Higher Cell Numbers Research Articles

Laser microgrooving and resorbable blast texturing for enhanced surface function of titanium alloy for dental implant applications

Long-term dental implant success is dependent on biocompatibility and osseointegration between the bone and the implant. Surface modifications such as laser-induced microgrooving which increase contact area can enhance osseointegration by establishing and directing a stable attachment between the implant surface and peri‑implant bone. The objective of this study was to evaluate pre-osteoblast proliferation, morphology, and differentiation on titanium alloy (Ti64) surfaces—Laser-Lok© (LL), resorbable blast textured (RBT), and machined (M)—compared to tissue culture plastic (TCP) control. We hypothesized the LL surfaces would facilitate increased cellular alignment compared to all other groups, and LL and RBT surfaces would demonstrate enhanced proliferation and differentiation compared to M and TCP surfaces. Surface roughness was quantified using a surface profilometer, and water contact angle was measured to evaluate the hydrophilicity of the surfaces. Cellular function was assessed using quantitative viability and differentiation assays and image analyses, along with qualitative fluorescent (viability and cytoskeletal) imaging and scanning electron microscopy. No differences in surface roughness were observed between groups. Water contact angle indicated LL was the least hydrophilic surface, with RBT and M surfaces exhibiting greater hydrophilicity. Cell proliferation on day 2 was enhanced on both LL and RBT surfaces compared to M, and all three groups had higher cell numbers on day 2 compared to day 1. Cell orientation was driven by the geometry of the surface modification, as cells were more highly aligned on LL surfaces compared to TCP (on day 2) and RBT (on day 3). At day 21, cell proliferation was greater on LL, RBT, and TCP surfaces compared to M, though no differences in osteogenic differentiation were observed. Collectively, our results highlight the efficacy of laser microgrooved and resorbable blast textured surface modifications of Ti64 for enhancing cellular functions, which may facilitate improved osseointegration of dental implants.

Salmonella Biofilm Formation under Fluidic Shear Stress on Different Surface Materials.

This study characterized biofilm formation of various Salmonella strains on common processing plant surface materials (stainless steel, concrete, rubber, polyethylene) under static and fluidic shear stress conditions. Surface-coupons were immersed in well-plates containing 1 mL of Salmonella (6 log CFU/mL) and incubated aerobically for 48 h at 37 °C in static or shear stress conditions. Biofilm density was determined using crystal violet assay, and biofilm cells were enumerated by plating on tryptic soy agar plates. Biofilms were visualized using scanning electron microscopy. Data were analyzed by SAS 9.4 at a significance level of 0.05. A surface-incubation condition interaction was observed for biofilm density (p < 0.001). On stainless steel, the OD600 was higher under shear stress than static incubation; whereas, on polyethylene, the OD600 was higher under static condition. Enumeration revealed surface-incubation condition (p = 0.024) and surface-strain (p < 0.001) interactions. Among all surface-incubation condition combinations, the biofilm cells were highest on polyethylene under fluidic shear stress (6.4 log/coupon; p < 0.001). Biofilms of S. Kentucky on polyethylene had the highest number of cells (7.80 log/coupon) compared to all other strain-surface combinations (p < 0.001). Electron microscopy revealed morphological and extracellular matrix differences between surfaces. Results indicate that Salmonella biofilm formation is influenced by serotype, surface, and fluidic shear stress.

A quantitative method for early-stage detection of the internal-short-circuit in Lithium-ion battery pack under float-charging conditions

Detecting the internal short circuit (ISC) of Lithium-ion batteries is critically important for preventing thermal runaway. Conventional approaches mainly focus on ISC detection for dynamic load profiles, while the commonly seen float-charging scenarios with a high risk of ISC are rarely considered. Technical challenges arise from not only the lack of models describing battery dynamics at fully charged conditions but also the computational burden caused by the high number of series-connected cells in a battery pack. To address these issues, we here propose a simple and accurate method to quantitatively identify the leakage current of the battery with ISC, by checking the behaviors of the battery equalization system. Battery-in-the-loop experiments are carried out to verify the proposed method under a wide range of the short circuit resistances (∼20 to ∼500Ω), using both LiFePO4 and Li(NiCoMn)0.33O2 batteries with different aging degrees. The typical errors of the identified leakage currents can be well-bounded within ±1 mA, including the cases of using both passive and active balancing. The proposed method is completely model-free and, therefore, can be transplanted into low-cost embedded systems to support broader applications.

Morphological Analysis of the Hemolymph Cell Composition in the Bivalve Mollusk &lt;i&gt;Anadara&lt;/i&gt; &lt;i&gt;broughtonii&lt;/i&gt; Schrenck, 1867 (Sea of Japan)

The hemolymph cells of the ark clam Anadara broughtonii were examined using light microscopy, flow cytometry and gradient centrifugation. All three methods of analysis made it possible to identify two main types of cells in the hemolymph of the ark clam Anadara broughtonii – large granular erythrocytes and small agra-nular amebocytes. Erythrocytes accounted for 95.6 ± 0.9% of the total number of hemolymph cells. Erythrocytes were hemoglobin-containing cells with a great number of granules in the cytoplasm, a low nuclear-cytoplasmic ratio (NCR) and a lower intensity of cellular respiration compared to amebocytes. Amebocytes are cells of predominantly irregular shape with a high number of cells that do not contain or contain no more than 10 granular inclusions in the cytoplasm. All types of hemocytes found in the ark clam hemolymph demonstrated the same ability to spontaneously produce reactive oxygen species. For the first time, it has been shown that red blood cells of the ark clam Anadara broughtonii are capable of phagocytosis. At the same time, amebocytes absorbed on the average two times more zymosan particles (10.3 ± 0.7 pcs.), compared to erythrocytes (5.3 ± 0.1 pcs.).

An optimized TRIzol-based method for isolating RNA from adipose tissue.

High-quality RNA isolation from recalcitrant adipose tissue with high lipid content and low cell numbers is difficult. Many studies have made efforts to optimize methods for isolating RNA from adipose tissue through combinations of column-based kits and phenol-chloroform methods, or through in-house protocols. However, the considerable complexity of these protocols and the various kits/materials required hamper their wide use. Herein, we describe an optimized protocol based on TRIzol reagent, which is the most accessible ready-to-use reagent for nucleic acid and/or protein isolation in laboratories. This article provides a step-by-step protocol yielding sufficient and qualified RNA from lipid-rich specimens for downstream applications.

Combined effects of intermittent fasting with swimming-based high intensity intermittent exercise training in Wistar rats

High caloric intake and physical inactivity are known precursors to the development of several chronic metabolic diseases. For obesity and sedentarism, High Intensity Intermittent Exercise (HIIE) and Intermittent Fasting (IF) have emerged as individual strategies to attenuate their negative effects by improving metabolism. To study their combined effects, Wistar male rats (n = 74, 60 days old) were divided into four groups: Sedentary Control (C), swimming-based HIIE only (HIIE), Intermittent Fasting only (IF), and swimming-based HIIE associated with Intermittent Fasting (HIIE/IF). Over an eight-week period swimming performance, body composition, weight and feeding behavior were analyzed. The final morphology of white adipose tissue showed a significant reduction in adipocyte size consistent with a higher number of cells per area in exercised animals (vs C and IF, p < 0.05), which also displayed characteristics of browning through UCP-1 levels and CD31 staining. These results suggest that the increased performance in the HIIE/IF group is, in part, by modifications of WAT metabolism through the browning process.

Hydrogels with stiffness-degradation spatial patterns control anisotropic 3D cell response

In nature, tissues are patterned, but most biomaterials used in human applications are not. Patterned biomaterials offer the opportunity to mimic spatially segregating biophysical and biochemical properties found in nature. Engineering such properties allows to study cell-matrix interactions in anisotropic matrices in great detail. Here, we developed alginate-based hydrogels with patterns in stiffness and degradation, composed of distinct areas of soft non-degradable (Soft-NoDeg) and stiff degradable (Stiff-Deg) material properties. The hydrogels exhibit emerging patterns in stiffness and degradability over time, taking advantage of dual crosslinking: Diels-Alder covalent crosslinking (norbornene-tetrazine, non degradable) and UV-mediated peptide crosslinking (matrix metalloprotease sensitive peptide, enzymatically degradable). The materials were mechanically characterized using rheology for single-phase and surface micro-indentation for patterned materials. 3D encapsulated mouse embryonic fibroblasts (MEFs) allowed to characterize the anisotropic cell-matrix interaction in terms of cell morphology by employing a novel image-based quantification tool. Live/dead staining showed no differences in cell viability but distinct patterns in proliferation, with higher cell number in Stiff-Deg materials at day 14. Patterns of projected cell area became visible already at day 1, with larger values in Soft-NoDeg materials. This was inverted at day 14, when larger projected cell areas were identified in Stiff-Deg. This shift was accompanied by a significant decrease in cell circularity in Stiff-Deg. The control of anisotropic cell morphology by the material patterns was also confirmed by a significant increase in filopodia number and length in Stiff-Deg materials. The novel image-based quantification tool was useful to spatially visualize and quantify the anisotropic cell response in 3D hydrogels with stiffness-degradation spatial patterns. Our results show that patterning of stiffness and degradability allows to control cell anisotropic response in 3D and can be quantified by image-based strategies. This allows a deeper understanding of cell-matrix interactions in a multicomponent material.

Analytics in Extrusion-Based Bioprinting: Standardized Methods Improving Quantification and Comparability of the Performance of Bioinks.

Three-dimensional bioprinting and especially extrusion-based printing as a most frequently employed method in this field is constantly evolving as a discipline in regenerative medicine and tissue engineering. However, the lack of relevant standardized analytics does not yet allow an easy comparison and transfer of knowledge between laboratories regarding newly developed bioinks and printing processes. This work revolves around the establishment of a standardized method, which enables the comparability of printed structures by controlling for the extrusion rate based on the specific flow behavior of each bioink. Furthermore, printing performance was evaluated by image-processing tools to verify the printing accuracy for lines, circles, and angles. In addition, and complementary to the accuracy metrics, a dead/live staining of embedded cells was performed to investigate the effect of the process on cell viability. Two bioinks, based on alginate and gelatin methacryloyl, which differed in 1% (w/v) alginate content, were tested for printing performance. The automated image processing tool reduced the analytical time while increasing reproducibility and objectivity during the identification of printed objects. During evaluation of the processing effect of the mixing of cell viability, NIH 3T3 fibroblasts were stained and analyzed after the mixing procedure and after the extrusion process using a flow cytometer, which evaluated a high number of cells. It could be observed that the small increase in alginate content made little difference in the printing accuracy but had a considerable strong effect on cell viability after both processing steps.

Microencapsulation and Application of Probiotic Bacteria Lactiplantibacillus plantarum 299v Strain.

Microencapsulation is an up-and-coming technology for maintaining the viability of probiotics. However, the effect of core-to-wall ratios and ratios of polysaccharides on the protection of the Lactiplantibacillus plantarum 299v strain has not been deeply discussed. Lyophilization of the Lp. plantarum 299v strain was conducted, and different core-to-wall ratios and ratios of maltodextrin (MD) and resistant starch (RS) were applied. Results demonstrated that the content of MD and RS had an influence on the yield and bulk density in both core-to-wall ratios (1:1 and 1:1.5). In addition, samples coated with a core-to-wall ratio of 1:1.5 had significantly higher viability than those coated with a core-to-wall ratio of 1:1. Moreover, samples coated with core-to-wall ratios of 1:1 and MD:RS 1:1, as well as core-to-wall ratios of 1:1.5 and MD:RS 3:1, had the highest cell number after simulated gastric fluid and simulated intestinal fluid testing, respectively. Furthermore, the optimal formulation for the application of microencapsulated Lp. plantarum 299v in apple juice (serving as a functional beverage) is listed as follows: core-to-wall ratios of 1:1 and MD:RS 1:1, with the fortification method, and stored at 4 °C. After 11 weeks of storage, the cell count was 8.28 log (CFU/mL). This study provided a strategy for Lp. plantarum 299v to achieve high viability in long-term storage and provides an application in functional apple beverages.

Abstract SY16-02: Generation of B cell immunity in tertiary lymphoid structures supports response to immunotherapy

Abstract SY16-02: Generation of B cell immunity in tertiary lymphoid structures supports response to immunotherapy

Abstract 4135: A novel plasma cell-based mechanism of action of adenosine immunomodulation and A2AR antagonism

Abstract High levels of extracellular adenosine, often found in the tumor microenvironment (TME), promote immune suppression mainly through the A2A receptor (A2AR) expressed by tumor-infiltrating immune cells. Inupadenant (formerly known as EOS100850) is an oral, non-brain penetrant, potent and highly selective small molecule antagonist of A2AR. In a Phase I/Ib clinical trial (NCT02740985), inupadenant as monotherapy showed initial evidence of clinical benefit in subjects with advanced solid tumors. In this study, tumor biopsies with a high number of A2AR-expressing immune cells at baseline were associated with response or stable disease.We now report that infiltration of A2AR+ cells was strongly correlated with the expression of B- and, more specifically, antibody-secreting cell (ASC)-related genes, as assessed by gene expression (Nanostring) and immunohistochemistry (IHC), hinting at a potential novel role for A2AR in B cell biology. Therefore, we explored the expression and function of A2AR in human B cells. Immunocytochemistry staining of A2AR on sorted tonsillar B cell subsets showed that A2AR was predominantly expressed on ASCs, including plasma cells and plasma blasts versus naïve or memory B cells. The preferential expression of A2AR by ASCs was confirmed on NSCLC tissues by multiplex immunofluorescence. In addition to A2AR, ASCs expressed other adenosine pathway markers such as CD39, suggesting that the adenosine pathway is a key mechanism through which ASC functions may be modulated. Using B cells derived from peripheral blood, the A2AR agonist CGS-21680 was shown to inhibit the maturation of B cells into plasma cells, and that maturation could be fully restored by inupadenant. CGS-21680 did not affect B cell or plasma cell viability, indicating that the effect of A2AR signaling on plasma cell differentiation is not due to preferential plasma cell death in culture. Importantly, baseline expression of B cell- and ASC-related markers measured by Nanostring and IHC was associated with response or stable disease to inupadenant monotherapy, supporting the notion that these cells may be a novel target of inupadenant. This is in line with recent reports showing that B cells, plasma cells and tertiary lymphoid structures are associated with favorable responses to cancer immunotherapy. Interestingly, four out of the five non-progressors treated with inupadenant as monotherapy and with available biomarker data showed a reduction in ASC infiltration after inupadenant treatment, suggesting that inupadenant may promote terminal plasma cell differentiation and migration out of the tumor tissue and to the bone marrow.Altogether, these data support a novel plasma cell-centric mechanism of action of inupadenant, which may complement its reported T cell-mediated anti-tumor activity. Citation Format: Chiara Martinoli, Paola Tieppo, Marjorie Mercier, Hussein Shehade, Noemie Wald, Anais Vezzu, Annelise Hermant, Boris Pirlot, Stephanie Ma, Maurizio Ceppi, Yvonne McGrath, Maura Rossetti. A novel plasma cell-based mechanism of action of adenosine immunomodulation and A2AR antagonism. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4135.

Ubiquitin-Specific Protease 43 Impacts Pancreatic Ductal Adenocarcinoma Prognosis by Altering Its Proliferation and Infiltration of Surrounding Immune Cells.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating cancer, and the therapy options for PDAC remain restricted. The distinctive tumor immunological microenvironment (TIME) of PDAC, comprising a high number of stromal cells and a limited infiltration of cytotoxic T lymphocytes (CTLs), rendered immunotherapy ineffective. The protein level of ubiquitin-specific protease 43 (USP43) was a prognostic predictor in numerous cancers; however, its function in PDAC is limited. This article focuses on the influence of USP43 expression on PDAC prognosis and TIME alteration. Based on TCGA database and tissue microarray staining, the expression of USP43 in PDAC was evaluated. The association between USP43 and prognosis was then investigated using tissue samples and online databases. In PDAC tumor tissues, the correlation between USP43 expression and clinicopathological characteristics, immune cell infiltration, and prognosis was investigated. The expression of USP43 in PDAC cell lines was evaluated using quantitative polymerase chain reaction. Using a cell counting kit-8 (CCK-8) and a cell colony formation test, the viability of the cells was determined. On the basis of online databases and tissue samples, the link between USP43 and immune cell infiltration around PDAC was also examined. For statistical analyses, the software GraphPad, R, and SPSS 26.0 were utilized. The expression of USP43 was considerably higher in PDAC compared to normal pancreatic tissue in both the TCGA database and the tissue microarrays of PDAC patients (P < 0.001). High USP43 expression was associated with poor overall survival in both the TCGA database and the tissue microarray of PDAC patients (P = 0.046 and 0.021, respectively). USP43 overexpression promoted PANC-1 cell proliferation (P = 0.0018), but USP43 knockdown decreased PANC02 cell proliferation (P < 0.001). According to the TCGA database, USP43 is associated with T cell activation and inhibits CD8+ T cell activation in PDAC, as proven by a study of cell lines. Moreover, in both TCGA and PDAC cell lines, USP43 expression was negatively linked with the chemokine signaling pathway. Overexpression of USP43 is a potential prognostic indicator for PDAC patients. USP43 is a potential biomarker associated with T cell activation, suppression of CD8+ T cell enrichment, and the cytokine signal pathway. Future multicenter studies are needed to confirm our findings and their potential application in the treatment of PDAC patients.

A Study on Breast Augmentation Using Fat Grafting With Stromal Vascular Fraction.

Stromal vascular fraction (SVF) and fat grafting are emerging alternatives to implants for breast augmentation. However, the lack of controlled clinical data has led to conflicting results regarding the effectiveness of surgical treatments. This study aimed to identify the key factors affecting the outcomes of fat grafting with SVF and to recognize novel methods to improve the retention rate. In total, 384 women underwent breast augmentation using fat grafting with SVF. The patients were preoperatively and postoperatively managed and recalled for follow-up at 3, 6, and 18 months. The average volume of the injection in the left breast was 162.35 mL (range, 50-260 mL). The postoperative retention rates were 78.65% in 384 patients at 3 months, 77.17% in 273 patients at 6 months, and 77.48% in 102 patients at 18 months. The retention rates were compared based on the number of SVF cells; patients with more than 60 million cells had a retention rate of 70.77%, and those with less than 60 million cells had a retention rate of 85.60% at 18 months. The retention rates at the 18-month follow-up were 65.62% and 85.09% in stiff and soft breasts, respectively. A higher number of cells in the SVF was associated with a greater retention volume, and the retention volume was greater in patients with soft breasts.Given the higher use of the right arm, after 18 months of the surgery, the retention rate of the right breast (60.35%) was lower than that of the left breast (77.48%) ( P < 0.05; t = -13.199). Limiting arm movement, increasing the number of cells in the SVF, and improving the skin tension might enhance the retention rate in patients undergoing breast augmentation.

5599228 PITFALLS IN DIAGNOSING THROMBOTIC THROMBOCYTOPENIC PURPURA IN SICKLE CELL DISEASE

Background: Thrombotic microangiopathies are diagnosed by thrombocytopenia, microangiopathic haemolytic anaemia and evidence of end organ damage. In TTP this includes neurological symptoms, renal impairment and cardiac microthrombi. Confirmation of the diagnosis is severe deficiency (<10%) of the metalloproteinase ADAMTS13 and/or the presence of an inhibitor. The diagnosis of TTP in sickle cell disease (SCD) can be challenging as SCD is characterised by a chronic state of haemolysis often with very abnormal peripheral blood morphology so clinical features suggestive of TTP can be due to complications of SCD. More specifically, the coexistence of neurological impairment and thrombocytopenia seen in fat embolism syndrome (FES) complicating SCD can lead to an erroneous diagnosis of TTP. FES typically affects non-homozygous patients and those with a previously mild course of their illness manifesting with respiratory failure, neurological signs, thrombocytopenia and potential involvement of any system leading to high morbidity and mortality. Peripheral blood morphology typically shows very high number of nucleated red blood cells (nRBC). Aims: To identify published cases of TTP in patients with SCD and review the diagnostic approach. Methods: A PubMed literature search was performed using the terms “sickle cell” and “thrombotic thrombocytopenic purpura”/”TTP”. Results: Of 19 cases identified, 9 were individual case reports and a case series of 10. All patients had very mild course of their SCD. The median nadir platelet count was 38 x 109/L (7-70). 84% had neurological involvement either at presentation or shortly afterwards; primarily altered sensorium/drop in GCS. 79% patients also had severe type I respiratory failure either preceding or occurring simultaneously with the development of neurological signs and associated with extensive changes on chest imaging. Schistocytes were identified in all cases but in the vast majority at relatively low levels; at the same time, all showed large numbers of nRBCs. 18 of the 19 cases were published after 1998; in 15 there was no mention of ADAMTS13 testing at all. In the 3 cases with ADAMTS13 levels, all were normal; 2 samples were obtained before therapeutic plasma exchange (TPE) and 1 after. 18 patients received TPE and 1 an infusion of donor plasma. However, prior to TPE, all patients had already received red cell transfusion achieving Hb S levels of 30% or less. There were 2 (11%) deaths while the remainder of patients achieved complete recovery. One due to intracranial haemorrhage and the other, sepsis in the context of immunosuppression for prevention of TTP recurrence. The presence of early respiratory failure, the inconclusive morphological findings, the degree of thrombocytopenia, and the complete absence of confirmatory ADAMTS13 results bring the diagnosis of TTP to question in all cases. Given the clinical presentations, we suspect that some of these patients had FES. The positive outcomes may be explained by the fact that all patients received adequate red cell transfusion, but also the possible beneficial effect of TPE in acute complications of SCD with a hyper inflammatory component such as FES. Conclusion: The diagnosis of TTP in patients with SCD is challenging due to many overlapping features. It should not be made without confirmatory ADAMTS13 results and the possibility of alternative diagnoses should be explored.

Gouqizi () seed oil reduces D-galactose induced inflammation in testis of rats Janus kinase 1/signal transducerand activator of transcription 1/nuclear factor-κB and.

To investigate the efficacy of Gouqizi () seed oil (FLSO) on D-gal induced inflammation in testis of rats and . In aging Sertoli cells (TM4 cells) induced by D-galactose (D-gal), the expression of upregulated aging-related proteins. The number of cells counted by cell counting kit (CCK)-8 assay showed a high number of cells disposed with FLSO at 50, 100 and 150 μg/mL compared to that for the aging model. , male Sprague-Dawley rats ( = 50, 8-week-old, 230-255 g) were randomly categorized into control, aging model, and FLSO (low-, medium-, and high-dose) groups. The expression of nuclear factor-κB (NF-κB) and its upstream factors [Janus kinase 1 (JAK1) and signal transducerand activator of transcription 1 (STAT1)] was detected by Western blot and immunofluorescence, related inflammatory factors quantified by enzyme-linked immunosorbent assay. Evaluation of testicular tissue by Johnsen score, the spermatogenic function was explored. The expression of interleukin-1β (IL-1β) ( < 0.05), IL-6 ( < 0.001), and tumor necrosis factor α (TNF-α) ( < 0.05) was decreased significantly, while that of heme oxygenase-1 (HO-1) ( < 0.001) and IL-10 ( < 0.05) was increased in cells disposed with FLSO 100 μg/mL. FLSO inhibited the expression of NF-B and declined p-p65/p65 ( < 0.01), as detected by Western blotting. In, the levels in serum of IL-1β ( < 0.001), IL-6 ( < 0.05), and TNF-( < 0.01) declined while IL-10 ( < 0.05) was upregulated post-FLSO treatment. In addition, the expression of JAK-1 and STAT1 increased significantly in testicular tissue of rats treated with FLSO as compared to the aging model of rats ( < 0.001), while the expression of NF-κB ( < 0.001) declined in the testis in the FLSO group, as assessed by immunofluorescence. The levels of inhibor B and testosterone in serum both increased (< 0.05). In conclusion, this study determined the protective effects of FLSO to tolerate inflammatory injury in the testis, indicating that FLSO alleviates inflammation JAK-1/STAT1/NF-κB pathway.

Optimization of Probiotic Lactobacilli Production for In-Feed Supplementation to Feedlot Cattle

The selection of probiotic bacteria based on their beneficial characteristics does not necessarily mean they can be later scaled up and used for technological applications and formula design. Three probiotic strains—Lactobacillus acidophilus CRL2074, Limosilactobacillus fermentum CRL2085, and Limosolactobacillus mucosae CRL2069, originally isolated from feedlot cattle feces—have demonstrated beneficial characteristics when used as in-feed probiotics. Therefore, the current study was conducted to develop a low-cost culture medium to optimize growth conditions to enhance biomass production. The study also sought to identify appropriate cryoprotective agents to sustain high functional cell numbers after freeze drying. A central composite design was applied to determine the optimal medium composition. This yielded a simplified, low-cost effective medium containing 3% molasses and industrial yeast extracts (0.5 to 2.5%) as carbon and nitrogen sources, which were added to a basal medium for each strain. Established production conditions at 37 °C, without agitation, and pH-controlled for the CRL2085 and CRL2069 strains, and free pH for the CRL2074 strain, allowed us to obtain biomass yields of 12.95, 18.20, and 12.25 g, respectively, at 24-h incubation, compared with the MRS medium. In addition, the cryoprotective effect of the selected agents was demonstrated to be strain-dependent. Thus, the highest viability (109–1010 CFU/g), stability during 30-d storage, and survival rate (88–99%) were achieved when 10% MSG (monosodium glutamate), sucrose + fructose + trehalose + WPC (whey protein concentrate) + 10% MSG, and 1.2% WPC + 10% trehalose, were used for freeze drying CRL2074, CRL2085, and CRL2069, respectively. Moreover, the probiotic strains retained their probiotic functionality when hydrophobic characteristics were evaluated. These results highlight the need to perform strain-specific evaluation of the critical factors involved in the large-scale production of probiotic lactobacilli to sustain viability and stability after the freeze drying and storage processes.

Buffy Coat Processing Impacts on Monocytes’ Capacity to Present Lipid Antigens

Buffy Coats, generated from a blood donor's whole blood bag unit, are commonly used in biomedical research as a source of leukocytes due to the high number of cells that can be recovered from each Buffy Coat. Buffy Coats are leukocyte-enriched residual units obtained by centrifugation of whole blood. At the blood bank, blood can be processed using two different protocols according to the time interval between blood collection and processing. When blood collection and processing occur on the same day, it gives rise to Fresh Blood Buffy Coats. Alternatively, if blood processing only happens on the day after blood collection, Overnight Blood Buffy Coats are created. In this study, we aimed to address whether these two different Buffy Coat-processing protocols could differently impact monocyte function as antigen-presenting cells. For this purpose, we analyzed in the same experiment monocytes isolated from Fresh Blood and from Overnight Blood Buffy Coats. We assessed lipid antigen presentation by CD1d to invariant Natural Killer T (iNKT) cells. CD1d is a non-polymorphic MHC class I-like protein, which facilitates the study of antigen presentation among allogeneic samples. The results show that monocytes from Fresh Blood Buffy Coats have a better capacity to present antigens by CD1d, and consequently to activate iNKT cells, when compared to monocytes from Overnight Blood Buffy Coats. The differences observed were not explained by disparities in monocyte viability, CD1d expression, or basal activation state (monocyte expression of CD40 and CD80). Buffy Coats are a valid source of blood cells available daily. Hence, the type of protocol for Buffy Coat processing should be carefully considered in day-to-day research, since it may lead to different outcomes.

Induction of systemic, mucosal, and cellular immunity against SARS-CoV-2 in mice vaccinated by trans-airway with a S1 protein combined with a pulmonary surfactant-derived adjuvant SF-10.

There is a need for vaccines that can induce effective systemic, respiratory mucosal, and cellular immunity to control the COVID-19 pandemic. We reported previously that a synthetic mucosal adjuvant SF-10 derived from human pulmonary surfactant works as an efficient antigen delivery vehicle to antigen presenting cells in the respiratory and gastrointestinal tracts and promotes induction of influenza virus antigen-specific serum IgG, mucosal IgA, and cellular immunity. The aim of the present study was to determine the effectiveness of a new administration route of trans-airway (TA) vaccine comprising recombinant SARS-CoV-2 spike protein 1 (S1) combined with SF-10 (S1-SF-10 vaccine) on systemic, local, and cellular immunity in mice, compared with intramuscular injection (IM) of S1 with a potent adjuvant AddaS03™ (S1-AddaS03™ vaccine). S1-SF-10-TA vaccine induced S1-specific IgG and IgA in serum and lung mucosae. These IgG and IgA induced by S1-SF-10-TA showed significant protective immunity in a receptor binding inhibition test of S1 and angiotensin converting enzyme 2, a receptor of SARS-CoV-2, which were more potent and faster achievement than S1-AddaS03™-IM. Enzyme-linked immunospot assay showed high numbers of S1-specific IgA and IgG secreting cells (ASCs) and S1-responsive IFN-γ, IL-4, IL-17A cytokine secreting cells (CSCs) in the spleen and lungs. S1-AddaS03™-IM induced IgG ASCs and IL-4 CSCs in spleen higher than S1-SF-10-TA, but the numbers of ASCs and CSCs in lungs were low and hardly detected. Based on the need for effective systemic, respiratory, and cellular immunity, the S1-SF-10-TA vaccine seems promising mucosal vaccine against respiratory infection of SARS-CoV-2.

Insights into the enumeration of mixtures of probiotic bacteria by flow cytometry

The use of flow cytometry to enumerate microorganisms is gaining traction over the traditional plate count technique on the basis of superior accuracy, precision and time-to-result. Here, we assessed the suitability of live/dead flow cytometry for the enumeration of mixed populations of probiotic bacteria (L. acidophilus, L. paracasei, L. plantarum, L. salivarius, B. lactis and B. bifidum) whilst comparing outcomes with plate counting. Using a novel gating strategy designed specifically for the enumeration of mixed populations, the application of flow cytometry resulted in the detection of higher numbers of viable bacteria with a greater level of repeatability than plate counting (RSD of 6.82 and 13.14% respectively). Across all multi-species blends tested, viable cell input was more accurately recovered by flow cytometry (101.8 ± 6.95%) than plate counts (81.37 ± 16.03%). However, when certain probiotic mixtures contained preparations with high numbers of non-viable cells in their total population, flow cytometry had the potential for overestimation of the viable population. Nevertheless, the comparative plate counts of these mixtures were low and variable, thus supporting the use of flow cytometry for the enumeration of viable bacteria in mixed populations.

A nano-scale n-bit ripple carry adder using an optimized XOR gate and quantum-dots technology with diminished cells and power dissipation

In the nano-scale era, quantum-dot cellular automata (QCA) technology has become an appealing substitute for transistor-based technologies. QCA will be the preferred technology for developing the next generation of digital systems. On the other hand, the full-adder and ripple carry adder (RCA) are the crucial building blocks of complex circuits, the most used structures in digital operations systems, and a practical part of the most well-known complex circuits in QCA technology. In addition, this technology was used to design the full adder for several procedures, like multiplication, subtraction, and division. For this reason, the full adder is generally investigated as a central unit and microprocessor in developing QCA technology. Furthermore, most previous QCA-based adder structures have suffered from some drawbacks, such as a high number of cells, high energy consumption, the high number of gates, and the placement of inputs and outputs in a closed loop; hence, the implementation of an efficient adder with only one gate and a low number of cells, such as exclusive-OR (XOR) gate, can solve all previous problems. Therefore, in this paper, a significantly improved structure of 3-input XOR is suggested based on the promising QCA technology. In addition, a QCA clocking mechanism and explicit cell interaction form the foundation of the proposed QCA-based XOR gate configuration. This gate can be easily converted into an adder circuit while containing a small number of cells and being extremely compressed. The suggested QCA-based XOR design is focused on optimizing a single-bit adder using cellular interaction. The suggested single-bit adder contains 14 cells. Based on this adder, several different RCAs, such as 4, 8, 16, and 32-bit, are designed. The comparison of the proposed single-bit adder to the best coplanar and multi-layer ones shows a 51.72% and 36.36% reduction of cells, respectively. In addition, all suggested designs are verified through simulation using QCADesigner and QCAPro. Finally, many physical validations are provided to approve the functionality of the suggested XOR design.