at 20–22°C, 60–70% relative humidity, and under a photoperiod of 16:8 h (light: dark). E. Lanigerum was collected from Malus pumila in the suburbs of Tianjin, China . Adult apterous viviparae were used in all experiments.Adult Antennae of aphids were excised from the base with fine forceps and dehydrated in ethanol serial solutions . After drying in a Critical Point Dryer , the samples were coated in gold by an ion sputtering device . Finally, the samples were examined with a HITACHI SU8010 scanning electron microscope  at 3–10 kV. Pictures were only adjusted for brightness and contrast using Adobe Photoshop CS6 (Adobe Systems). Sensilla types were classified according to methods used by previous studies . An electronic circuit was established by these electrodes to extracellularly record ORN action potentials -2-hexenal, belongs to the green leaf volatiles and is one of the most abundant volatiles found in numerous plants. Methyl salicylate, on the other hand, is a known HIPV. In addition, three compounds, namely nepetalactol, nepetalactone, and allyl isothiocyanate, are associated with non-host-plants of the tested aphids. The study aims to determine the function of ORNs from three aphid species in response to compounds from different sources.In total, 18 volatile plant compounds with different chemical structures were chosen for the study . Common −7 to 10−3 g, respectively. A sample containing only paraffin oil served as a control, tested at the beginning and at the end of each recording  were purchased from Sigma-Aldrich Co.  and used for single sensillum recording . Each oft-test (α = 0.05). Multiple comparisons of data were assessed by one-way analysis of variance (ANOVA) following Duncan's multiple range test (α = 0.05).Graphs and statistics about sensilla responses and dose responses were generated by GraphPad Prism 7.0 . All data were calculated as mean ± SEM. A heat map was generated in HemI 1.0 , pedicel (Pe), and flagellum (Fl)  observation revealed that the antennae of lum (Fl) –C. The tC. cedri from the distal to the proximal end, showing an obvious primary rhinarium at the distal end -citronellal and (–)-piperitone with no significant differences observed (P > 0.05) (P < 0.05). Furthermore, neuronal responses in LP6 to (+)-limonene and (±)-citronellal both showed dose-dependent patterns at a range of dose from 10−7 to 10−3 g, with EC50 values of 4.31 × 10−6 g and 2.62 × 10−6 g, respectively  in the primary rhinaria of  > 0.05) . The ORNectively –C.trans-2-hexenal (30.8 ± 2.6 spikes/s), (±)-citronellal (30.2 ± 3.9 spikes/s), and α-terpinene (29.0 ± 2.9 spikes/s), but were inclined to α-pinene (16.8 ± 1.6 spikes/s), (–)-β-pinene (18.7 ± 2.9 spikes/s), nepetalactol (15.5 ± 1.2 spikes/s), and nepetalactone (10.0 ± 1.5 spikes/s) compared to the other three sensilla (P < 0.05). We recorded the responses of ORNs in LP5I to α-pinene and (–)-β-pinene at a range of dose from 10−7 to 10−3 g. The results demonstrated that ORNs were more sensitive to (–)-β-pinene (EC50 = 8.40 × 10−7 g) than to α-pinene (EC50 = 1.10 × 10−6 g)  showed similar sensitivity to that in LP5I (EC50 = 3.13 × 10−5 g)  –F. We al 10−5 g) –I.50 = 8.36 × 10−6 g). As the dose increased from 10−7 to 10−3 g, the neuronal firing rate increased from 6.5 to 28 spikes/s, while the neuronal firing rate decreased to 24.25 spikes/s at a higher dose of 10−3 g (The ORNs in LP5II showed excitatory responses to α-terpineol (33.2 ± 3.0 spikes/s), α-terpinene (33.0 ± 2.8 spikes/s), and geranyl acetate (34.0 ± 3.8 spikes/s). The response of neurons in LP5II to α-terpinene also exhibited a dose-dependent pattern . β-citronellol, sabinene, methyl salicylate, (±)-citronellal, and AITC also activated ORNs significantly more in LP5 than in LP6 with mean values of 23.0 ± 3.8 (P < 0.01), 21.3 ± 3.2 (P < 0.01), 19.7 ± 1.2 (P < 0.05), 19.3 ± 3.1 (P < 0.05), and 17.7 ± 2.3 spikes/s (P < 0.05) respectively. However, ORN responses to the remaining components showed no difference between LP5 and LP6 (P > 0.05)  , B.T. trifolii, methyl salicylate significantly elicited higher responses in ORNs in LP6 (18.8 ± 2.7 spikes/s) than in LP5 ORNs (3.5 ± 2.2 spikes/s) (P < 0.01), while responses of ORNs to other components did not show any significant differences between LP5 and LP6 (P > 0.05) , α-terpinene (21.0 ± 3.8 spikes/s), and (±)-citronellal (17.0 ± 3.5 spikes/s). ORNs in LP5 mainly responded to α-terpinene, (+)-limonene, (±)-citronellal, AITC, and (-)-linalool, which elicited firing frequencies of 22.6 ± 2.4, 20.3 ± 4.2, 20.0 ± 3.6, 17.5 ± 2.0, and 16.8 ± 3.4 spikes/s, respectively (In  > 0.05) , D. Moreectively .T. trifolii to 9 tested chemicals  were lower than in C. cedri and E. lanigerum (P < 0.05) (E. lanigerum produced the largest responses to (–)-linalool and α-terpineol compared to C. cedri and T. trifolii, while C. cedri had the strongest response to geranyl acetate compared to T. trifolii and E. lanigerum (P < 0.05). There were no significant differences in neuronal responses among C. cedri, T. trifolii, and E. lanigerum induced by AITC, (±)-citronellal, and nepetalactone (P > 0.05)  . Additio > 0.05) .C. cedri have stronger activities compared to the other two species in most cases, especially in response to trans-2-hexenal, (–)-piperitone, α-terpineol, (–)-linalool, β-citronellol, (±)-citronellal, (+)-limonene, and sabinene (P < 0.05) (E. lanigerum showed the highest responses to (–)-myrtenal and α-pinene (P < 0.05), while nepetalactone elicited the strongest responses in T. trifolii (P < 0.05)  , 8. Howe < 0.05) .C. cedri, E. lanigerum, and T. trifolii) from three subfamilies of Aphididae, which suggests a role of olfactory detection in the host-plant selection.Identifying the antennal structure and sensilla types of aphid species is needed to understand olfactory perception mechanisms in aphids. In this study, we observed and compared the morphology, distribution, and function of the antennal sensilla of three aphid species  on the 6th antennal segment. The shape of SPs on the 6th antennal segments varied in different aphid species. For example, SPs on the 6th antennal segments of T. trifolii were replaced by 2 stellate sensilla, which have only been identified in the Drepanosiphinae subfamily of aphids -limonene, sabinene, α-pinene, and (–)-β-pinene, and monoterpenoids (terpineol) are the major volatiles of the essential oil of Cedrus spp. -limonene, terpineol, and sabinene. This demonstrates that the strongest responses of C. cedri to monoterpenes and monoterpenoids were due to the detection of host plant volatiles. Moreover, we found that ORNs in the primary rhinaria of all three aphid species were strongly stimulated by the green leaf volatile trans-2-hexenal, which is one of the most abundant volatiles of plants  using 18 plant volatile compounds, including monoterpenes, monoterpenoids, and aliphatics. We found that most of the 18 chemicals could induce neuronal responses in the primary rhinaria of Brevicoryne brassicae (L.), A. fabae, and A. pisum -linalool, which is a plant volatile released from both intact and caterpillar-damaged apple foliage , while the neuronal activities in the primary rhinaria broadly respond to both plant volatiles and sex pheromone components (Dawson et al., In general, neuronal responses in the secondary rhinaria of male and gynoparae aphids are sensitive to the well-known sex pheromones of several aphid species, such as C. cedri, E. lanigerum, and T. trifolii from three subfamilies of Aphididae were identified and functionally characterized. The response profiles of ORNs in the placoid sensilla of primary rhinaria respond to plant-derived volatiles and reveal the intraspecific and interspecific variation of neuronal functions in aphids. This result provides a good basis to compare inter-species olfactory detection and host plant selection in different aphid species.In conclusion, the antennal sensilla on the primary rhinaria of The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors.G-RW, BW, and L-LY contributed to conception and design of the study and wrote the first draft of the manuscript. L-LY performed the experiments. BW and L-LY performed the statistical analysis. G-RW, BW, JS, and L-LY wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.


Background: Sepsis is one of the leading contributors to global mortality and morbidity, causing multi-organ failure, mainly involving cardiovascular failure, both systolic and diastolic dysfunction, leading to adverse clinical outcomes. There is little clinical data on the correlation with the mortality of patients with type 2 diabetes mellitus (T2DM) with sepsis and septic shock and left ventricular diastolic dysfunction. Our study sought to assess whether the severity of diastolic dysfunction could predict 28-day mortality.Methodology: The study included T2DM patients admitted to the intensive care unit (ICU) with sepsis and septic shock defined according to the Third International Consensus Definitions for Sepsis and Septic Shock at a tertiary care center in northern India. A total of 132 patients  score = 25.74 ± 4.79; Sequential Organ Failure Assessment (SOFA) score = 12.34 ± 3.36) underwent transthoracic echocardiography within two hours of ICU admission till 28 days of admission or till mortality occurred. Clinical variables (APACHE II and SOFA score) and cardiac biomarkers, such as N-terminal pro-B-type natriuretic peptide (NT-pro-BNP), troponin I, and creatine phosphokinase-MB, were measured at the time of admission and after 72 hours to compare with mortality. Diastolic dysfunction was defined according to the American Society of Echocardiography (ASE) 2009 guidelines, classifying subjects into grade 0 , if early diastolic velocity (e') ≥ 8 cm/s; grade 1 (impaired relaxation), if e' < 8 cm/s and early (E) to late (A) ventricular filling velocities (E/A) ratio < 0.8; grade 2 , if e' < 8 cm/s, E/A = 0.8-1.5, and peak E-wave velocity by the peak e' velocity (E/e') ratio = 9-12; and grade 3 (restrictive), if e' < 8 cm/s, E/A > 2, deceleration time (DT) < 160 ms, and E/e' ≥ 13.Results: Thirty-seven (40.65%) out of 132 patients had diastolic dysfunction on initial echocardiography, while 54 (59.34%) had diastolic dysfunction on at least subsequent echocardiography. Total mortality was 68.93% with the highest mortality (100%) observed among those with grade 3 diastolic dysfunction. The 28-day mortality with diastolic dysfunction in sepsis and septic shock patients showed significant results (p < 0.001), indicating that with a higher E/A ratio or higher grade of diastolic dysfunction with the increase in SOFA score, the early ICU mortality is the highest and have the shortest duration of ICU stay with mean ± SD = 6.2 ± 2.48, as compared to other grades with 100% mortality. Also, the cardiac biomarker NT-pro-BNP was markedly elevated with a mean ± SD value of 503 ± 269.3 pg/ml, indicating early predicted mortality. No correlation was detected between mortality and the mean levels of fasting blood sugar, postprandial blood sugar, and glycosylated hemoglobin.Conclusion: Our study concluded that diastolic dysfunction is an important and strongest independent mortality predictor in patients with T2DM with severe sepsis and septic shock, and the higher the grade of diastolic dysfunction, the higher the mortality with the lowest mean ICU stay. Sepsis affects 48.9 million people per year globally, of which 11 million die, representing a mortality rate of 19.7% . CardiovThis observational cohort study was conducted at a tertiary care center in northern India over 18 months from July 2022 to November 2022 after obtaining approval from the Institutional Ethics Committee, All India Institute of Medical Sciences, Rishikesh (AIIMS/IEC/2/321). T2DM was defined based on the American Diabetes Association (ADA) criteria, which include an elevated glycated hemoglobin A1c (HbA1c) level of 6.5% or higher, a random blood sugar (RBS) measurement equal to or exceeding 200 mg/dL, a fasting blood sugar (FBS) reading of 126 mg/dL or higher, and an oral glucose tolerance test (OGTT) result that reveals a two-hour glucose level in venous plasma of 200 mg/dL or greater. All subsequent patients aged > 18 years having T2DM (ADA criteria) with sepsis and septic shock (according to surviving sepsis guidelines) were included. Severe sepsis was defined as having any of the following: (i) evidence of an infection or clinical suspicion of infections; (ii) leukocytosis or leukopenia with total leucocyte count <4000 or >12,000; (iii) tachypnea with respiratory rate > 22/minute or needing mechanical ventilation; (iv) systolic blood pressure < 90 mmHg; (v) temperature > 38°C or < 36°C; or (vi) any evidence of any end organ failure. Septic shock was defined as "severe sepsis with severe hypotension after failed fluid resuscitation lasting for more than one hour, or requirement of a vasopressor support" [Clinical dataAll demographics and clinical parameters, daily fluids resuscitation, vasopressor requirement, hematological and biochemical data, including FBS, PPBS, and HbA1c, arterial lactate levels, sepsis biomarkers, cardiac biomarkers , blood cultures, and cultures of specimens from the site of infection were measured since the time of diagnosis of severe sepsis or septic shock and on subsequent days of echocardiography and the mean, minimum, and maximal were calculated. Disease severity by SOFA and APACHE II scores was assessed on the day of ICU admission, 72 hours after admission, and soon to 28 days after admission or till mortality occurred.EchocardiographyAll patients underwent serial transthoracic echocardiography using an echocardiographic machine, with an echo probe of 2-4 MHz. The initial echocardiogram was done after the diagnosis of sepsis in the ICU. The subsequent echocardiogram was performed on the next day to confirm the stability or any changes in the echocardiogram. Diastolic dysfunction was assessed by measuring E and A peak velocities using spectral Doppler of mitral inflow and e’ and a’ velocities using tissue Doppler of the septal mitral annulus in the apical four-chamber view. We defined diastolic dysfunction according to the American Society of Echocardiography (ASE) 2009 guidelines, classifying subjects into grade 0 , if e' ≥ 8 cm/s; grade 1 (impaired relaxation), if e' < 8 cm/s and E/A < 0.8; grade 2 , if e' < 8 cm/s, E/A = 0.8-1.5, and E/e' = 9-12; and grade 3 (restrictive), if e' < 8cm/s, E/A > 2, deceleration time (DT) < 160 ms, and E/e' ≥ 13  and analysis was done using MS Excel 2016 and SPSS version 23 . Assessment of diastolic dysfunction and its correlation with the 28-day mortality of the patient was done by chi-square test. The SOFA and APACHE II scores were analyzed by the Mann-Whitney test. The inotropes requirement was analyzed by Pearson's chi-square test. The cardiac biomarkers Trop-I, CPK-MB, and NT-pro-BNP were analyzed by the Mann-Whitney test. FBS, PPBS, and HbA1C were analyzed by unpaired Student's T-test. All the categorical variables are reported as percentages and continuous or discrete variables are reported as means ± standard deviation (SD). A p-value < 0.05 was considered significant.Acinetobacter baumannii (26.5%), Escherichia coli (16.6%), Klebsiella (12.80%), Pseudomonas (6.80%), and Streptococcus pneumonia (2.20%). A total of 88 patients (66.6%) experienced septic shock despite fluid resuscitation requiring inotropes: single inotropes, i.e., norepinephrine , dual inotropes, i.e., norepinephrine and vasopressin , and triple inotropes, i.e., norepinephrine, vasopressin, and dobutamine/dopamine  , complicated urinary tract infections (21.20%), hospital-acquired pneumonia (12.80%), gastrointestinal infections (6%), skin and soft tissue infections (4%), and others (14%). The source of positive infections was confirmed by culture-positive organisms isolated in 86 (65.15%) patients, of which, the most common organisms were All enrolled patients were under mechanical ventilator at the time of echocardiography due to respiratory dysfunction or failure or low Glasgow Coma Scale score. In all the patients, after admission to the intensive care unit, echocardiography was performed; 37 (40.65%) had diastolic dysfunction at the time of initial echocardiography and 54 (59.34%) had diastolic dysfunction on subsequent echocardiography. The diastolic dysfunction parameters were assessed, left ventricular ejection fraction was calculated, and the studies were averaged and used for further analysis.Predictors of mortalityA total of 132 patients were enrolled in this study, of which 88 (66.67%) died in the ICU and 44 (33.3%) survived. Among the non-survivors, the highest mortality was found in patients with grade 3 of LVDD. Patients who had normal LVDD showed the lowest mean ICU stay (6.26.2 ± 2.48) as compared to those who had grade 2 LVVD (9.07 ± 4.06) and grade 3 LVVD (12.65 ± 10.68), respectively.The mortality at 28 days was analyzed by chi-square test, which shows 13 (100%) mortality in grade 3 LVDD, 19 (86.37%) mortality in grade 2, 41 (73.22%) in grade 1, and 15 (36.7%) in normal LVDD patients, suggesting that rise in mortality at 28 days as the grades of diastolic dysfunction increase indicates diastolic dysfunction is a major important indicator for early mortality in sepsis  as compared to survivors , which was statically significant .The cardiac biomarker NT-pro-BNP (mean ± SD) calculated by the Mann-Whitney test was found to be higher in non-survivors (503.24 ± 269.3) as compared to survivors (503.24 ± 269.3) , indicating an increase in cardiac enzyme showing the myocardium involvement in sepsis and septic shock .FBS, PPBS, and HbA1C were analyzed by the unpaired Student's T-test. The mean ± SD of FBS in the survivors was 164.2 ± 68.9 mg/dl. In the non-survivors, the FBS was 173.5 ± 70.1 mg/dl; however, no statistically significant association was found (p = 0.466). The mean ± SD of PPBS in the survivors was 279.2 ± 66.9 mg/dl. In the non-survivor group, the PPBS was 293.5 ± 69.1 mg/dl, and there was no statistically significant association (p = 0.254). The mean ± SD of HbA1C level in survivors was 8.3 ± 1.2%, and in the non-survivors group, the HbA1C level was 8.5 ± 1.3%, which was statically not significant (P = 0.388)  .Since the ground-breaking research by Parker and colleagues who employed cineangiography over two decades ago to reveal the prevalence of systolic dysfunction in septic shock, the intriguing connection between this dysfunction and improved survival rates has remained shrouded in mystery . SubsequThe cardiac biomarker NT-pro-BNP was the highest in non-survivors, with a mean ± SD value of 503 ± 269.3 pg/ml, with lower NT-pro-BNP in survivors with mean ± SD of 286.40 ± 206.9 pg/ml, calculated by Mann-Whitney test, which shows left ventricular heart failure is more prevalent in death group, i.e., those who have diastolic dysfunction with sepsis . HoweverThe SOFA scores calculated at admission and at 72 hours of admission, comparing the live patients and dead patients, were statistically significant and were calculated by the Mann-Whitney test, showing the mean increase in SOFA score plays an important role in 28-day mortality and also with a higher grade of LVDD, the SOFA score increases, compared to other grades of LVDD. The APACHE II scores were calculated at the time of admission and at 72 hours of admission, and the results calculated by the Mann-Whitney test show significant results, showing a rise in the APACHE II score is an important predictor of mortality . The carDiastolic dysfunction is the strongest independent early mortality predictor in severe sepsis and septic shock patients with diabetes mellitus. Many physicians are concerned about ventricular systolic function, cardiac output, and pressure, here our study shows that diastolic dysfunction plays an important role in contributing to early mortality, hence it should not be overshadowed. No correlation was detected between mortality and the mean levels of FBS, PPBS, and HbA1c. Further studies with large sample sizes are required to test whether the severity of grade of diastolic dysfunction is correlated with severe sepsis and studies for any therapeutic interventions that might improve and increase survival.


Results: Compared to the single treatments, the combination of o-Vanillin and RG-7112 significantly reduced the amount of senescent IVD cells, proinflammatory cytokines, and neurotrophic factors. Moreover, both single and combination treatments significantly reduced neuronal sprouting in rat adrenal pheochromocytoma (PC-12 cells). Conclusions: Combining o-Vanillin and RG-7112 greatly enhanced the effect of either senolytic alone. Together, these results support the potential of senolytics as a promising treatment for IVD-related low back pain.Background: Low back pain is a global health problem directly related to intervertebral disc (IVD) degeneration. Senolytic drugs (RG-7112 and o-Vanillin) target and remove senescent cells from IVDs in vitro, improving tissue homeostasis. One drawback of using a single senolytic agent is the failure to target multiple senescent antiapoptotic pathways. This study aimed to determine if combining the two senolytic drugs, o-Vanillin and RG-7112, could more efficiently remove senescent cells and reduce the release of inflammatory factors and pain mediators in cells from degenerating human IVDs than either drug alone. Methods: Preliminary data evaluating multiple concentrations of o-Vanillin and RG-7112 led to the selection of four treatment groups. Monolayer and pellet cultures of cells from painful degenerate IVDs were exposed to TLR-2/6 agonist. They were then treated with the senolytics o-Vanillin and RG7112 alone or combined. p16 Low back pain is experienced by approximately 80% of individuals at some point in their lifetime. Globally, it is the number one cause of years lived with disability . DespiteHealthy, painless IVDs are largely avascular and aneural. Evidence shows that degenerating, painful IVDs are innervated , inferriTargeting cellular senescence  is a novel venue currently being investigated to treat chronic lower back pain. To date, the literature suggests that senescent cell accumulation during tissue degeneration contributes directly to the onset and progression of degenerative musculoskeletal illnesses such as osteoarthritis  and IVD The senescence-associated secretory phenotype (SASP) refers to the secretion of cytokines, chemokines, neurotrophins, growth factors, and proteases by senescent cells ,11. ThesIn human IVD, there is an expression of Toll-like receptor (TLR)-1, -2, -3, -4, -5, -6, -9, and 10, and the expression of TLR-1, -2, -4, and -6 are increased with the degree of disc degeneration and pain ,14. OverSynthetic drugs that selectively target and remove senescent cells (senolytic) have recently been identified ,17. Two Senescent cells are heterogenous, and one drawback of using a single senolytic agent is the failure to target multiple senescent antiapoptotic pathways in the same cell type or different cell populations within a target tissue. Concurrently targeting multiple and indirectly related antiapoptotic pathways may result in increased targeting of senescent cells in the absence of toxicity for normal proliferating or quiescent cells. Successful combination therapy is exemplified by the combination of Dasatinib and Quercetin that targets antiapoptotic networks instead of a single target . The lowCreating environments that reflect the true milieu in humans is critical for translation from the bench to medical practice. Moving away from 2D monolayer cultures, which do not provide a realistic environment seen in humans , is critical. This is especially important when investigating senolytics as, in reality, drugs do not overlay on cells but instead need to infiltrate through multiple regions to provide an effect, in this case, on the intervertebral disc. As such, this has led to increased use of 3D in vitro pellet or organoid cell cultures.The current manuscript used a 3D culture model of cells from painful degenerate IVD and stimulated them with a TLR-2 agonist to heighten the senescent phenotype. The objectives were a) to determine if treatment with o-Vanillin and/or RG-7112 has the potential to alter factors that directly affect innervation, b) to determine if a combination treatment with o-Vanillin and RG-7112 was more potent than single treatment at reducing senescent phenotypes and SASP factors.TM  and Fungiozone . Then, the matrix was minced and digested in 0.15% collagenase type II (Gibco) for 16 h at 37 °C. Cells were passed through both a 100 μm filter and 70 μm filter before being resuspended in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum , PrimocinTM, Glutamax , and maintained in a 5% CO2 incubator at 37 °C.All procedures performed were approved by the ethical review board at McGill University (IRB # 2019-4896). Degenerate IVDs were obtained from chronic low back pain patients who received discectomies to alleviate pain. Donor information is presented in Monolayer culture: Experiments were performed with degenerate IVDs (NP and AF cells) within passages 1 to 2. Then, 20,000 cells were seeded in 8-well chamber slides (Nunc™ Lab-Tek™ II Chamber Slide™ System) for immunocytochemistry experiments following treatment. Three hundred thousand cells were seeded in 6-well plates  for ELISA and RNA extraction following treatment. All cells were left to adhere for 12 to 24 h and then serum-starved in DMEM with 1X insulin–transferrin–selenium  for 6 h prior to treatment. Induction of senescence was performed by exposing cells to 100 ng/mL of Pam2CSK4  for 48 h. Cells were treated with senolytic compounds for the last 6 h of the incubation . Serial 2. Pellets were left in DMEM for two days to form and stabilize, followed by two days of induction with 100 ng/mL of Pam2CSK4 and then treated with either 100 μM of o-Vanillin, 5 μM of RG-7112, 5 μM of RG-7112 and 100 μM of o-Vanillin, 2.5 μM of RG-7112 and 100 μM of o-Vanillin, 5 μM of RG-7112 and 50 μM of o-Vanillin or 2.5 μM of RG-7112 and 50 μM of o-Vanillin for four days; meanwhile pellets in the control group stayed in DMEM  (Combination treatment with o-Vanillin and RG-7112 results in additive apoptotic and proliferative activity in pellet cultures. A). Following the treatment period, pellets from all groups were cultured for 16 days, and their culture media were collected every 4 days and pooled as posttreatment media (Combination treatment with o-Vanillin and RG-7112 results in additive apoptotic and proliferative activity in pellet cultures. A). After day 24, cell pellets were washed in PBS, fixed with 4% paraformaldehyde , cryoprotected in 10–30% sucrose, and then transferred via spatula into a plastic mold for embedment in Optimum Cutting Temperature compound , and finally flash-frozen at −80 °C and kept at −20 °C. Sections were cut 5 µm thick with a cryostat  and placed on slides for immunostaining.Pellet culture: 100,000 cells/tube were collected by centrifugation at 300 g for 5 min. Pellets were incubated in 300 μL of DMEM, 2.25 g/L of glucose , 5% FBS, 5 μM of ascorbic acid, 1% GlutaMAX, 0.5% Gentamicin  at 37 °C and 5% COn = 5 with no senolytic treatment, n = 5 treatment with 5 μM of RG-7112 and 100 μM of o-Vanillin, n = 5 with 2.5 μM of RG-7112 and 100 μM of o-Vanillin and n = 5 with 5 μM of RG-7112 and 50 μM of o-Vanillin).PC12 culture: Rat adrenal pheochromocytoma (PC12) cell line expresses the receptor for and responds to NGF. When exposed to NGF, they take on a neuronal-like phenotype. They are commonly used to study neuronal differentiation and neurite sprouting ,25. PC12ink4a , Ki-67  and caspase-3  primary antibodies. After washing, cells were incubated with the appropriate Alexa Fluor® 488- or 555-conjugated secondary antibody  for 2 h at room temperature and then counterstained with DAPI for nuclear staining. Photomicrographs were acquired with a fluorescent Olympus BX51 microscope equipped with an Olympus DP71 digital camera . Over 1000 cells were quantified for each pellet. Positive cell percentage was quantified by Fiji ImageJ (version: 2.1.0/1.53c). Briefly, the number of cells stained positive for one of the target proteins  was counted and compared to the total number of cells positive for DAPI staining.Pellet sections were heated to 60 °C for 30 min, then washed and permeabilized with PBS 1% Tween and 0.1% Triton. Cells were blocked with 0.3% Triton X-100 in PBS, saturated with 1% BSA, 1% serum, and 0.1% Triton X-100 for 2 h and then incubated for 2 h at room temperature with p16Each medium was applied in duplicate wells, and two random images per well were taken using a Zeiss Axiovert 40 C inverted light microscope  with a Canon PowerShot A640 camera and 52 mm Soligor adaptor tube . The graph is generated by counting 10 images from 7 different donors per group. This would equal over 2000 cells being quantified for each condition. The percentage of cells with neurites was determined and then averaged for each experimental condition.ink4a CINtec PLUS Kit , according to the manufacturer’s instructions. Slides were also exposed to primary antibodies specific to Ki-67  and caspase 3  overnight at 4 °C. A mouse- and rabbit-specific HRP/DAB (ABC) Detection IHC Kit  was used for p16ink4a, caspase-3 and Ki-67 staining. Images were processed using AxioVision LE64 software 4.9.1. Ten fields, randomly distributed across the well, were analyzed, total cells were counted, and the percentage of positive cells was calculated.Monolayer cultures were washed with PBS, fixed with 4% paraformaldehyde , and blocked in PBS with 1% BSA , 1% serum, and 0.1% Triton X-100  for 1 h. Slides were then incubated with the p16ink4a, TNF-α, CXCL-10, CXCL-1, CXCL-8, GM-CSF, TGF-β, CCL-2, CCL-5, CCL-7, CCL-8, NGF, BDNF, TLR-1, -2, -4, -6, neurofilament light chain (NF-L), plasminogen activator urokinase receptor (Plaur), Polo-like kinase 2 (Plk2), poliovirus receptor (PVR), vaccinia growth factor (VGF)) and the housekeeping gene (GAPDH), can be found in −ΔΔCt method after normalizing to GAPDH and nontreated samples [RNA was extracted using the TRIzol chloroform extraction method previously described for surgical samples and PC12 cells . Then, 5 samples .Cell culture media from degenerate IVD cells cultured in monolayer and pellets were used to assess the concentrations of IL-6, IL-8, IL-1β, TNF-α, NGF and BDNF. Next, 150 μL of monolayer culture media and day 12 pellet culture media were used. ELISAs were performed as per the manufacturer’s instructions . Colorimetric absorbance was measured with a Tecan Infinite M200 PRO  spectrophotometer and analyzed with i-control 1.9 Magellan software 1.68. Protein levels of the treated conditions and controls were then compared.Dimethylmethylene Blue (DMMB) assays were conducted as previously described  to quantMetabolic activity was assessed by the Alamar Blue assay . Brieflyt-test or one-way ANOVA. Specific tests are indicated in the figure legends with the corrections. A p-value <0.05 was considered statistically significant. Data are presented as mean ± SD.Data were analyzed using Graph Prism 9 . Analysis was performed using the paired Student’s ink4ap16 and TLR-2 [p = 0.004860), CCL5 from 49.03 ± 11.49 to 15.00 ± 6.14 (p = 0.015869), CCL7 from 9.30 ± 1.43 to 4.96 ± 2.20 (p = 0.015001), CCL8 from 28.40 ± 4.94 to 2.91 ± 2.40 (p = 0.003127), GM-CSF from 118.55 ± 10.07 to 7.15 ± 4.83 (p = 0.000239), BDNF from 1.77 ± 0.13 to 1.07 ± 0.16 (p = 0.002507), NGF from 1.35 ± 0.11 to 0.61 ± 0.19 (p = 0.000954), TNF-α from 7.36 ± 2.36 to 1.77 ± 0.77 (p = 0.022738), CXCL1 from 594.16 ± 44.72 to 38.66 ± 36.54 (p = 0.000002), CXCL8 from 594.50 ± 98.64 to 209.32 ± 157.89 (p = 0.022738), and CXCL10 from 745.23 ± 107.79 to 74.22 ± 89.64 (p = 0.001659) (ink4a expression from 3.83 ± 1.05 in the control to 1.21 ± 0.22 in RG-7112 (p = 0.004324) (p = 0.009338) was also observed in RG-7112 (2.69 ± 0.98)-treated cells compared with the control (9.17 ± 1.29), as has previously been reported for o-Vanillin [IVD cells from patients with low back pain and IVD degeneration were exposed to a TLR-2/6 agonist for 48 h in the presence or absence of RG-7112 (5 μM) during the last 6 h. We have previously observed that exposure of IVD cells from patients with low back pain and IVD degeneration to a TLR-2/6 agonist significantly increases the expression of SASP factors, nd TLR-2 . We havend TLR-2 . Here, wnd TLR-2 . Like o-.001659) A. In add.004324) B. Lastlyp = 0.000068 in RG-7112-treated cells, IL-1β expression decreased from 40.21 ± 3.53 pg/mL in the control to 20.68 pg/mL ± 3.86, p = 0.000098 in RG-7112-treated cells, IL-8 expression decreased from 410.01 pg/mL ± 30.47 in the control to 147.92 pg/mL ± 16.56, p = 0.000030 in RG-7112-treated cells and NGF expression decreased from 358.43 pg/mL ± 19.85 in the control to 262.32 pg/mL ± 6.96, p = 0.000227 in RG-7112-treated cells  during the last 6 h of culture. As previously reported , treatmeed cells D.ink4a-positive cells, nuclear localization) was observed with all four dilutions of RG-7112  and three dilutions of o-Vanillin . Representative images can be seen in ink4a-positive senescent cells with RG-7112 was 18.50% ± 1.61 at 0.25 μM (p = 0.0437), 14.58% ± 0.42 at 0.5 μM (p = 0.004), 13.25% ± 0.46 at 2.5 μM (p < 0.0001), and 10.57% ± 0.48 at 5 μM (p < 0.0001). With o-Vanillin, the remaining percentage of p16ink4a-positive cells was 15.59% ± 1.42 at 10 μM (p = 0.0076), 14.21% ± 1.41 at 50 μM (p = 0.0013) and 11.17% ± 1.01 at 100 μM (p < 0.0001) (p = 0.0149) increased to 21.50% ± 0.75 following treatment with 5 μM RG-7112 treatment and to 21.61% ± 1.22 (p = 0.0093) following 100 μM o-Vanillin treatment as compared to the untreated control (18.36% ± 0.96) (p = 0.0141) for 5 μM RG-7112 and to 10.87% ± 1.03 (p = 0.0144) for 100 μM o-Vanillin compared with the untreated control (9.09% ± 0.77).Given the role of o-Vanillin in TLR-2/6-induced senescence and the findings above, which demonstrate a similar role to RG-7112, we investigated whether there is an additive effect of combining the two senolytic compounds. First, to determine the lowest concentration with senolytic activity, IVD cells from patients with low back pain and IVD degeneration were exposed to the TLR-2/6 agonist for 48 h in the presence of serial dilutions of either RG-7112 or o-Vanillin during the last 6 h of the treatment. Control samples only received induction with the TLR-2/6 agonist. A significant decrease in senescent cells  B. The nuink4a. Three combinations were assessed: 5 μM of RG-7112 and 100 μM of o-Vanillin; 2.5 μM of RG-7112 and 100 μM of o-Vanillin; and 5 μM of RG-7112 and 50 μM of o-Vanillin.Given that 5 μM of RG-7112 and 100 μM of o-Vanillin resulted in the most significant decrease in senescence, combinations of these concentrations were selected for future experiments. Moreover, 2.5 μM of RG-7112 and 50 μM of o-Vanillin were further investigated, given that they both resulted in a significant decrease in the expression of p16To further assess the combination of RG-7112 and o-Vanillin, pellet cultures of IVD cells from patients with low back pain and IVD degeneration were exposed to the TLR-2/6 agonist for 48 h, treated with senolytics (in combination or single drugs) for four days and then cultured for a total of 24 days A. The coink4a, apoptotic , and proliferative  capacities were assessed. All treatments significantly reduced the number of senescent cells compared to the control. The largest decrease in ink4ap16-positive cells was observed in pellet cultures treated with the combination of 5 μM of RG-7112 and 100 μM of o-Vanillin. Control cultures had 18.97% ± 1.89 while treated with combination cultures had 3.74% ± 1.52 (p = 0.0004) senescent cells (p = 0.0002), followed by 5 μM of RG-7112 and 50 μM of o-Vanillin, which reduced senescent cells to 9.26 ± 1.35 (p = 0.0092) and 5 μM of RG-7112 and 100 μM of o-Vanillin applied as single drugs, which reduced the number of senescent cells to 11.09% ± 0.98 (p < 0.0001), and 10.31% ± 0.84 (p < 0.0001), respectively  and 2.5 μM of RG-7112 and 100 μM of o-Vanillin  significantly enhance the effect.The pellet cultures’ expression of p16ectively D. Comparp = 0.0011). Treatment with 2.5 μM of RG-7112 and 100 μM of o-Vanillin showed 29.27% ± 2.35 proliferating cells, and 5 μM of RG-7112 and 50 μM of o-Vanillin showed 20.93 ± 2.99 proliferating cells (p = 0.0415). The percentage of proliferating cells was 22.52% ± 3.08 (p = 0.0132) and 21.40% ± 4.17 (p = 0.0588) in RG-7112- and o-Vanillin-treated cultures. Compared to RG-7112 and o-Vanillin alone, only the two combinations of 100 μM of o-Vanillin with either 5 μM  or 2.5 μM  of RG-7112 significantly enhance the effect (p = 0.0057)  had a significantly greater number of senescent cells undergoing apoptosis relative to the RG-7112 and o-Vanillin single senolytic groups  and o-Vanillin 38.04% ± 7.52 (p = 0.0357) (The effect on proliferation with the greatest increase in Ki-67-positive cells was observed when treating cells with 5 μM of RG-7112 and 100 μM of o-Vanillin relative to the control. Proliferating cells increased from 15.24% ± 1.10 to 36.74% ± 3.25 (p = 0.0002) (p = 0.0231) and the single treatment with o-Vanillin , the combined treatment still had higher sGAG released into the culture media  I. Similare media I.p = 0.0034), IL-8 to 400.2 pg/mL ± 31.14 (p = 0.0003), IL-1β to 70.86 pg/mL ± 4.399 (p = 0.0002), TNF-α to 157.7 pg/mL ± 19.47 (p = 0.0109), BDNF to 808.5 pg/mL ± 62.77 (p < 0.0001) and NGF to 166.1 pg/mL ± 8.89 (p = 0.0001)  and NGF protein expression   A–F. Howe 0.0001) A–C. TNF- 0.0001) D. Relati 0.0083) E,F.p = 0.001669; Plaur: 2.10 ± 0.61, p = 0.001669; Plk2: 5.05 ± 0.57, p = 0.002624; PVR: 5.88 ± 0.42, p = 0.001669; VGF: 2.07 ± 0.72, p = 0.001669)  (p < 0.0001) (p < 0.0001), and to 47.74% ± 4.65 (p < 0.0001) for o-Vanillin  and  A. Intere.002073) A. Next w 0.0001) . When coVanillin .Previously, we demonstrated that TLR-2/6 activation induces cell senescence and SASP factor release in IVD cells . We furtink4a-positive cells, increase proliferation and increase apoptosis) were reduced once the concentration of o-Vanillin is below 50 μM and the concentration of RG-7112 is below 2.5 μM. Being that this is the first time that the combination of o-Vanillin and RG-7112 is being assessed, we decided only to use concentrations at which the senolytic compounds alone demonstrated senolytic activity, which is why we did not go below 50 μM for o-Vanillin and 2.5 μM for RG-7112 in the treatment groups (single or combined treatment).Cellular senescence can be induced by replicative senescence or stress-induced premature senescence . In low ink4a and SASP expression levels in the IVD [The assessment of monolayer culture is an excellent model to determine if compounds have senolytic effects; however, to better understand if there is any physiological relevance to these treatments, a model that mimics the physiological environment of the human disc is required ,23,29. A the IVD . Dasatin the IVD . A phase the IVD . Of note the IVD . CollectWith the growing body of evidence supporting the critical value of senolytic compounds in musculoskeletal diseases, there has been an increasing interest in evaluating the effect of senolytic compounds in chronic pain. An association between chronic pain and senescence has previously been suggested in mice subjected to experimental nerve damage. Here, reduced telomere length and p53-mediated cellular senescence in the spinal cord resulted in mice maintaining a high pain level . Low bacFuture studies should use in vivo models of disc degeneration to better represent the physiological environment of the IVD and to utilize pain behavior techniques to better understand if o-Vanillin and RG-7112 can lower pain levels in a live animal model. Moreover, a greater understanding of the molecular mechanisms of how o-Vanillin and RG-7112 work in combination for IVD degeneration may lead to improved therapies and quality of life for individuals with discogenic pain.In conclusion, the present study shows that combining o-Vanillin and RG-7112 greatly enhanced the effect of either drug alone in reducing the number of senescent cells. Additionally, we demonstrated that combination treatment reduced gene expression of markers involved in neurite sprouting and decreased sprouting in PC-12 cells. Our results support the potential of senolytics to reduce chronic pain.


Replacement heifer development is a critical component in beef production. The identification of the ideal uterine environment will greatly benefit reproductive efficiency. Additionally, the elucidation of how diet can influence this environment could alter management strategies. Therefore, our hypothesis was that different levels of protein supplementation would affect the uterine environment of beef heifers without inhibiting development or the ability to conceive. Supplemental protein did not greatly affect the uterine environment as it pertained to amino acid concentrations. However, uterine amino acid concentrations did change throughout development, and protein supplementation can influence uterine luminal fluid composition on d 14 post-insemination, which may affect conception rates.n = 60) were blocked by body weight (BW) and randomly assigned to one of three supplemental protein treatment groups (10% (CON), 20% (P20), and 40% (P40)). Mixed model ANOVAs were used to determine whether protein supplementation treatments, time, and the interaction or protein supplementation, semen exposure, and the interaction influenced uterine luminal fluid (ULF) and pregnancy outcomes. Amino acids (AAs) were impacted (p < 0.001), specifically, the essential AAs: Arg, Iso, Leu, Val, His, Lys, Met, Phe, Trp. Protein supplementation influenced multiple AAs post-insemination: Arg (p = 0.03), CC (p = 0.05), 1-MH (p = 0.001), and Orn (p = 0.03). In conclusion, protein supplementation did not affect the reproductive development via puberty attainment or the timing of conception even with alterations in growth. However, uterine AA concentrations did change throughout development and protein supplementation influenced ULF d 14 post-insemination, which may affect the conception rates.Replacement heifer development is one of the most critical components in beef production. The composition of the ideal uterine environment could maximize fertility and reproductive efficiency. Our hypothesis was that protein supplementation would affect the uterine environment of beef heifers without inhibiting development or reproduction. To test the effects of dietary supplementation on these outcomes, a randomized complete block design with repeated measures was implemented. Angus heifers ( Replacement heifer development is one of the most critical components of beef production with nearly 6 million entering the United States beef herd annually . The oveIn addition to influencing growth and development, supplementation may also influence the uterine environment of replacement heifers ,15. By iSeveral recent studies have analyzed amino acid (AA) concentrations within ULF throughout the estrous cycle , during n = 60), one month after weaning (266.2 ± 12.2 kg), were utilized for the completion of objectives and sample collection. All heifers had ad libitum access to mixed grass hay , and a ®, Boehrenger Ingleheim, Duluth, GA) and a controlled internal drug release (CIDR) device  was inserted into the vagina for 7 days. Upon CIDR removal, heifers received an im injection of 500 µg prostaglandin-F2α . Fixed-time artificial insemination was performed by a single technician beginning approximately 54 h after CIDR removal, concurrent with an im injection of GnRH. At the time of breeding, one animal from each pen was randomly selected to not be inseminated and serve as a synchronized but unexposed control for all pregnancy-associated analyses. Immediately following the last uterine flush, all animals were given another injection of PGF2α im to terminate any conceptions that may have survived the 14 d post-insemination flush. Seven days after the end of treatments, all animals underwent another synchronization protocol, AI breeding, and were exposed for 100 d to a fertile bull as per the standard operating procedure of the research unit. Ultrasonography was conducted 35 days after AI and 35 days after bull removal for pregnancy determination. The resulting pregnancies were utilized for the long-term impacts of protein supplementation during development.Before the final sample collection, all heifers underwent a 7-day CO-Synch + CIDR estrus synchronization protocol  concentration via radioimmunoassay using the double-antibody radioimmunoassay kit  as described previously [Plasma samples were collected via jugular venipuncture into 10 mL vacutainer tubes  and allowed to clot at room temperature before being centrifuged at 5000× eviously  to estimTwenty-five AAs and intermediary metabolites, namely alanine (Ala); arginine (Arg); aspartic acid (Asp); citrulline (Cit); cysteine (CC); glutamine (Gln); glycine (Gly); histidine (His); isoleucine (Ile); leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); valine  and intermediates 1-methyl-histidine (1 MH); alpha-aminoadipic acid (AAAA); alpha-aminobutyric acid (AABA); delta-hydroxylysine (DHL); ornithine (Orn); and sarcosine (Sar), were quantified within ULF via liquid chromatography–mass spectrometry (LC/MS). Prior to LC/MS, solid phase extraction was performed by the EZfaast Amino Acid Analysis Kit . Following solid phase extraction, AAs were derivatized in solution for further separation from components that may interfere with quantification. After derivatization, samples were re-dissolved in an aqueous mobile phase before analysis and quantification on an LC/MS system. The column used for LC/MS was the EZ:faast AAA-MS column 250 × 2.0 mm with a flow rate of 0.25 mL/min at 35 ℃. Concentrations were determined by comparing the area under the curve for each peak to certified standards, included with the extraction kit .n = 12; 4 per treatment). Separate mixed-model ANOVAs and mean separation via PROC GLIMMIX  were used to determine whether the protein supplementation, time, or the interaction influenced BW, BCS, pH, age at pubertal onset, or AA concentrations. Separate mixed-model ANOVAs and mean separation via PROC GLIMMIX were used to determine whether protein supplementation, exposure to semen, or the interactions influenced AA concentrations after undergoing synchronization and insemination. Crude protein level and sample day were included in the model as fixed effects. Weight class, the interaction of weight class × crude protein level, and the 3-way interaction of weight class × crude protein level × sample day were included in the model as random effects. Bodyweight, BCS, pH, and P4 levels were included in all AA models as covariates and removed if not significant (p > 0.05). Log transformation was performed when AA concentrations did not yield normally distributed residuals. Shapiro–Wilk’s statistic (W > 0.8), the Kolmogorov–Smirnov test (K > 0.2), and the visual assessment of residual plots were assessed to assess normality. Reported concentrations and standard error of the mean were back calculated and reported in µM. A correlation analysis, for any AAs found to be impacted by protein supplementation or from exposure to semen, was utilized to assess the connections between concentrations during development and after insemination. The frequency procedure within SAS was used to determine whether protein supplementation influenced the breeding season following the end of treatments. Due to the limitations of frequency analyses, a survival analysis was also performed to determine whether the time to conception was impacted by previous protein supplementation. Means were considered different when p ≤ 0.05 with a tendency at p ≤ 0.10.To test the effects of dietary supplementation on heifer BW, BCS, the incidence of puberty, ULF pH, and ULF AA profile, a randomized complete block design with repeated measures was utilized. Because BW, BCS, and ULF were collected from each animal, an individual heifer represents the sampling unit. However, the experimental unit was the pen (p < 0.001) for BW during development. The BW of CON heifers was lower  than P20 or P40 heifers  beginning on day 42 and continued through day 163  but were similar on d 42 (p = 0.63). On d 56, P20 supplemented heifers were intermediary (p = 0.05) between CON and P40 groups  which tended to continue through d 70 . Heifers provided P20 and P40 had a greater BCS (5.8 ± 0.1) than CON heifers (5.5 ± 0.1) on d 84 and maintained this difference through d 98 (p = 0.002). Supplementation with P20 resulted in higher BCS (5.7 ± 0.1) compared with CON or P40 groups  on d 112 (p < 0.001). Heifers in the P20 and P40 had a higher BCS  versus CON heifers (5.4 ± 0.1) on d 126 (p < 0.001). Heifers supplemented with the P40 supplement had a greater (p < 0.001) BCS (5.9 ± 0.1) compared with the CON or P20 groups  on d 140. On the final day of treatment, BCS for P20 and P40 heifers  was greater (p < 0.001) compared with heifers on the CON supplement (5.5 ± 0.1 There was a significant treatment × day interaction (p > 0.98). The onset of puberty occurred at 340.9 ± 16.3, 344.3 ± 15.0, and 344.0 ± 14.4 d of age for CON, P20, and P40 heifers, respectively, after approximately 100 days of supplementation. While still above the threshold for a functional corpus luteum (1 ng/mL) for all treatments, plasma P4 levels at the time of estimated puberty (p = 0.02) in P20 heifers compared with P40 heifers and CON heifers being intermediate. These data indicate that, while growth may have been impacted by protein supplementation, reproductive maturation and thus heifer development were not impacted.Although heifers receiving the CON supplement weighed less than contemporaries throughout most of the study, treatment did not influence age at puberty ( puberty  were grep = 0.23). There was an interaction between treatment and time for the relative change in uterine pH , whereas on d 56, the P40 heifers (0.23 ± 0.1) had a more basic uterine environment compared to the P20 (−0.22 ± 0.1) and CON (−0.13 ± 0.1) groups (p = 0.005). Over the final four sampling periods (d 84–d 163), all heifers exhibited a basic relative pH but no differences were observed amongst treatments (p > 0.10). Relative uterine pH at the time of detectable puberty was also not different among treatments (p = 0.18). The lack of impacts on uterine pH could be a consequence of differing estrus status as these females reached puberty or that overall protein levels in the diet were not different enough to have an impact on the reproductive tract.Due to the variability of pH amongst the 34—1 L bags of saline, pH was normalized based on flush saline pH. Overall, the pH (6.61 ± 0.2) among samples did not differ (p ≥ 0.16). However, Cit and DHL tended to differ  depending on the CP supplementation level. Heifers on the CON supplement tended (p = 0.08) to have less Cit (0.07 ± 0.03 µM) compared with heifers given the P40 supplement (0.14 ± 0.03 µM) with P20 heifers being intermediate (0.09 ± 0.03 µM). Uterine DHL tended (p = 0.10) to be greater in the ULF of P40 heifers (0.08 ± 0.01 µM) compared with P20 and CON heifers .Among the free AAs and intermediates quantified from the EZ:faast Amino Acid Analysis Kit, most were not influenced by protein supplementation and none exhibited a treatment × day interaction (p < 0.001) on d 56 and decreased slightly thereafter. Citrulline was present in low levels throughout development (Cit < 0.19 ± 0.04 µM) but increased (p < 0.001) after 28 d (0.11 ± 0.04 µM) of supplementation, except on d 112 (0.01± 0.04 µM), which was similar to initial concentrations. Concentrations of Gln decreased (p < 0.001) from d 28 to 112 (1.50 ± 0.22 µM to 0.05 ± 0.22 µM) but were highest at d 140 (1.32 ± 0.22 µM). Free Orn concentrations varied over time (p < 0.001), but only exceeded 0.1 µM on d 28. Proline ranged from 0.04 to 0.69 ± 0.09 µM  are presented in p < 0.001). Glycine concentrations were increased (p < 0.001) on d 84 and 140  early (d 28 and 84) and decreased during the remainder of the development. Leucine was the highest (p < 0.001) at d 28 (0.62 ± 0.09 µM) and remained elevated for the remainder of development (p < 0.001) during development. Valine concentrations were greatest (p < 0.001) on d 28 (1.35 ± 0.17 µM) but relatively stable (0.76–1.24 µM) except for on d 112  but peaked at d 84 (110.91 ± 21.4 µM). Free DHL was very low throughout development but differed across time (p < 0.001). Histidine concentrations oscillated (p < 0.001) during development with d 28, 84, and 140 being elevated compared to d 0, 56, and 112 (p < 0.001) on d 28 of supplementation, remained elevated through d 56, and decreased thereafter  and Gln  in heifers that were not exposed to semen (0.41 ± 0.3 µM) compared with heifers that were inseminated (0.03 ± 0.3 µM). Interestingly, Gln tended to increase (p = 0.10) in heifers that were inseminated (3.22 ± 1.0 µM) compared with heifers not exposed to semen during breeding (1.30 ± 1.0 µM). Protein supplementation did impact multiple AA post-insemination. Arginine was dramatically increased (p = 0.03) in the P40 heifers (18.75 ± 7.74 µM) over CON supplemented heifers (1.88 ± 7.74 µM) and P20 heifers (5.82 ± 7.74 µM) were intermediate (p = 0.05) in P40 compared with CON and P20. Heifers supplemented with P40 supplementation (0.27 ± 0.07 µM) had increased (p = 0.001) 1 MH over CON and P20  in P40 heifers (0.43 ± 0.18 µM) compared with CON heifers (0.16 ± 0.18 µM) and P20 were intermediate (0.13 ± 0.18 µM). Lysine tended to be increased (p = 0.10) in P40 supplemented heifers (2.89 ± 1.41 µM) over CON and P20 heifers  with concentrations on any d during development or the average concentration. On d 0, Arg tended to be negatively correlated (R = −0.39) with concentrations on d 163 for heifers receiving the P20 supplement. Arginine concentrations on d 56 were positively correlated with d 163 Arg concentration for P20  and P40  but only tended to be correlated in CON heifers  were utilized in a correlation analysis to determine whether uterine concentrations of AAs during development  were indicative of AAs concentrations in ULF post-insemination d 163; . Concentp ≥ 0.22) with Arg concentrations after breeding. Citrulline concentrations on d 163 were not related (p ≥ 0.41) to any Cit concentrations during development for heifers on the P40 treatment. For heifers on the P20 treatment, concentrations of Cit on d 56 were positively correlated . On d 56 and the overall average concentrations of Cit were positively correlated with Cit concentrations on d 163 (p ≤ 0.05) on d 28, 56, 112, 140, and the overall average and negatively correlated on d 84  with concentrations after breeding but were not related (p ≥ 0.21) on d 0, 28, 56, 84, or 112 in P20 heifers. For heifers receiving the CON supplement, CC concentrations after insemination were positively correlated (p < 0.05) with CC concentrations on d 28 (R = 0.45) and d 112 (R = 0.59) and tended to be correlated (p < 0.10) with concentrations on d 56 (R = 0.37) and the overall average  to be negatively correlated (R = −0.40) with concentrations on d 112 but was not related to concentrations at any other time point or the average (p ≥ 0.17). Concentrations of 1 MH after insemination were highly correlated to 1 MH concentrations on d 56 , d 84  and the overall average , but only in heifers on the CON supplement. Heifers on the P20 and P40 treatments were not found (p ≥ 0.31) to have any relationships in 1 MH concentrations during development and after breeding  in the frequency distribution of when conception occurred amongst treatments. Interestingly, double the number of CON heifers were conceived under AI, and all were bred by the second cycle, whereas both the P20 and P40 conceptions occurred relatively linearly  for heifers on the CON supplement (24.0 ± 4.5 d) compared to P20 heifers (46.1 ± 7.1 d) with P40 being intermediate (39.5 ± 5.8 d; The survival analysis did confirm what the frequency analysis did not have the power to determine. The average d to conception was shorter (In the current experiment, heifers receiving CON supplement achieved puberty at approximately the same age as contemporaries, despite having a lower BW. This is in agreement with a previous study where heifers receiving a high starch intake had a lower BW at puberty . Age at In order for proper embryo development to take place, pH within the uterine environment must be closely regulated to maintain developmental competence . A majorClassical studies originally reported an impact of dietary protein on uterine pH, in a study that supplied cycling dairy heifers with 150% of crude protein requirements . High prOverall, AA concentrations in the present study were much lower than those reported in previous studies using similar flush methods to analyze ULF ,20,21,43The composition of the histotroph, specifically in terms of AAs, is important in fetal development ,46. ThesThe importance of early conception in the first breeding season largely affects the lifetime production of replacement heifers. Day and Nogueira  determinIn summary, protein supplementation influenced the BW and BCS but did not affect age at puberty. Variations in the final BW or BCS were not intended; however, these results are useful in signifying the importance of the overall nutritional plane since CON heifers were gaining weight and maintained an adequate BCS for successful heifer development. Protein levels in the CON diet do appear to be limiting growth. However, it is likely that increased supplementation to CON heifers to compensate for decreased growth would elevate the overall feed costs without an increased return on production efficiency. Further research is necessary to determine whether a lower BW at puberty, achieved by a lower ADG, can be achieved using supplements similar to P20 or P40 without compromising the reproductive success but decreasing the cost of development. The supplement type did affect the amount of P4 at the attainment of puberty and altered the concentrations of Trp and Orn in ULF of developing heifers. Thus, this supports the hypothesis that supplements with different characteristics, in this case, CP, are transported post-digestion in varying amounts into the uterine environment. Further studies are needed to determine whether AA concentrations are consistently affected by different feed types and stages of development. Additionally, identifying the times of development and AAs that may affect fertility could be beneficial to reproductive and production efficiencies in beef cattle. These effects could be elucidated to develop management strategies to optimize heifer development by decreasing the cost associated with development while maximizing reproductive and productive outputs.


After optimizing the feeding strategy, the concentration of viable bacteria reached 5.80 × 109 cfu/mL. In the application effect verification experiment, the degradation rates of NH4+-N, TN, TP, and COD in the experimental group reached 96.69%, 75.18%, 73.82%, and 90.83%, respectively, showing a significant improvement compared to the results of the control group. The main components in the control group were Dokdonella, Brevundimonas, Alishewanella, Rhodobacter, Pseudoxanthomonas, and Thauera, whereas those in the experimental group were Dokdonella, Proteocatella, Rhodobacter, Dechlomonas, and Nitrospira. Proteocatella, Dechlomonas, and Nitrosra, which were unique to the experimental group, are common bacteria used for nitrogen and phosphorus removal. This explains the difference in the sewage treatment capacity between the two groups. This study provides an alternative sewage treatment microbial flora with a reasonable production cost and high degradation efficiency for NH4+-N, TN, TP, and COD.Microbial flora plays an important role in microorganism-enhanced technology. The pollutant degradation ability and viable counts of these agents are crucial to guarantee their practical application. In this study, an efficient pollutant-degrading microbial flora was screened, its medium components and culture conditions were optimized, and its effect was verified in zeolite trickling filter towers. After a 24 h culture under the optimal conditions, the viable count reached 4.76 × 10The online version contains supplementary material available at 10.1186/s13568-023-01604-0. 4+-N, TN, TP, and COD was screened.A mixed-bacteria group with a high degradation rate of NHThe viable cell concentration and pollutant degradation efficiency of the screened microbial flora was increased significantly by optimizing its cultivation conditions.Increased pollutant degradation efficiency was verified in a zeolite trickling filter tower and explained by the microbial composition of the screened microbial flora.The online version contains supplementary material available at 10.1186/s13568-023-01604-0. Additionally, the bacterial flora was used to treat the simulated sewage to evaluate its pollutant degradation efficiency comprehensively. Finally, the composition of the bacterial flora was analyzed to explain its excellent pollutant degradation ability.Our study lays a solid foundation for the production of microbial agents with high pollutant degradation efficiencies at a low cost.o42′-114o14′ E, 34o16′-34o58′ N), xinmi city Jinmen Sewage Treatment Co., Ltd. , Henan Chengsheng Sewage Treatment Co., Ltd.  and Zhengzhou Tongji Environmental Protection Engineering Co., Ltd. (110o42′-116o17′ E and 36o18′-36o58′ N) respectively. Their water sources are domestic sewage, landfill leachate, sewage from chemical industry park and sewage from food Industry Plant.Samples A, B, C and D were taken from Zhengzhou Sewage Treatment Co., Ltd. 2SO4, 50.0 mL/L Vickers salt solution, 5.62 g/L C4H4Na2O4. 6H2O and 10.0 mL/L trace elements.Heterotrophic nitrification liquid medium (HNLM) contains 0.5 g/L 2MoO4, 1.57 g/L CuSO4, 50.0 g/L EDTA, 2.2 g/L ZnSO4, 5.0 g/L FeSO4 and 1.61 g/L CoCl2.Trace elements contains 5.5 g/L CaClThe composition of heterotrophic nitrification solid medium (HNSM) was the same as that of HNLM with the addition of 15.0 g/L agar.3COONa.3H2O, 50.0 mL/L Vickers salt solution, 1.0 g/L (NH4) 2SO4 and 10.0 mL/L trace elements.The inorganic salt medium contains 10.297 g/L CH4, 0.05 g/LMnSO4, 5.0 g/L K2HPO4, 2.5 g/L MgSO4 and 2.5 g/L NaCl.The Vickers salt solution comprises 0.05 g/L FeSO4,30.0 mg/L CaCl2, 30.0 mg/L KH2PO4, 500.0 mg/L KHCO3, 100.0 mg/L NaNO3, 30.0 mg/L (NH4)2SO4 and 100.0 mg FeCl3.The simulated wastewater contains 500.0 mg/L glucose, 200.0 mg/L MgSOAll above-mentioned solutions were adjusted to pH 7.0 and sterilized for 20 min at 121 °C.4+-N, TN, TP, NO3-N, and COD after a 24 h treatment at 30 °C and 150 r/min.Four samples, A, B, C, and D, were diluted 10 folds with 0.9% physiological saline and then shaken with glass beads for 1 h. Microorganisms in the samples were activated and acclimated by culturing for 24 h at 30 °C and 150 r/min in the broth medium and HNLM successively. The domesticated microorganisms were inoculated into simulated wastewater at a 0.1% ratio to evaluate their pollutant degradation capacity by determining the degradation ratios of NH4+-N to optimize culture medium components. The concentration of viable bacteria is the sole evaluation criterion to optimize culture conditions.Pollutant-degrading enzymes are induced enzymes. The composition and concentration of the culture medium affect the growth of microorganisms and their ability to reduce pollutants. However, the culture conditions mainly affect the growth of microorganisms. Therefore, the evaluation criteria were the concentration of viable bacteria and the ability to degrade NH4+-N degradation ability were studied using a single-factor experiment. These factors were studied successively, and the optimized results were used in the subsequent steps. Sodium citrate, sodium succinate, sodium acetate, and glucose were used as alternative carbon sources. Beef paste, soybean meal, corn steep liquor powder, yeast extract powder, fish meal, peptone, tryptone, and urea were used as alternative sources of organic nitrogen. Ammonium sulfate and ammonium chloride were used as alternative sources of inorganic nitrogen. Based on the single-factor experiment, an Taguchi L9 (33) orthogonal experiment was designed using SPSS software (version 20.0) to optimize the concentrations of the carbon and nitrogen sources.Based on the composition of HNLM, the effects of the carbon and nitrogen sources on the viable bacterial concentration and NH7 cfu/mL, and cultured at 150 r/min and 30℃ for 24 h to evaluate their ability to degrade NH4+-N.Broths cultured in different media were centrifuged for 10 min at 5000 r/min. The precipitated PDBF cells were washed with PBS three times, inoculated into an inorganic salt medium at a concentration of 1.52 × 10Based on the initial culture conditions , the effects of temperature, pH, loading ratio, rotating speed, and inoculation ratio on the concentration of viable bacteria in culture broth were studied using a single factor experiment successively in a shake flask. All optimized results were used in the subsequent steps. Alternative temperatures were 20, 25, 30, 35, and 40 ℃. Alternative pH values of 5.0, 6.0, 7.0, and 8.0 were used. Alternative loading ratios of 2.5, 5.0, 10.0, 15.0, and 20.0% were used. Alternative rotating speeds of 90, 120, 150, 180, and 210 r/min were used. The alternative inoculation ratios were 1, 3, 5, 7, and 9%.Before optimization of the feeding strategy Table  for fed-The PDBF application verification experimental systems were constructed as 1200 mm × 150 mm diameter vertical Perspex tubes  mixed with mixed liquor volatile suspended solids  was pumped into the system to stimulate microorganism growth and biofilm formation. The experimental conditions for the two groups were the same, except that the experimental group was inoculated with the studied microbial flora in addition to activated sludge. After the bacteria film was formed on the surface of the zeolite filler, simulated sewage containing 300.0 mg/L COD and 75.0 mg/L NH4+-N was pumped into the system to evaluate the pollutant degradation efficiency of the studied PDBF. The effluents were sampled from the sampling port E5 at 0 and 24 h to analyze the pollutant degradation ratio. The intermittent aeration mode was applied during the start-up and experimental processes, that is, aeration for 4 h to maintain the dissolved oxygen at 4–6 mg/L, then non-aerarion for 4 h. This process was repeated.Control and experimental groups were established. In the 10-d start-up procedure, a high-concentration nutrient solution . Three replicates were used for each dilution.4+-N, TN, TP, and COD degradation rates were determined to evaluate the pollutant treatment efficiencies of the PDBFs. The concentrations of NH4+-N, NO3−-N, TN, TP, and COD were analyzed according to the China Standard Test Method for Water and Wastewater . The degradation rates of the pollutant indices were calculated as the ratio of the concentration of the treated sample to that of the untreated sample. Three parallel experiments were conducted for each experimental group.The NHThe DNA of the biofilm obtained from the zeolite trickling filter was extracted using the PowerSoil DNA Kit  according to the manufacturer's instructions. PCR amplification was performed using universal primers 341F (50′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GGAC-TACHVGGGTWTCTAAT-3′). Polymerase chain reaction (PCR) amplification was performed with Pfu polymerase using the following thermocycling program: denaturation at 95 °C for 5 min followed by 30 cycles of 95 °C for 30 s, 55 °C for 60 s, and 72 °C for 90 s, with a final incubation at 72 °C for 7 min. Purified PCR products were sequenced using the Illumina MiSeq platform . The 16sRNAs of the samples  mentioned in the experiment have all been uploaded to the National Center for Biotechnology Information, and the associated BioProject accession numbers is PRJNA534376.4+-N, NO3−-N, TN, and TP. Therefore, it was used in subsequent experiments.The pollutant degradation parameters of the samples are listed in Table Optimizing the medium components and culture conditions of the microbial flora can improve the concentration of viable bacteria and pollutant degradation by liquid microbial agents, further promoting the degradation of pollutants in sewage. On the other hand, a high fermentation level is the key to reduce the preparation cost of solid sewage treatment microbial agents.4+-N degradation rate, sodium succinate (81.5%) and sodium acetate (80.4%) were more effective than sodium citrate (77.2%). Considering both the viable bacterial concentration and the NH4+-N degradation rate, sodium acetate was used for further experiments.The microbial flora grew well when the carbon sources were sodium acetate, citrate, or succinate  orthogonal experiment . The improvements in the degradation effect of NH4+-N and TP were particularly significant.The pollutant degradation results of the control and experimental groups for the simulated wastewater after 24 h of treatment are shown in Fig. Dokdonella, Brevundimonas, Alishewanella, Rhodobacter, Pseudoxanthomonas, and Thauera, while those of samples E1 and E2 were Dokdonella, Proteocatella, Rhodobacter, Nitrospira, and Thauera. Proteocatella, Azospira, and Nitrospira only existed in the biofilms of the experimental group. In addition to the above bacteria with high content, the experimental group also contained small amounts of Acinetobacter, Acidovorax, Clostridium, etc.  and mixed flora (Pseudomonas geniculata ATCC 19374 and Bacillus cereus EC3 Mixture), the number of viable bacteria is significantly higher.Excessive nutrients in the initial medium cause the overgrowth of bacteria and rapid consumption of nutrients, leading to premature aging and autolysis of bacteria. Moreover, the growth of the flora is limited because of a lack of nutrition during the late fermentation stage. Proper feeding can prolong the logarithmic growth period, improve the final fermentation level, and achieve high-density fermentation  sodium succinate (2) sodium citrate (3) sodium acetate (4) glucose (5) blank group; b Organic sources:(1) beef paste (2) soybean meal powder (3) Corn syrup dry powder (4) yeast extract powder (5) fish meal (6) peptone (7) urea (8) tryptone (9) blank group; c Inorganic nitrogen sources (1) 0.5 g/L ammonium sulfate; (2) 0.4 g/L ammonium chloride; (3) 1.0 g/L ammonium sulfate; (4) 0.8 g/L ammonium chloride; (5) blank group; d Complex nitrogen sources (1) ammonium sulfate 0.45 g/L + soybean meal powder 0.35g/L; (2) ammonium sulfate 0.45 g/L + fish meal 0.23g/L; (3) ammonium sulfate 0.5g/L; (4) ammonium sulfate 0.9 g/L + soybean meal powder 0.7g/L; (5) ammonium sulfate 0.9 g/L + fish meal 0.46g/L; (6) Ammonium sulfate is 1.00 g/L; Figure S2. Optimization results of culture conditions in shaking flask. Figure S3. The growth curve of the screened PDBF.


In this work, the possibility of managing the rheological and mechanical parameters of composites based on PLA with the use of cubic structures of organofunctional spherosilicates was verified. To accurately observe the effect of various organosilicon modifier substitutions on changes in composites’ properties, we synthesized and used monofunctional octasubstituted derivatives as reference systems. The OSS/PLA systems were tested with concentrations of 0.1–2.5% (w/w) using extrusion to obtain a filament with a diameter of 1.75 mm. The printed samples underwent comprehensive tests including microscopic , rheological, thermal , mechanical (impact and strength) as well as water contact angle tests. The work is interdisciplinary in nature and combines elements of organosilicon synthesis, materials engineering, and materials processing and characterization technology. Additive technologies are recognized as one of the key elements of the new generation of the 4.0 Industry. SmartTech Analysis is currently a leader among analytical companies in the field of additive technologies. It mainly focuses on forecasting the economic impact of 3D printing, which in 2020 was 11.7 billion dollars. In subsequent years, it is forecasted at USD 24 billion in 2024 and USD 55 billion in 2030, respectively. The development of additive technologies depends on the industry's demand, mainly in terms of automation and optimization of processes. The most progress has been made in the sector of designing and manufacturing new materials. The latest SmartTech Analysis report shows that the largest revenues until 2019 were generated by FDM technology2. It is expected that the greatest revenues among the known and applied incremental methods are generated by the FDM technology, mainly due to its low complexity and commercial access to raw materials (filaments)2. Currently, FDM printers are available not only for large corporations but also for micro-enterprises and individual customers. Device prices come in a very wide range, on the market There is also a wide range of products (in terms of color and flexibility modification) and a database of millions of ready-made designs, which greatly simplifies the process.The dynamic development of additive techniques occurred in the second half of the 80s. Then, after the expiry of the original patent on FDM (2009) and SLS technology (2014), there was a wave of new companies offering equipment and materials dedicated to 3D printing. It was also associated with a decrease in 3D printer prices and the related increase in the number of scientific publications and new patents in this area. Currently, 3D printing is one of the most dynamically developing industries. Both competitive pressures and increasingly sophisticated business models mean that companies need to shorten development cycles and move in the right direction with speed and flexibility. 3D printing in many industries is revolutionizing production thanks to more materials available and a greater ability to supply parts that match their mechanical properties. Currently, incremental techniques are increasingly replacing other mature processing methods, while the demand for 3D printing results is constantly growing3. The general scheme of a typical FDM 3-D printer was presented in Fig. 7 poly  glycol modified (PET-G)9, amorphous acrylonitrile butadiene styrene (ABS)12 and polyamide-12 (PA-12)14.The FDM technique is a kind of micro-extrusion, it consists in feeding molten thermoplastic material from the printer's nozzle and then applying it layer by layer on a movable table15, shortening the supply chain, and enabling spare parts manufacturing in production plants16, and the ability to produce even very complex structures (without the need to create many expensive forms)17.The importance of 3D printing to other technologies is very significant due to the many benefits that the technique brings, e.g. short time from design to prototype execution18. The addition of glass fibers and a plasticizer/compatibilizer as a modifier of the mechanical properties of composites based on ABS (the addition of the fibers themselves causes too rigid a structure and contributes to the difficulty of the printing process—clogging and abrasion of the nozzle)19 or use to improve the mechanical properties at the boundary of the matrix and glass fibers, carbon or Kevlar20. To a large extent, the improvement of the properties of materials popularly used in 3D printing is based on physical modification with the use of reinforcements in the form of synthetic or natural fibers21. Metal nanoparticles are also often used as reinforcing additives23. The addition of graphite improved the rheological and tribological properties24. In our previous work, we also examined the effect of the addition of MoS225 which showed a change in tribological properties, and TiO2, which improved the mechanical properties of the obtained composites26. So far, however, there are no solutions that would enable the improvement of several parameters at the same time or would increase the desired parameters without drastically lowering others.The greatest limitation of the FDM technique is the lack of highly specialized materials that would allow the elimination of such limitations as adhesion to the layer and the printer bed, or improvement of performance properties without compromising others. Printing issues often arise from imperfections in print tightness and hydrophobic and/or mechanical and thermal properties. So far, the greatest emphasis has been placed on the development of new devices with higher printing speeds, larger working area, etc. There are several cases in the literature in which thermoplastic polymers used in the FDM technique have been modified to improve the quality of printing or the properties of model objects. There are known works where the authors created a SiC/C/PLA composite characterized by good electrical conductivity and shape memory1,5]n27. Thanks to their structure, i.e. an inorganic core made of Si–O–Si connections and organic substituents at the silicon atom, they have organic–inorganic hybrid properties29. Silsesquioxanes can be used as additives in the production of thermoplastic or thermosetting polymers as well as composite materials. They are used in optoelectronics, microelectronics, pharmacy (drug carriers), medicine, and dentistry32. Octasubstituted silsesquioxanes are an especially interesting group. They are obtained by a simple hydrolytic condensation reaction of such compounds as trichloalkoxysilanes or trialkoxysilanes34. Silsesquioxanes, due to their compact molecular structure and nanometric dimensions, are characterized by good dispersion properties. A special subgroup of cage silsesquioxanes, sometimes treated as a separate class of compounds, are spherosilicates, which are characterized by an additional D-type siloxane linker, separating the R functional group from the siloxane core, composed of Q-type units. These structures are characterized by high activity in catalytic reactions, among which the hydrosilylation of olefins is of the greatest importance, due to the large variety and availability of substrates , the quantitative nature of the reaction, and the ease of the synthetic procedure. These characteristics determine the growing interest in compounds of the spherosilicate type as functional additives in materials engineering36 or building blocks in polymer chemistry37.One of the most attractive and increasingly used groups of modifiers of various classes of polymers is polyhedral oligomeric silsesquioxanes with the general formula [RSiOThe purpose of this work was to assess the properties of spherosilicate/PLA composite materials obtained by polymer compounding methods and dedicated to the FDM 3D printing technique. The material was modified with a series of mono- and bifunctional octaspherosilicate (OSS) additives obtained by a hydrosilylation reaction. Modifiers were tested and characterized by means of spectroscopy and spectrometry . The conducted tests of composite materials revealed that the additives strongly affect the PLA melt rheology, polymer fusion during printing, material delamination, fracture mode under mechanical load, and therefore mechanical strength of the printed samples. Additionally, the thermal and surface properties of the obtained materials have been affected by the OSS compounds.Polylactide (PLA) type Ingeo 2003D was purchased from NatureWorks . The chemicals were purchased from the following sources: Tetraethoxysilane (TEOS), chlorodimethylsilane, tetramethylammonium hydroxide (TMAH) 25 wt% methanol solution from ABCR, vinyltrimethoxysilane, octadec-1-ene from Linegal Chemicals  chloroform-d, 2 wt% Karstedt’s catalyst xylene solution from Aldrich , P2O5, toluene, from Avantor Performance Materials Poland S.A. . Toluene was degassed and dried by distilling it from P2O5 under an argon atmosphere.1H, 13C, and 29Si Nuclear Magnetic Resonance (NMR) spectra were recorded at 25°C on Bruker Ascend 400 and Ultra Shield 300 spectrometers using CDCl3 as a solvent. Chemical shifts are reported in ppm concerning the residual solvent (CHCl3) peaks for 1H and 13C.MALDI-TOF mass spectra were recorded on an UltrafleXtreme mass spectrometer , equipped with a SmartBeam II laser (355 nm) in the 500–4000 m/z range. 2,5-Dihydroxybenzoic acid  served as a matrix. Mass spectra were measured in reflection mode. The data were analyzed using the software provided with the Ultraflex instrument—FlexAnalysis (version 3.4).−1.Fourier Transform-Infrared (FT-IR) spectra were recorded on a Nicolet iS 50 Fourier transform spectrophotometer (Thermo Fisher Scientific) equipped with a diamond ATR unit with a resolution of 0.09 cmWater contact angle analyses (WCA) were performed by the sessile drop technique at room temperature and atmospheric pressure, with a Krüss DSA100 goniometer Three independent measurements were performed for each sample, each with a 5 µl water drop, and the obtained results were averaged to reduce the impact of surface non-uniformity.2O3 crucibles. Measurements were conducted under nitrogen (flow of 20 mL/min) in the range of 30 ÷ 800 °C and a 10 °C/min heating rate, Sensitivity: 0.1 µg.Thermogravimetry (TG) was performed using a NETZSCH 209 F1 Libra gravimetric analyzer. Samples of 5 ± 0.2 mg were cut from each granulate and placed in AlDifferential scanning calorimetry (DSC) was performed using a NETZSCH 204 F1 Phoenix calorimeter Samples of 6 ± 0.2 mg were cut from each granulate and placed in an aluminum crucible with a punctured lid. The measurements were performed under nitrogen in the temperature range of − 20 ÷ 290°C and at a 10 °C/min heating rate., Sensitivity: 0.1 µg.The effect of the modifier addition on the mass flow rate (MFR) was also determined. The measurements were made using an Instron plastometer, model Ceast MF20 according to the applicable standard ISO 1133. The measurement temperature was 190 ± 0.5 °C, while the piston loading was 2.16 kg.For flexural and tensile strength tests, the obtained materials were printed into type 1B dumbbell specimens by EN ISO 527:2012 and EN ISO 178:2006. Tests of the obtained specimens were performed on a universal testing machine INSTRON 5969 with a maximum load force of 50 kN. The traverse speed for tensile and for flexural measurements was set at 2 mm/min. Charpy impact test (with no notch) was performed on an Instron Ceast 9050 impact machine according to ISO 179−1. For all the series, 6 measurements were performed for each material.Scanning Electron Microscopy with Energy Dispersive Spectroscopy (SEM/EDS) analyses were recorded on a Quanta FEG 250 (FEI) instrument; SEM at 5 kV and EDS at 30 kV.Surface structure and breakthroughs were analyzed under Digital Light Microscope Keyence VHX 7000 with 100- × 1000 VH-Z100T lens. All of the pictures were recorded with a VHX 7020 camera.38. In a typical procedure, a 500 mL three-neck, round-bottom flask was charged with 25.00 g of Octahydrospherosilicate (24.56 mmol), 250 ml of toluene, and olefins, that is, vinyltrimethoxysilane (VTMOS) and octadec-1-ene (OD) in a molar ratio according to the stoichiometry of the product to be obtained . A magnetic stirring bar was added. A thermometer and condenser equipped with an argon inlet and oil bubbler were attached, the flask was placed in a heating mantle and the system was purged with argon. The reaction mixture was set at 110°C and before reaching boiling, 25 µl of Karstedt’s catalyst solution was added, which resulted in a quick increase of temperature and the system starting to reflux. The reaction mixture was kept at reflux and samples were taken for FT-IR control until full Si–H group consumption was observed. After reaction completion  solvent was evaporated under a vacuum to dry to obtain the final product. OSS-8TMOS and OSS-2OD-6TMOS were obtained as clear liquids of relatively low viscosity, OSS-4TMOS-4OD, and OSS-3TMOS-5OD as soft, white waxes, and OSS-8OD as hard, white wax.The hydrosilylation reaction was performed according to a previous report1H NMR : d (ppm) = 1.31–1.26 , 0.88 , 0.59 , 0.12 ;13C NMR : d (ppm) = 33.70, 32.11, 32.10, 29.99, 29.96, 29.94, 29.93, 29.92, 29.91, 29.87, 29.86, 29.65, 29.55, 23.18, 22.87, 18.08, 17.89, 14.28 (–CH2CH3), − 0.17 (SiMe2);29Si NMR : d (ppm) = 12.58 (OSiMe2), -108.88 (core).1H NMR : d (ppm) = 3.56 (OMe), 1.30–1.25 , 0.88 , 0.61–0.56  0.13 ethyl SiMe2), 0,12  ;13C NMR : d (ppm) = 51.00, 50.75, 50.69, 50.50 (OMe), 33.68, 32.10, 29.95, 29.92, 29.90, 29.84, 29.64, 29.54, 23.16, 22.86, 17.88, 14.27 (octadecyl), 8.64 (Si–CH2CH2-Si), 7.41, 5.27 (SiCH(CH3)Si), 0.40 (Si–CH2CH2-Si), − 0.18, − 0.21, (octadecyl SiMe2), − 0.95, − 0.98, − 1.00 ((trimethoxysilyl)ethyl SiMe2);29Si NMR : d (ppm) = 13.21–12.59 (OSiMe2), − 41.55 (Si(OMe)3), − 108.88-(−108.98) (core).1H NMR : d (ppm) = 3.55 (OMe), 1.30–1.25 , 0.88 , 0.61–0.56  0.13 ethyl SiMe2), 0,12  ;13C NMR : d (ppm) = 50.99, 50.73, 50.69, 50.51 (OMe), 33.68, 32.11, 29.96, 29.94, 29.91, 29.86, 29.66, 29.55, 23.16, 22.87, 17.86, 14.28 (octadecyl), 8.63 (Si–CH2CH2-Si), 7.42, 5.29 (SiCH(CH3)Si), 0.41 (Si–CH2CH2-Si), − 0.18, − 0.21, (octadecyl SiMe2), − 0.95, − 0.97, − 1.02 ((trimethoxysilyl)ethyl SiMe2);29Si NMR : d (ppm) = 13.22–12.59 (OSiMe2), -41.55 (Si(OMe)3), -108.88-(-108.98) (core).1H NMR : d (ppm) = 3.56 (OMe), 1.30–1.25 , 0.88 , 0.61–0.56  0.13 ethyl SiMe2), 0,12  ;13C NMR : d (ppm) = 50.98, 50.75, 50.68, 50.50 (OMe), 33.67, 32.10, 29.90, 29.84, 29.64, 29.54, 23.16, 22.86, 17.86, 14.26 (octadecyl), 8.94, 8.63(Si–CH2CH2-Si), 7.41, 5.27 (SiCH(CH3)Si), 0.41 (Si–CH2CH2-Si), − 0.18, − 0.21, − 0.23 (octadecyl SiMe2), − 0.95, − 0.98, − 1.00, − 1.06 ((trimethoxysilyl)ethyl SiMe2);29Si NMR : d (ppm) = 13.20–12.58 (OSiMe2), − 41.57 (Si(OMe)3), − 108.95–(− 108.98) (core).1H NMR : d (ppm) = 3.55 , 1.11  0.59 , 0.13 ;13C NMR : d (ppm) = 50.67 (OMe), 8.58 (Si–CH2CH2-Si), 7.36, 5.25 (SiCH(CH3)Si), 0.41 (Si–CH2CH2-Si), − 1.05 (SiMe2);29Si NMR : d (ppm) = 13.23 (OSiMe2), − 41.66, − 42.78 (OSi(OMe)3), − 108.95 (cage).The polymer and the chosen additive were homogenized using a laboratory two-roll mill ZAMAK MERCATOR WG 150/280. A portion of 500 g PLA Ingeo™ 2003 D was heated on the rolls until molten, and then the additive was slowly added in portions, until the final concentration of 5.0 wt.% (with respect to the whole system mass). The mixing was performed at the roll temperature of 210 °C for 15 min., getting to full homogeneity of the composition. The resulting polymer system was then granulated using a SHINI SG-1417-CE grinding mill and dried at 55 °C/24 h. The granulates were diluted with neat PLA up to the final additive loading of 0.10, 0.25, 0.50, 1.0, 1.5, 2.5 wt.% upon screw extrusion with subsequent cold granulation on the single-screw extrusion setup FILABOT EX6, and dried for 24 h at 40 °C.The granulates obtained as above were used for the extrusion of filaments of 1.75 mm diameter by a single-screw extrusion setup FILABOT EX6. Extrusion temperatures were sequentially from the nozzle to the feed zone respectively: 170 °C, 195 °C, 190 °C, and 60 °C.Using a 3D printer Prusa i3 MK3S + two types of samples were printed by FDM: dumbbells and bars, according to PN-EN- ISO 527−2. The parameters of printing are given in Table -1 and 889 cm-1, associated with stretching and bending of the Si–H bond, respectively. The hydrosilylation proceeded with the completion of the reaction (~ 99%). The structure and purity of the compounds were confirmed by 1H, 13C, and 29Si NMR. Hydrosilylation of VTMOS resulted in the formation of 15–20% of α isomer of the corresponding (trimethoxysilyl)ethyl group formed in all the examples, while OD hydrosilylation gave exclusively β isomer of the corresponding octadecyl group attached to the spherosilicate siloxy moiety. The MALDI-TOF mass spectrometry analysis confirms the statistical distribution of hydrosilylation products formed. For example, for OSS-5OD-3TMOS, a series of congeners were observed, from a product containing 7 (trimethoxysilyl)ethyl groups and 1 octadecyl group to a product containing 8 octadecyl groups 40, and polylactide (PLA) matrices35, allowing to study structure-, state of matter-, and processing methodology-related aspects of cage siloxane-polymer systems miscibility. The EDS images were taken from a cross-section of the extruded granulate. It can be seen that, besides OSS-8TMOS MOS Fig. .Figure 5Thermal analysis). Under the polymer melt conditions, many additives for plastics show limited miscibility with the polymer phase in the form of migration capabilities41. Based on the physical appearance of OSS-8TMOS, the cause may be its low viscosity and fairly low molecular weight, the latter rising significantly with the number of octadecyl groups in the product molecule.The linear scans confirmed that OSS-8TMOS shows the best additive distribution, while PLA composition with OSS-8OD was characterized by significant additive concentration inconsistencies. This effect was present to a much smaller extent for OSS-4OD-4TMOS due to the presence of both well-dispersed and aggregated phases. It is important to note that as for OSS-8TMOS, the additive distribution was seemingly very uniform, however, as further explained for DSC analysis, it does not translate to full miscibility of PLA and the compound itself . The analysis of the sample cross-sections after mechanical fracture was an essential tool to understand the impact of the additives on PLA compositions behavior during 3D printing such as interlayer fusion or voids formation, and their modes of mechanical failure  that translate into changes in mechanical strength of the samples under load  provides them lubricating action for the polymer melt under load, as well as miscibility thereof in the polymer melt, and therefore thinning and plasticizing properties.Melt flow ratio (MFR) was used as a simple tool for the assessment of polymer melt flowability under static load Fig. . It is vOptical microscopy) revealed that the OSS compounds affect several aspects of the material microstructure and mechanical behavior under load, e.g. interlayer fusion, delamination, cracking, phase separation, and polymer phase plasticization  require the development of materials dedicated strictly to the processing properties of 3D printers. For this reason, each time when designing a material, it is necessary to characterize the mechanical and thermal properties as comprehensively as possible or to assess the quality of the printout. The introduction of silsesquioxanes with variable functional group stoichiometry, presented in this study, is a useful tool for precise modeling of the indicated properties. Materials with a concise internal structure of the printout are a product for maintenance systems, production of spare parts, and parts of machines and devices in short-series production. In particular, objects with a solid internal structure are used in printing elements for the transport of gases or liquids (e.g. hydraulic fittings). This work presented the attempts at the application of novel compounds of octaspherosilicate (OSS) type as the additives for controlling the properties of PLA for FDM 3D printing, as a continuation of our previous studies on this matter. The effects of the additive molecular structure and loading were discussed with respect to the additive-polymer compatibility (miscibility), and the thermal, mechanical, and surface properties of such obtained materials, as well as their print quality and rigidity. The alkoxysilyl groups provided good miscibility of the additives with PLA, while on the other hand, long alkyl chains were too apolar for suitable compatibility with the polyester-type polymer. However, the right proportion of the two substituents within an OSS compound allowed for obtaining an additive of superior properties when introduced into PLA. Especially interesting properties were obtained for OSS-2OD-6TMOS, where improved interlayer fusion within the printed material, plasticization of the material, and therefore increased mechanical toughness were achieved, on top of enhanced crystallization ability and hydrophobic properties.