The 1918 H1N1 influenza pandemic was among the most severe in history, taking the lives of approximately 50 million people worldwide, and novel prophylactic vaccines are urgently needed to prevent another pandemic. Given that macaques are physiologically relevant preclinical models of human immunology that have advanced the clinical treatment of infectious diseases, a lethal pandemic influenza challenge model would provide a stringent platform for testing new influenza vaccine concepts. To this end, we infected rhesus macaques and Mauritian cynomolgus macaques with highly pathogenic 1918 H1N1 influenza virus and assessed pathogenesis and disease severity. Despite infection with a high dose of 1918 influenza delivered via multiple routes, rhesus macaques demonstrated minimal signs of disease, with only intermittent viral shedding. Cynomolgus macaques infected via intrabronchial instillation demonstrated mild symptoms, with disease severity depending on the infection dose. Cynomolgus macaques infected with a high dose of 1918 influenza delivered via multiple routes experienced moderate disease characterized by consistent viral shedding, pulmonary infiltrates, and elevated inflammatory cytokine levels. However, 1918 influenza was uniformly nonlethal in these two species, demonstrating that this isolate is insufficiently pathogenic in rhesus and Mauritian cynomolgus macaques to support testing novel prophylactic influenza approaches where protection from severe disease combined with a lethal outcome is desired as a highly stringent indication of vaccine efficacy.IMPORTANCE The world remains at risk of an influenza pandemic, and the development of new therapeutic and preventative modalities is critically important for minimizing human death and suffering during the next influenza pandemic. Animal models are central to the development of new therapies and vaccine approaches. In particular, nonhuman primates like rhesus and cynomolgus macaques are highly relevant preclinical models given their physiological and immunological similarities to humans. Unfortunately, there remains a scarcity of macaque models of pandemic influenza with which to test novel antiviral modalities. Here, we demonstrate that even at the highest doses tested, 1918 influenza was not lethal in these two macaque species, suggesting that they are not ideal for the development and testing of novel pandemic influenza-specific vaccines and therapies. Therefore, other physiologically relevant nonhuman primate models of pandemic influenza are needed. Influenza is one of the major causes of worldwide morbidity and mortality, claiming approximately 500,000 lives annually. Influenza A virus has caused multiple human pandemics over the past century, including in 1918, 1957, 1968, and 2009. The most severe pandemic was the 1918 pandemic caused by an avian-origin H1N1 influenza virus, which was responsible for the deaths of 50 million to 100 million people worldwide. Recently, sporadic bird-to-human transmissions have been reported . This in6 TCID50 (50% tissue culture infective doses). While small-animal models of influenza possess many advantages, they do not recapitulate human disease pathology, and many human-specific therapeutics cannot be tested. Therefore, the nonhuman primate (NHP) model of influenza is crucial for the development of novel therapeutic and prophylactic treatments for influenza.Our understanding of influenza pathology and transmission is based predominantly on animal model systems. The 1918 influenza strain is highly virulent in small-animal models of mice  and ferr7 TCID50, of the influenza virus A/Netherlands/18/94 (H3N2) to test for vaccine effectiveness (4 TCID50), tonsillar (2.5 × 103 TCID50), and conjunctivas bilaterally (1.25 × 103 TCID50) (6 PFU of virus) inoculated through a combination of intratracheal, intranasal (i.n.), intraocular, and oral routes  of the 1918 influenza virus stock used for macaque infection was determined prior to use to be 103.2 PFU, which was consistent with data from previous reports for this virus  decreased in animal 30519 to 93% on day 4 p.i. but returned to 100% by day 6 . Taken together, these results show that 1918 influenza virus replicates variably and poorly in rhesus macaques, with negligible signs of clinical disease.Viral loads in the nasal , throat 6 PFU via intranasal, ocular, oral, and intratracheal routes of exposure (50).The limited replication observed in rhesus macaques led us to investigate the replication of 1918 influenza virus in another widely used nonhuman primate species, cynomolgus macaques. Based on previous studies describing a lethal outcome in cynomolgus macaques infected with a dose of 7 × 104 PFU (n = 4) or 5 × 105 PFU (n = 4) of 1918 influenza virus via intrabronchial inoculation. We believed that direct inoculation to the lower respiratory tract would initiate replication sufficient to cause severe disease. Animals were initially sampled every 2 days until day 12 p.i. for blood, bronchoalveolar lavage fluid, radiographs, and swabs to monitor disease progression and viral replication. Other vitals such as weight, heart rate, respiratory rate, oxygen saturation, temperature, and clinical scores were also monitored. A schematic outline of the experimental groups and sampling schedule is shown in n = 4) that were infected with a higher dose of 7 × 106 PFU, which matched the dose used previously to elicit a lethal outcome in the same animal model . No differences in the numbers of white blood cells, monocytes, and platelets in the whole blood were noted. The number of lymphocytes increased later on day 10 p.i. for group 1 and group 3, while animals in group 2 appeared to have low levels of lymphocytes detected in the blood. There was also an increase in neutrophils found in the blood of animals from group 2 beginning on day 8 p.i. until day 16 p.i., while group 3 animals showed a spike in neutrophils only on day 8 p.i. Serum biochemistry data were also collected; however, no discernible differences were found across the three groups .10-lower infection dose than group 3, it was not unexpected that the clinical disease observed in group 2 was milder than that in group 3, suggesting that the differences in virus doses and routes of exposure between the two groups contributed to the differences in disease outcomes.A comparison of the vitals of the three groups of infected cynomolgus macaques showed that the greatest disease severity was observed in the animals from group 3. These animals received the highest dose of inoculum and were infected via multiple routes of exposure, which included exposure to the upper and lower respiratory tracts. Rates of weight loss were comparable between groups 2 and 3, having maximum weight losses of 13.7% and 11.2%, respectively . In contLevels of viral shedding were monitored in the animals during the course of infection by RT-qPCR. Of the swabs that were collected, nasal and throat swabs were most consistently positive for viral RNA  and B, w50 assays. Infectious virus was present in nasal swabs collected from both group 2 and group 3, although some animals in group 3 reached higher titers than those for group 2  values ranging from 34.5 to 37. These results suggest that viral clearance in the infected tissues was occurring as early as day 14 p.i. and was mostly nondetectable by day 28 p.i.Various lung and other respiratory tract tissues, organs, and lymph nodes were collected at the endpoint of each study. Not surprisingly, very limited viral RNA was detected from the tissues that were collected from the group 1 and 2 animals on day 28 p.i., with only 1 and 2 positive tissue samples out of 17, respectively . For group 3 animals that were necropsied earlier on day 14 p.i., 10 out of 17 tissue samples were positive for viral RNA. All of the tissue samples from every group contained very low levels of viral RNA, having threshold cycle , interleukin-1β (IL-1β), IL-12, RANTES, macrophage inflammatory protein 1α (MIP-1α), IL-15, epidermal growth factor (EGF), IL-5, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), interferon gamma (IFN-γ), macrophage-derived chemokines (MDC), interferon-inducible t cell alpha chemoattractant (I-TAC), IL-2, IL-4, IL-8, and MIP-1β . HoweverInterestingly, unique to group 1 was the elevated expression of tumor necrosis factor alpha (TNF-α), granulocyte colony-stimulating factor (G-CSF), and IL-17 at day 8 p.i. TNF-α and IL-17 are proinflammatory cytokines, while G-CSF is a chemoattractant for neutrophils. In contrast, nearly baseline levels of TNF-α, G-CSF, and IL-17 were observed for group 2 and group 3 animals on day 8 p.i. These results suggest that the activation of a different set of proinflammatory cytokines occurred in animals from group 1 compared to groups 2 and 3 and that these differences may have contributed to the varying disease severity observed among the groups.This study compared the disease progression of 1918 influenza virus in two different nonhuman primate models of disease. Our initial assessment of rhesus macaques showed that 1918 influenza virus infection results in insignificant to mild clinical disease despite inoculation with a high titer and exposure via multiple routes. Inconsistent viral replication was observed in these animals, with only two out of four animals showing detectable levels of viral RNA during the acute phase of infection. Taken together, these results show that 1918 influenza virus replicates variably and poorly in rhesus macaques, resulting in minimal to negligible signs of clinical disease. These results may be due to the lower expression levels of sialyl-α-2,6-Gal saccharides, the main receptor for human influenza A viruses, in the lungs of rhesus macaques .10 PFU lower than the one reported previously  cells. MDCK cells were grown in minimum essential medium  supplemented with 5% fetal bovine serum  and 1× l-glutamine .Influenza virus A/South Carolina/1918 (H1N1) was generated by reverse genetics -treated trypsin (Sigma-Aldrich). This stock was used for animal inoculation. The mouse 50% lethal dose (MLD50) for this stock was determined previously to be 103.2 PFU (A passage 2 (P2) virus stock was prepared using MEM supplemented with 0.1% bovine serum albumin (BSA) , 1× 03.2 PFU ; this va50 of the 1918 influenza virus, 6-week-old female BALB/c mice  were anesthetized using isoflurane and infected intranasally (i.n.) with 10-fold serial dilutions of virus (n = 5 mice/dilution) in 50 μL MEM supplemented with 0.1% BSA. Mice were monitored daily and scored for signs of illness, including weight loss, ruffled fur, hunched posture, and rapid or labored breathing. Mice were euthanized when they lost ≥25% of their initial weight. The MLD50 value was calculated using the method of Reed and Muench  assays. Animals were screened for antibodies against 2009 H1N1 pandemic influenza viruses, which are antigenically related to the 1918 pandemic virus, by standard HI assays using turkey red blood cells as previously described (6 PFU via multiple routes: 1 mL ocular (0.5 mL/eye), 1 mL intranasal (0.5 mL/naris), 1 mL oral, and 4 mL intratracheal. After infection, animals were scored daily, and weight loss, temperature, respiratory rate, heart rate, and levels of SPO2 were monitored. Animals were sampled once before infection and then on days 2, 4, 6, 8, 14, and 22 postinfection. Swabs  and serum samples were collected to monitor viral loads. Thoracic radiographs, serum biochemistry, and hematology were performed.In the first NHP study, four male rhesus macaques (aged 4 to 7 years) were transferred to the NML in Canada from the Oregon National Primate Research Center in the United States. Macaques were free of escribed . Animalsn = 4), and subsequently infected with the same 1918 influenza virus stock as the one used in rhesus macaques. Each group contained two males and two females, and their ages varied from 5 months to 12.5 years. Group 1 was infected with a dose of 5 × 104 PFU by intrabronchial inoculation to the left side. Group 2 was infected with a dose of 5 × 106 PFU by intrabronchial inoculation to the left side. Group 3 was infected with a dose of 7 × 106 PFU via multiple routes: 1 mL ocular (0.5 mL/eye), 1 mL intranasal (0.5 mL/naris), 1 mL oral, and 4 mL intratracheal. After infection, animals were scored daily, and weight loss, temperature, respiratory rate, heart rate, and levels of SPO2 were monitored for the duration of each study. Animals from groups 1 and 2 were euthanized on day 28, while animals from group 3 were euthanized on day 14. Blood, serum, and swab samples were collected on days 0, 2, 4, 6, 8, 10, 14, 16, 21, and 28 for analyses of viral loads, serum biochemistries, hematology, and cytokine responses. Bronchoalveolar lavage fluid (BALF) was collected on days 0, 8, 14, 16, 21, and 28. Thoracic radiographs  were taken on sampling days to monitor for pulmonary infiltrates. Tissues from the lungs, bronchiole lymph nodes, tonsils, bronchi, trachea, bronchioles, liver, kidney, spleen, and heart were also collected on necropsy days for analysis of viral loads.In the second NHP study, 12 Mauritian cynomolgus macaques confirmed to be serologically negative for H1N1 influenza via standard HI assays as described above were purchased from an accredited vendor, randomly divided into three groups  (8 to 11 mL) was infused into the catheter, followed by aspiration and collection of the BALF. The same procedure was repeated two to three times. A pulse oximeter was used throughout the procedure to monitor the animal’s heart rate and oxygen saturation, and the animal was provided supplemental oxygen. BALF was spun down at 600 × Clinical scoring was assessed based on changes in the cardiovascular, respiratory, digestive, nervous, and musculoskeletal systems of the animals from their normal preinfection baseline values . ScoringRadiographs are evaluated and scored for the presence of pulmonary infiltrates by two board-certified clinical veterinarians according to a standard scoring system . BrieflyThoracic radiograph findings are reported as a single radiograph score for each animal on each examination day. To obtain this score, the scores assigned to each of the six lung lobes are added and recorded as the radiograph score for each animal on each examination day. Scores may range from 0 to 18 for each animal on each examination day.Whole-blood samples were analyzed for complete blood counts using the HM5 hematology system (Abaxis Veterinary Diagnostics). Levels of white blood cells, lymphocytes, monocytes, neutrophils, and platelets were determined. A VetScan VS2 chemistry analyzer (Abaxis Veterinary Diagnostics) and VetScan comprehensive diagnostic profile reagent rotors  were used to measure the levels of alkaline phosphatase (ALP), alanine transaminase (ALT), amylase (AMY), blood urea nitrogen (BUN), albumin (ALB), creatinine (CRE), and total bilirubin (TBIL).Viral loads in blood, swab, and tissue samples were determined by RT-qPCR detecting the 1918 M gene as described previously . A QIAam50 assays were performed on all RT-qPCR-positive samples using MDCK cells. Tissue samples were weighed and homogenized in 1 mL of MEM–0.1% BSA–l-Glu–2× penicillin-streptomycin (PS) with a 5-mm stainless steel bead using a Bead Ruptor Elite tissue homogenizer (Omni) for 30 s at a frequency of 4 m/s. Cell debris was pelleted by centrifugation at 1,500 × g for 10 min, and the supernatant was used for TCID50 assays. Samples were serially diluted (1:10) in MEM supplemented with 0.1% BSA, l-Glu, and TPCK-trypsin (MEM–BSA–l-Glu–trypsin), and 100 μL of each dilution was added to a 96-well plate of MDCK cells in triplicate. The plates were incubated at 37°C with 5% CO2, and the presence of cytopathic effect (CPE) was observed at 4 to 5 days postinfection. The TCID50 titer per milliliter or gram of tissue was determined using the method of Reed and Muench (TCIDd Muench .Cytokine responses in BALF samples from NHPs were examined using the cytokine monkey magnetic 29-plex panel for the Luminex platform (Thermo Fisher Scientific). Cytokines that were analyzed included G-CSF, IFN-γ, IL-10, IL-12, IL-17A, IL-2, IL-4, IL-6, IL-8, MCP-1, MIP-1α, MIP-1β, RANTES, TNF-α, EGF, eotaxin, FGF-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), HGF, IL-1β, IL-1RA, IL-15, IL-5, IP-10, I-TAC, MDC, MIF, MIG, and VEGF-A. The procedure was performed according to the manufacturer’s recommendations; BALF samples were tested undiluted, and test plates were run using a Luminex Magpix instrument.P value of <0.05 is marked by *, #, and § in the figures.Data are presented as the means and standard errors of the means (SEM) for each group. Statistical analysis was performed within the group and between the groups at each time by two-way analysis of variance (ANOVA) with Tukey’s multiple-comparison tests using GraphPad Prism software v9.2 . Significance at a


Serratia marcescens is a Gram-negative bacterium found in terrestrial and aquatic environments and studied for its polysaccharide utilization capabilities as part of larger efforts to discover novel carbohydrate-active enzymes. Here, we announce the genome sequence of an S. marcescens strain (PIC3611) that is able to utilize complex polysaccharide substrates. Serratia marcescens is a Gram-negative bacterium known for its red pigmentation and potent degradation of marine polysaccharides, particularly chitin , has a robust ability to degrade various complex chitin-containing substrates  at −80°C. The strain was grown to full density  in a morpholinepropanesulfonic acid (MOPS)-glucose (0.2% [wt/vol]) broth at 30°C for 48 h, and cell pellets were collected as described previously (De novo genome assembly was completed with the SPAdes de novo assembler (v3.10) with the following parameters: –k 21,33,55,77,99,127 and –careful . There was a total of 62,571,120 reads from the MiSeq sequencing, which corresponds to ~2,800× genome coverage ((number of reads x 250 bp read length)/genome size). Sequencing and assembly found that the S. marcescens PIC3611 genome was 5,531,323 bp with an average G+C content of 59%, both of which were in agreement with other sequenced S. marcescens strains (S. marcescens strain JWCZ2 (GenBank Accession: CP055161.1) as the top hit, with an E value of 0.0 (100% coverage and 100% identity), as further confirmation. Quality assessment of the genome assembly used the getorf function in QUAST (v4.2) (eviously . The peleviously . Reads wmbler v3. with theT (v4.2) . FunctioT (v4.2) . PGAP foPRJNA802829, and raw data files can be obtained from the NCBI SRA under accession number SRX14024400. The genome sequence for S. marcescens PIC3611 can be found in the NCBI GenBank database under accession number JAKQYC000000000 and assembly number ASM2260299v1.The NCBI BioProject accession number for this genome is


Diabetes is a metabolic disease that currently affects nearly half a billion people worldwide. β-cells dysfunction is one of the main causes of diabetes. Exposure to endocrine-disrupting chemicals is correlated with increased diabetes incidence. We hypothesized that treatment with bisphenol A (BPA) induces endoplasmic reticulum (ER) stress that activates the unfolded protein response (UPR), leading to impaired function of the β-cells, which over time, can cause diabetes. In this study, we aimed to evaluate UPR pathways activation under BPA treatment in β-cells and possible recovery of ER homeostasis. MIN6 cells (mouse insulinoma cell line) and isolated pancreatic islets from NOR (non-obese diabetes resistant) mice were treated with BPA. We analyzed the impact of BPA on β-cell viability, the architecture of the early secretory pathway, the synthesis and processing of insulin and the activation of UPR sensors and effectors. We found that the addition of the chemical chaperone TUDCA rescues the deleterious effects of BPA, resulting in improved viability, morphology and function of the β-cells. In conclusion, we propose that modulators of UPR can be used as therapeutic interventions targeted towards regaining β-cells homeostasis. Diabetes is characterized by β-cell failure to meet insulin demand due to either β-cell dysfunction or loss of β-cell mass, which leads to ineffective insulin secretion (for type 1 diabetes—T1D), or by the development of insulin resistance due to abundant insulin secretion, but which for various reasons cannot be properly used by the body T2D). Risk factors that lead to the appearance of diabetes can be genetic factors, but also a sedentary lifestyle or an unhealthy diet D. Risk f. In receBisphenol A (BPA) is a synthetic compound used in the manufacturing of polymers that are found in bottles, electronics, sports equipment, and in epoxy resins coating the interior of food cans  or as a ER is the organelle where correct folding and processing of proteins occurs through chaperons and enzymes involved in post-translational modifications. However, in conditions that disrupt metabolic homeostasis, there is an increase in misfolded proteins leading to ER stress. The UPR is the ER reaction to an accumulative stress. UPR is a well-conserved molecular mechanism comprising of three ER sensors—protein kinase RNA (PKR)–like ER kinase (Perk), inositol requiring enzyme 1 (Ire1) and activating transcription factor 6 (Atf6)—activated by the build-up of misfolded proteins . The thrProinsulin is produced in the rough ER lumen from pre-proinsulin, travels through the Golgi apparatus and matures into active insulin with the help of cellular endopeptidases that cleave a fragment called C-peptide, and other two peptide chains, B- and A- that oligomerize. All these processes take place in distinct regions of the trans-Golgi apparatus called insulin granules ,24,25. TRoutinely, β-cells manage up to 20% of newly synthesized misfolded proinsulin, which represents 30–50% of the total translational load of the cell, mostly due to a well-adapted UPR . HoweverSeveral studies provided information that BPA activates an ER stress in different types of cells, such as mouse non-parenchymal hepatocytes  and humaTo study the effect of BPA on β-cells’ viability, we have treated MIN6 cells with increasing concentrations of BPA. Untreated and vehicle (ethanol)-treated cells grow in small clusters that tend to fuse to enlarged aggregates, without reaching full confluency, with very few cells that start to detach from the cluster and die (Cells exposed for 24 h to stressors were labeled with Annexin V and propidium iodide (PI) to mark the apoptotic and the dead cells, respectively, then analyzed by flow cytometry. Viable cells were considered all the cells gated as in Next, we analyzed if the BPA effects are similar on the viability of cells from freshly isolated islets of Langerhans from NOR mice. After 24 h of treatment with up to 250 µM BPA and with 5 µg/mL Tm, respectively, islets were stained with PI and quantified. Representative images of islets are presented in Most extracellular proteins are processed through the secretory pathway to becoming biologically active. As a secreted hormone, insulin follows this pathway being produced by the β-cells in the ER and transported to the Golgi apparatus, where it is further processed and packaged in the secretory granules (SG). After glucose stimulation, SG merges with the plasma membrane to release insulin into circulation . As the In the cells exposed to the vehicle, calnexin displays a network pattern accompanied by puncta and localized accumulations, which are characteristic of this marker. The Golgi apparatus in control cells showed a specific appearance, a ribbon-like, compact structure located on one side of the cell. Upon 24 h of treatment with concentrations of BPA higher than 100 µM, alterations in both these structures were observed. As such, the fluorescence intensity of calnexin is decreased at 100 µM BPA and becomes diffuse at 250 µM BPA, indicating the disassembly of the ER network. Similarly, treatment with 100 µM BPA produced a scattered pattern for the GM130 staining, while at 250 µM BPA, the GM130 labeling was almost lost, suggesting the disintegration of the Golgi complex . All theTo investigate in more detail how MIN6 cells are affected by BPA at the ultrastructural level, we performed conventional electron microscopy (EM). MIN6 cells treated with the control vehicles displayed a normal β-cell morphology. The insulin secretory granule showed a distinctive appearance, where the electron-dense crystalline core was surrounded by an electron-lucent halo and enclosing membrane ; Figure , BPA. FuIns2, mRNA expression at different time points  . In addition, for the 24 h time point, we analyzed Ins2 gene expression both in MIN6 cells and in isolated islets upon exposure to concentrations of BPA ranging between 10 and 250 µM and 5 µg/mL Tm, respectively, used as an ER stress control  A. We fou control B,C. In back loop B,C, Tm.n = 2359 cells (vehicle) to 39.0 ± 19.7, n = 4743 cells (BPA 10 µM); to 43.8 ± 26.5, n = 2353 cells (BPA 50 µM); to 47.8 ± 28.5, n = 2575 cells (BPA 100 µM) (n = 2359 cells (vehicle) to 3.3 ± 7.7, n = 692 cells (n = 2277 cells (DMSO) to 68.4 ± 40.0, n = 1457 cells (n = 2359 cells (vehicle) to 8.7 ± 5.9, n = 4743 cells (n = 2359 cells (vehicle) to 3.8 ± 7.0, n = 692 cells) expressions were almost completely blocked by the drug  and insulin (green) D. For ce 100 µM) D,E. Howe92 cells E. The ex43 cells F. The mothe drug D–F.Atf6 gene expression and its effectors Edem1 and Dnajc3, as well as Ire1 downstream targets uXbp1 and sXbp1 (Atf4 (Atf4) and Ddit3 (Chop), upon exposure to 100 and 250 µM BPA, respectively. BPA 100 µM induces a similar trend of gene expression as the controls (not treated (NT) and vehicle) for both Atf4 and Ddit3 genes. On the other hand, treatment with 250 µM BPA strongly increased the expression of these two genes. At 2 h after exposure, we observed a twofold increase in Atf4 and a sevenfold increase in Ddit3 compared to the control. After 12 h of treatment, Atf4 was increased from twofold to fourfold, while Ddit3 increased from sevenfold to 28-fold, reaching the peak expression of this gene in these conditions. From this point on, Atf4 expression remained constant at 24 h and increased from fourfold to almost eightfold at 48 h, compared to the control. Meanwhile, Ddit3 expression decreased at 24 h from 28-fold to 18-fold and remained constant onwards , total eIF2α and Chop after 8 h BPA treatment  to 72.9 ± 41.1, n = 1275 cells (BPA 100 µM + TUDCA), and from 3.2 ± 7.7, n = 692 cells (BPA 250 µM) to 13.8 ± 14.4, n = 210 cells (BPA 250 µM + TUDCA) (n = 2575 cells (BPA 100 µM) to 19.5 ± 11.9, n = 1275 cells (BPA 100 µM + TUDCA) and from 3.8 ± 7, n = 692 cells (BPA 250 µM) to 7.7 ± 6.4, n = 210 cells (BPA 250 µM + TUDCA)  and insulin (green) fluorescence intensities in the cells treated with vehicle and BPA in the presence or absence of TUDCA. The results revealed that the presence of TUDCA in cells treated with BPA significantly improved the expression levels of both proinsulin  A,B, and + TUDCA) A,C. We n+ TUDCA) E. Taken Previous studies showed that pancreatic β-cells can be a target for the toxic effects of BPA ,39,40. HIn this study, we hypothesized that BPA interferes with insulin processing in the ER and thus activates a pro-apoptotic UPR. By stimulating the cells with a chemical that acts as a chaperone, TUDCA, we intended to improve the fault in protein processing and restore insulin homeostasis. To test this hypothesis, we exposed MIN6 cells and isolated pancreatic islets to different concentrations of BPA in the absence or presence of TUDCA and evaluated β-cells viability, UPR activation, morphology and function. Our study showed that treatment with a high concentration of BPA resulted in increased apoptosis through the activation of the pro-apoptotic UPR, as well as impaired insulin synthesis and processing. Co-treatment with TUDCA improved the cell function and viability.We evaluated the cytotoxicity of BPA on two different models, MIN6 cells and freshly isolated mouse pancreatic islets, by flow cytometry upon staining MIN6 cells with Annexin V and PI  and by fbona fide ER stress inducer, produced a dramatic effect on the ER and the Golgi apparatus, inducing well-documented cisternal distention and swelling of the mitochondria, suggesting a somewhat different mechanism than that of BPA  displayed a peak in expression at 12 h of BPA exposure, also found significantly at the corresponding Chop protein level (at 8 h), and by 48 h it returned to the 2 h values  nor Bax and Bad (pro-apoptotic factors) varied significantly , bovine serum albumin  from Sigma-Aldrich ; Tunicamycin  and tauroursodeoxycholic acid  from EMD Millipore, Sigma-Aldrich; Annexin V-APC (#640941) from BioLegend  and propidium iodide  from ThermoFisher Scientific . All the other chemicals were purchased from Carl Roth . Dimethyl sulfoxide  was used as solvent for Tm.The following antibodies were used for immunofluorescence: mouse monoclonal anti-human/mouse proinsulin biotinylated antibody , #BAM13361), mouse monoclonal anti-calnexin antibody , #NB300-518), rabbit polyclonal anti-GM130 antibody , guinea pig polyclonal anti-insulin antibody , #GTX27842). Secondary antibodies were from ThermoFisher Scientific: anti-streptavidin AlexaFluor-568 , AlexaFluor-488  and AlexaFluor-568 .α (1:500), rabbit polyclonal anti-eIF2α (1:500) and mouse monoclonal anti-Chop (clone L63F7) (1:200) from Cell Signaling , rabbit polyclonal anti-Atf4 (1:1000) from GeneTex. Secondary antibodies conjugated with HRP were from Biolegend (anti-mouse and anti-rabbit) and from Santa Cruz Biotechnology (anti-rat). Standard blocking conditions (5% milk in TBS-T) were used throughout, except when anti-P-eIF2α antibody was used, and 1% BSA in TBS-T was utilized.The antibodies used for immunoblotting were the following: mouse monoclonal anti-actin antibody (C4) (1:500), rat monoclonal anti-GRP78/Bip (1:250) and mouse monoclonal anti-vinculin (1:1000) from Santa Cruz Biotechnology , rabbit polyclonal anti-Phospho-eIF2http://n2t.net/addgene:115969, accessed on 2 May 2022; RRID: Addgene_115969) [MIN6 cells were a kind gift from Pedro Herrera’s lab and used at passages between 21–29. Cells were cultured in DMEM high glucose , supplemented with 15% FBS (PAN-Biotech), 1% penicillin/streptomycin (PAN-Biotech) and 71 μM β-mercaptoethanol (culture medium). Media was changed every 2–3 days and cells were passaged once per week. For treatments, cells were passaged and left to adhere overnight. The next day, the medium was replaced with a culture medium containing different BPA concentrations , 5 µg/mL Tm, 250 µM TUDCA, 100 µM BPA + 250 µM TUDCA, 250 µM BPA + 250 µM TUDCA. The vehicles used to dissolve the drugs were the following: 96% ethanol for BPA, distilled water for TUDCA and DMSO for Tm, respectively, and it never exceeded 0.5% from the culture medium. Treatments were maintained for 24 h or otherwise as stated in the results and figures sections. MIN6 cells were transfected 24 h after passage. Plasmid pLVX-ATF4 mScarlet NLS was a gift from David Andrews   and was ad libitum access to food and water. All experimental procedures involving animals were conducted in accordance with the European Union Directive 2010/63/EU and approved by the national competent authority (Authorization No. 590/13.01.2021).Male NOR (non-obese diabetic resistant) mice were obtained from The Jackson Laboratory   and housed under specific pathogen-free conditions in our Animal Facility. Mice were kept under controlled temperature (21 °C), humidity (55–60%) and light conditions (12 h light:12 h darkness cycle) and with 2+, Mg2+  and 0.08% BSA. The harvested pancreas was incubated in a water bath at 37 °C for 15 min. The digestion was stopped by the addition of cold RPMI medium , #10–040-CV) supplemented with 10% FBS. Following digestion, the pancreas was transferred through a metallic strainer and washed two times with RPMI. Islet separation was performed with 10 mL of Histopaque-1077  overlaid with 5 mL RPMI and centrifuged at 850× g for 15 min. Islets are transferred from the gradient with a pipette to a new tube with RPMI. Islets were washed three times with RPMI, resuspended in RPMI with 10% FBS and hand-picked. For treatments, islets were cultured in DMEM low glucose (PAN-Biotech), supplemented with 10% FBS and 1% penicillin/streptomycin, for the indicated time.Islets were isolated from NOR mice. Briefly, the pancreas was injected through the papilla of Vaters with 2.5 mL Collagenase XI solution: Collagenase XI  dissolved in HBSS buffer supplemented with Ca5 cells/well. After 24 h treatment with various concentrations of BPA (10–500 µM), 5 µg/mL Tm, 250 µM TUDCA, 100 µM BPA + 250 µM TUDCA, 250 µM BPA + 250 µM TUDCA, respectively, the culture medium containing the floating and dead cells was collected and the remaining cells were detached with 0.125% Trypsin (PAN-Biotech) and pooled with the collected medium. After centrifugation, cells were double stained with Annexin V-APC and PI, following manufacturer’s instructions. Cells were evaluated by flow cytometry with CytoFLEX . In total, 50,000 events were counted for every sample. The analysis consisted in gating out the debris, followed by evaluation of the percentages of cell populations equivalent for each group as follows: dead (PI positive), late apoptotic (PI and Annexin V positive), early apoptotic (Annexin V positive) and viable cells (negative to PI and Annexin V). Islets from NOR mice were cultured in 6-well plates (50 islets/plate) with BPA (10–250 µM) and 5 µg/mL Tm overnight. Islets were stained with PI and the dye intensity was quantified using Fiji/ImageJ software 1.51J8.MIN6 cells were cultured in 6-well plates at a density of 3 × 104Cl for 10 min and permeabilized with 0.5% Triton X-100 for 20 min. In total, 1% BSA in PBS was used as a blocking buffer for 30 min, while the endogenous biotin was blocked with a biotin/avidin kit  for the cells stained with anti-proinsulin antibody. Cells were incubated with primary antibodies for 1 h at room temperature, washed 3 times with PBS and stained with secondary antibodies for 45 min at room temperature, followed by 3 washes with PBS. Cells were stained with DAPI (ThermoFisher Scientific) for 5 min, washed with PBS and rinsed with distilled water. Vectashield , H-1000) was used as a mounting medium. Images of cells labeled for proinsulin and insulin were obtained on a fluorescence microscope Leica DMi8  and were minimally processed using LasX software (version 3.7.6.25997). Fluorescence quantification was performed using QuPath software (version 0.3.2) [Cells were cultured on glass coverslips in 6-well plates. After 24 h of exposure to various reagents (as reported in the figure legends), cells were washed with PBS, fixed with 4% PFA in PBS and processed for immunofluorescence. Briefly, cells were washed with PBS, treated with 50 mM NHn 0.3.2)  on imageg at 4 °C. The aqueous phase was transferred to a new tube and incubated with isopropanol for 30 min on ice. Samples were centrifuged for 15 min, at 12,000× g in a centrifuge kept at 4 °C. The RNA pellet was washed with 70% ethanol and with 100% ethanol, respectively. After air drying, the pellet was resuspended in 20 μL DEPC-water. RNA purity and concentration were evaluated by NanoDrop (ThermoFisher Scientific). For reverse-transcription reaction, 1 μg RNA was used and the cDNA was obtained with 9902 Veriti PCR  by using a qScript cDNA SuperMix  kit or iScript Reverse Transcription Supermix . To evaluate gene expression, qPCR was performed, where the equivalent of 12.5 ng of cDNA was used with a Roche LightCycler 480 System  and PerfeCTa SYBR Green SuperMix kit (Quantabio). The primer sequences for the genes analyzed are found in For total RNA isolation, cells and islets were collected in 1 mL TRItidy G , #A4051) and stored at −80 °C. Samples were mixed with 0.2 mL chloroform, kept on ice for 10 min, and then centrifuged for 15 min at 12,000× Cells cultured in 6-well plates were lysed using RIPA buffer  supplemented with proteases inhibitors  and phosphatases inhibitors . Total protein was quantified using the ROTI Quant Universal kit. After normalization, samples were mixed with Laemmli 5× (S × 5) supplemented with 5% β-mercaptoethanol, boiled for 5 min at 95 °C and loaded in 10% SDS-PAGE gels. Proteins were transferred on a PVDF membrane using a Trans-Blot Turbo Transfer System (BioRad). Membranes were blocked with 5% skimmed milk in TBS-Tween 0.05% (TBST) or 1% BSA in TBST for 1 h at room temperature. Membranes were incubated with the antibodies presented above, then with ECL solution  and visualized using LAS-4000 FujiFilm .For ultrastructural analysis, MIN6 cells treated as described were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, post-fixed in 1% osmium tetroxide and stained “en bloc” with 1% uranyl acetate. Cells were dehydrated through a graded series of ethanol, transferred to propylene oxide and embedded in Epon 812. Ultrathin 70 nm sections were cut with an ultramicrotome, placed on formvar-coated copper grids (200 mesh) and double-stained with uranyl acetate and lead citrate before examination on an electron microscope  at 100 kV. Images were acquired with an Eagle 4K bottom-mounted CCD camera and analyzed using Fiji/ImageJ (v1.53) software . For eacp < 0.05 . The results are shown as means ± SD, exceptions are graphs in The results were quantified and the mean or median of 3 or more biological replicates was evaluated. Statistical analysis was performed using GraphPad Prism software. One-way ANOVA was performed and the differences between groups were obtained with the Tukey test. A significant difference was considered to be for


Background: Endolichenic fungi (ELF), which live the inside the lichen thallus, contain many secondary metabolites that show various biological activities. Recent studies show that lichen and ELF secondary metabolites have antioxidant, antibacterial, antifungal, cytotoxic, and anticancer activities.Purpose: Here, the effects of an ELF extract and its bioactive compounds were investigated on the H1975 cell line focusing on immune checkpoint marker inhibition.Methods: An ELF was isolated from the host lichen Bryoria fuscescens (Gyelnik) Brodo and D. Hawksw and identified the species as Nemania sp. EL006872. The fungus was cultured on agar medium and acetonic extracts were obtained. Secondary metabolites radianspenes C and D, and dahliane D, were isolated from the crude extract. The biological effects of both the crude extract and the isolated secondary metabolites were evaluated in cell viability, qRT-PCR assays, flow cytometry analysis and western blotting.Results: The cell viability assay revealed that extracts from Nemania sp. EL006872 and the isolated secondary compounds had low cytotoxicity. The crude extract, radianspenes C and D, and dahliane D, suppressed expression of mRNA encoding PD-L1 and aromatic hydrocarbon receptor (AhR), and surface expression of PD-L1 protein by cells exposed to benzo[a] pyrene. Radianspenes C and D, and dahliane D, reduced expression of AhR, PD-L1, ICOSL, and GITRL proteins by H1975 lung cancer cells, as well as exerting anti-proliferative effects.Conclusion: Radianspenes C and D, and dahliane D, bioactive compounds isolated from Nemania sp. EL006872 ELF, have the potential for use as immunotherapy and immunoncology treatments. Exposure to environmental pollutants and tobacco smoke is an important public health problem involving major pollutant benzo[a]pyrene (BaP) . SourcesLichens, symbiotic organisms comprising fungi and algae (or cyanobacteria), have attracted much attention as a source of therapeutic agents for the treatment of numerous diseases, including cancer . SimilarCancer immunotherapy aims to strengthen the human immune system, thereby enabling it to eliminate cancer cells . The funNemania sp. EL006872 extracts, and its diterpenoids metabolites, radianspenes C and D, and dahliane D, particuraly in the context of immune checkpoint inhibition. To the best of our knowledge, few studies have examined the efficacy of radianspenes and dahlianes as potential immunotherapy agents.In this study, the aim was to identify the immunotherapeutic potential of Bryoria fuscescens (Gyelnik) Brodo and D. Hawksw were collected in 2019 during the field trips in Mt. Uludağ , Bursa, Turkey, organized by Prof. Sesal from Dept. of Plant Diseases and Microbiology, Marmara University, Istanbul, Turkey. Voucher samples (KoLRI 052660) has been deposited at the Korean Lichen and Allied Bioresource Center (KOLABIC) in the Korean Lichen Research Institute (KoLRI), Sunchon National University, Korea (https://cc.aris.re.kr/kolabic). Endolichenic fungi were isolated with the surface sterilization method . The ITS sequence of Nemania sp. EL006872 is available in Fungus internal transcribed spacer sequencing conducted as already depicted . The endPotato dextrose agar (PDA)  powder (39 g) was prepared into 1 L distilled water, which was then boiled and mixed to dissolve the powder. The mixture was autoclaved. The autoclaved PDA solution was cooled down to 55–60°C and then directly transferred into the petri dishes. Petri dishes were cooled to room temperature. ELFs were transferred to be cultured on agar in each petri dish. The dishes were incubated at 25°C for 3–4 weeks. ELF mycelia were grown on PDA and then frozen with agar and lyophilized. The lyophilization was separated into water. Then, acetone was added to samples tubes, and the tubes were shaken for around 2 h. Each sample was then filtered to separate from solid particles. Crude extracts of ELFs were then obtained by rotary evaporator. Finally, the crude extracts were dissolved in 100% DMSO for use.n-hexane and ethyl acetate (EtOAc). A Waters 600 HPLC system  equipped with a Hector-M C18 column  or a Spursil C18 EP column  was used for the purification of compounds 1–3 from the EtOAc fraction. The EtOAc fraction (78.9 mg) was further separated by preparative HPLC  into five subfractions (E1– E5). Compounds 1  and 2  were purified from the subtraction E4 by preparative HPLC . Compound 3  was obtained from the subfraction E4 by preparative HPLC . NMR spectra were obtained using a Bruker Avance III HD 500 spectrometer  (The dried crude extract (2.7 g) was suspended in water, then successively extracted with  States) .2 in a humidified atmosphere at 37°C.H1975 non-small lung cancer cell line was used in this research. H1975 cell was cultured in RPMI  supplemented with 10% fetal bovine serum  and 1% penicillin streptomycin solution. Cells were incubated in 5% CO3 cells/well in 96-well plates, grown overnight, and then treated with crude extract and pure compounds. After incubation, 15 μL methyl thiazolyl tetrazolium (MTT) reagent (Sigma-Aldrich) was added to each well, and the samples were incubated for an additional 4 h. After 4 h, the supernatants were removed, and formazan crystals were dissolved in 150 µL DMSO. Absorbance was measured at 570 nm and was determined using a microplate reader with Gen5 (v2.03.1) software .Extract and pure compounds were dissolved in DMSO . Cells were seeded at a density of 3 x 10H1975 cells were seeded at a density of 1000–1500 cells/well in RPMI 1640 and incubated overnight. After 72 h, media containing the crude extract and indicated compounds or DMSO was replaced with fresh medium for 12 days. Colonies were fixed with methanol, and stained with 0.5% crystal violet. Then, the plating efficiency and survival rate of the control group and treatment group cells were determined. Next, the plating efficiency of the control group and treatment groups were determined .5 cells/well on 6-well plate and grown overnight. Cells were treated with crude extract, pure compounds, and benzo[a]pyrene or DMSO. Total RNA was isolated from H1975 cells using RNAiso Plus . 1 mg of RNA was converted to cDNA using M-MLV reverse transcriptase . qPCR was performed using SYBR Green . qRT-PCR reactions and analyses were performed on a CFX instrument . Primers used for qRT-PCR were as follows: GAPDH (forward) 5′-atcaccatcttccaggagcga-3′ and (reverse) 5′-agttgtcatggatgaccttggc-3`; PD-L1 (forward) 5`-ggagattagatcctgaggaaaacca-3` and (reverse) 5`-aacggaagatgaatgtcagtgcta-3`; AhR (forward) 5`-attgtgccgagtcccatatc-3` and (reverse) 5`-aagcaggcgtgcattagact-3`.Quantitative RT-PCR (qRT-PCR) was conducted as already depicted. H1975 were plated at a density of 2 x 105 cells/well on a 6-well plate and grown overnight. Then, cells were treated with crude extract, radianspenes C and D, and dahliane D 4 h prior to exposure benzo[a]pyrene (1 µM) and then cells were incubated for 72 h. H1975 cell line was incubated for 72 h and collected in PBS by trypsin digestion. After washing three times, cell pellets were resuspended in 2% BSA and 100 μl aliquot (1 cells  ×  106 cells) was used for antibody labeling with anti-PD-L1 antibody  at 4 °C for 30 min in the dark. After washing with FACS buffer twice and cell was incubated with secondary antibody anti-Rabbit IgG (H + L)  at 4 °C for 30 min in the dark. After washing steps, the labeled cells were analyzed by flow cytometry using Cytoflex flow cytometer  and CytExpert 2.0.0.152 software.H1975 were plated at a density of 2 × 10Cells were cultured in a 6-well plate for 12 h, treated with 7.5, 15, and 30 µM concentrations of radianspenes C and D, and dahliane D for 72 h, washed twice with cold PBS, and lysed in lysis buffer. Primary antibodies , ICOSL , GITRL , AhR , and GAPDH ) against were used to probe membranes. After membranes incubated with secondary antibodies (Thermo Fisher Scientific) and detected with using an Immobilon Western Chemiluminescent HRP Substrate Kit , and luminescence imaging . Multi-Gauge 3.0 was used to assess band densities, which were then normalized to GAPDH levels in each sample. The values are given in arbitrary densitometric units that correlate to the signal intensities.Nemania sp. EL006872 culture. First of all, cell viability assay using acetonic extract of Nemania sp. EL006872 was performed to determine the cytotoxic activity of the extract (10 μg/mL). As a result, acetonic extracts of the Nemania sp. EL006872 that maintained H1975 cell viability were tested for future experiments (eriments .Exposure to benzo[a]pyrene (BaP) increases expression of PD-L1 by lung epithelial cells, likely mediated via AhR . Recent Nemania sp. EL006872 crude extract on expression of AhR and PD-L1 mRNA in BaP exposed H1975 cells were examined. qRT-PCR revealed that expression of PD-L1 and AhR mRNA in BaP exposed H1975 cell was downregulated by pretreatment of acetone extracts of Nemania sp. EL006872  were evaluated. Expression of PD-L1, GITRL, and ICOSL were downregulated by radianspenes C and D, and dahliane D, in a dose-dependent manner. In addition, the expression of the transcriptional regulator AhR was also investigated. The level of AhR protein decreased upon treat to ELF-derived compounds. Thus, radianspenes C and D, and dahliane D, exert potent inhibitory effects against multiple immune checkpoints .in vitro, a clonogenic assay was performed in H1975 cells. Cancer cell proliferation and tumor growth are the defining features of cancer progression  Brodo and D. Hawksw; 2) Nemania sp. EL006872 showed immune checkpoint inhibition activity through suppressing AhR and PD-L1 mRNA and surface protein expression in BaP exposed H1975 cells; 3) radianspenes C and D, and dahliane D were isolated from Nemania sp. EL006872 crude extract and found to have bioactivity; 4) radianspenes C and D, and dahliane D suppresses the expression of multiple immune checkpoints including PD-L1, ICOSL, and GITRL; 5) radianspenes C and D, and dahliane D showed anti-proliferative activity.Here, the effects of an ELF extract and isolated secondary metabolites on immune checkpoints in non-small lung cancer cells were investigated. This study identified  1) the ECoprinus radians showed cytotoxic effect on MDA-MB-435 human breast cancer cells with an IC50 value of 0.91 μM   found in fungi . A previ 0.91 μM . Anothernes A–K) . Previounes A–K) . Here, ines A–K) . The nonNemania sp. EL006872, and isolated secondary metabolites, inhibit expression of PD-L1 and AhR on non-small lung cancer cell exposed to BaP.Surgery, radiotherapy, and chemotherapy are the standard treatments for cancer; however, their effects are limited, necessitating development of new therapeutic options such as antitumor immunotherapy . Studies+, CD8+, and FoxP3+ regulatory effector cell populations. Overexpression of ICOS ligand is associated with reduced patient survival  pathway regulates the Treg population and, subsequently, tumor development . Expresssurvival . Many stsurvival . Anothersurvival . Targetipression . StudiesNemania sp. EL006872 crude extract and its secondary metabolites have the potential for use as support therapies, and to act as templates for development of new target molecules to overcome drug resistance. Our study shows that radianspenes C and D, and dahliane D, can be isolated from ELF and used to conduct research into the treatment of cancer and other diseases related to immune checkpoints. Future studies will examine the molecular mechanisms of action and in vivo effects of Nemania sp. EL006872 secondary metabolites on immunoregulation and tumor growth.The ELF extract inhibit immune checkpoint ligands in cancer cells exposed to BaP, an environmental pollutant. Radianspenes C and D, and dahliane D, suppress multiple immune checkpoint markers in lung cancer cells. The


Nanopesticides were chemically synthesized as rods (Fe2O3), stars (ZnO), and spheres (MgO) and were characterized by XRD and SEM analysis. The in vitro bioassays included adult immersion, larval immersion, and larval packet tests. Non-target effects of the nanopesticides were evaluated using snails. The LC90 values of Fe2O3 NPs  were lowest followed by MgO  and ZnO , for the tick adult, larval and egg stages, respectively. Fe2O3 NPs were capable of arresting oviposition and larval hatching in the study ticks in vitro. The snail toxicity experiments revealed minimum to mild off-target effects for all nanopesticides tested. This study is the first to report the comparative efficacy of magnesium, iron, and zinc nanomaterials for toxicity in egg, adult and larval stages of Hyalomma ticks. Further studies of NPs on establishing the efficacy against ticks and safety at host-human-environment interface could lead to promising nanopesticde applications.Ticks (Acari: Ixodidae) are blood-feeding parasites capable of transmitting diseases to animals (Piroplasmosis) and humans . The non-judicious use of chemical acaricides has led to the development of acaricide-resistant ticks, making the control of ticks and tick-borne diseases difficult. This study reports the efficacy of magnesium oxide (MgO), iron oxide (Fe Ticks are blood-feeding arthropods known to transmit lumpy skin disease, Q fever, rickettsiosis, ehrlichiosis, Boutonneuse fever and Lyme disease ,2. LivesHyalomma ticks are one of the most significant disease-transmitting genera of Ixodidae due to their vector potential for livestock and impact on public health [c health . A growic health . To thisc health .2O3 NPs against the major stages of Hyalomma ticks and determines their toxicity in snails (being one of suitable candidates for pesticide and ecotoxicology testing).Metallic and non-metallic nanoparticles (NPs) have shown great promise for rapidly inducing toxicity and reducing lethal concentrations at various stages of the parasite’s life cycle . NPs hav2·6H2O), sodium dodecyl sulfate (SDS), sodium hydroxide (NaOH), zinc acetate dihydrate (Zn(CH3COO)2·2H2O), polyethylene glycol (PEG), urea (NH2CONH2), iron chloride tetrahydrate (FeCl2·4H2O) and ammonia were purchased from Sigma-Aldrich USA.Magnesium chloride 2·2H2O and 1 g urea were dissolved in 65 mL distilled water to prepare solution A. One gram of urea and 3 g PEG were dissolved in 65 mL distilled water to prepare solution B. Solutions A and B were mixed, and 13 mL of concentrated ammonia was dropwise added to maintain a pH of 12. The solution was poured into a Teflon vessel and heated in an autoclave reactor at 110 °C for 5 h. The product was washed, dried, ground to a fine powder, and calcinated at 550 °C for 5 h.2O3 NPs:Synthesis of Fe Two grams of FeCl2·4H2O were dissolved in 6 mL distilled water, and 42 mL of concentrated ammonia was added. The reaction mixture was heated in an autoclave at 140 °C for 3 h. The product was washed, dried, and ground to a fine powder before application.NP characterization: A Rigaku D/max Ultima III X-ray powder diffractometer operated at 40 kV and 0.130 A current and equipped with a Cu-Kα radiation source (wavelength 0.15406 nm) was used for the XRD analysis of NP products. An SEM Quanta 250 operated at 30 kV was used to scan the NPs.Engorged ticks were collected from the ears, eyelids, lips, and tails of bovines  with no reported history of acaricidal exposure within the previous 30 days . Ticks wHyalomma ticks using the following bioassays: egg immersion (EIT), larval packet (LPT), larval immersion (LIT) and adult immersion (AIT). All tick rearing and bioassays were performed within a biological oxygen demand incubator, at a temperature of 28–30 °C and 80–90% relative humidity, for the required time [We evaluated the efficacy of various concentrations of NPs against egg, larval and adult stages of collected red time ,21. Briered time . After 1red time .2O3 NPs), and Group 7 . The five treatment groups  were further divided into four sub-groups receiving the following NP concentrations: 0.01 mg/mL, 0.1 mg/mL, 1 mg/mL, and 10 mg/mL, with n = 5 snails randomly assigned to each subgroup. A 50 µL of solution from each of 0.01 mg/mL, 0.1 mg/mL, 1 mg/mL, and 10 mg/mL NPs solution was poured onto the anterior mouth portion side of each snail, using the method described by Radwan et al. [For the toxicity evaluation, 120 apparently healthy and active land snails were collected regardless of gender from the organic garden at the University of Agriculture, Faisalabad, Pakistan, where chemicals and pesticides had not been used in the previous 30 days. There were seven groups of snails, including two control (negative and positive) and five treatment groups. The seven groups were designated as follows: Group 1 , Group 2 (cypermethrin), Group 3 (deltamethrin), Group 4 (MgO NPs), Group 5 (ZnO NPs), Group 6  with probability ≤0.05 considered as significant.The LC2O3 for diffraction peaks at 2 theta (θ) 22°, 33°, 35°, 46°, and 56° were (012), (104), (110), (124), and (116), respectively (JCPDS # 33-0664) [2O3 NPs. The XRD pattern showed that the peaks were not sharp, indicating that the product was not completely crystalline. XRD patterns of the synthesized products were plotted as 2 theta (θ) values vs. intensity . Miller 33-0664) . The set2O3 are shown in An SEM image of MgO NPs synthesized hydrothermally is provided in 2O3 NPs (2O3 NP treatment groups was approximately 80%, and the lowest lethal Fe2O3 NP concentration required to arrest larval hatching was LC90 = 1.68 mg/L. Mortality data in terms of LC50 and LC90 along with associated confidence intervals have been mentioned in The preliminary stereomicrographs showing adult, larval and egg stages of Hyalomma ticks are shown in 2O3 NPs . The mor2O3 NPs . The egg2O3 NPs a. The eg2O3 NPs, MgO NPs, and ZnO NPs. Mortality was directly proportional to NPs concentrations. All the snails exposed to ZnO NPs survived for 24 h against all four concentrations. In the control group, only one of the snails was dead on the fifth day of the trial. Most snails died on day 3 of the trial.As a preliminary investigation, all three NPs demonstrated minimum to mild off-target effects on the snails . The higHistopathology: Snails exposed to metallic NPs were found to be illustrating pathologies at various sites of digestive glands (e glands . The gla2O3 NPs, researchers determined that the LC50 was 50 µg/mL at 60 min and the LC99 was 150 µg/mL at 30 min against Hyalomma spp. The authors also showed 85.7% Hyalomma mortality in 0 min exposure to 250 µg/mL of Fe2O3 NPs [50 values of 4.76 mg/L and 4.92 mg/L and LC90 values of 8.87 mg/L and 9.1 mg/L, respectively, against Hyalomma ticks [2O3 NPs and ZnO NPs are safer and more effective NPs nickel NPs than titanium NPs [Hyalomma tick. Similarly, the LC50 for plant-derived ZnO NPs tested in Rhichichephalus ticks was 6.87 mg/L.ZnO NPs are one of the safest nanomaterials and have been successfully applied in the areas of food, textiles, medicine and healthcare, agriculture, and engineering ,30. In oe2O3 NPs . In our ma ticks . Mortalima ticks ,34. Prevnium NPs ,35 againIt has been proposed that NPs induce toxicity by accelerating or slowing certain cellular mechanisms within host cells . The uniMgO NPs have been declared relatively safe materials by the FDA. They are also easier to procure and versatile . AlthougSnails are considered model animals for ecotoxicity assessment of nanomaterials due to their propensity to bioaccumulate and their importance in land and aquatic ecosystems . Snail dHyalomma ticks. Iron oxide demonstrated the lowest lethal concentrations, followed by magnesium oxide and then zinc oxide nanopesticides. The results of toxicity assays in snails indicate minimal ecotoxicity compared to commonly used acaricides. Further study is recommended for exploring in vivo effects of applying nanopesticides on the tick hosts and environment.Zinc oxide, magnesium oxide, and iron oxide nanopesticides demonstrated significant anti-tick activity in vitro against


However, amendment of salt-affected soil with combined phosphogypsum and seed inoculation with PGPR under deficient irrigation conditions was more effective than singular application and control plots in ameliorating the harmful effects of water stress and soil salinity. Additionally, combined application limited Na uptake in leaves and increased K uptake and leaf chlorophyll a, b, and carotenoids as well as improved SOD, CAT, and POD activities to ameliorate oxidative stress indicators , which eventually positively reflected on productivity and quality in quinoa. We conclude that the potential utilization of phosphogypsum and PGPR are very promising as sustainable eco-friendly strategies to improve quinoa tolerance to water stress under soil salinity.The aim of the study was to estimate the impact of soil amendments ) separately or their combination on exchangeable sodium percentage (ESP), soil enzymes’ activity (urease and dehydrogenase), pigment content, relative water content (RWC), antioxidant enzymatic activity, oxidative stress, productivity, and quality of quinoa under deficient irrigation conditions in two field experiments during the 2019–2020 and 2020–2021 seasons under salt-affected soil. Results revealed that ESP, soil urease activity, soil dehydrogenase activity, leaf chlorophyll a, b, and carotenoids, leaf K content, RWC, SOD (superoxide dismutase), CAT , and POD (peroxidase) activities were declined, resulting in overproduction of leaf Na content, proline content, and oxidative stress indicators (H Chenopodium quinoa Willd.) is a seed crop of the Chenopodiaceae family belonging to the C3 group of plants [Quinoa  [4) or as fertilizer [2+), P, and sulfur (S). It helps in augmenting aeration, water holding capacity, oxygen penetration, and nutrient uptake in soil or when coupled with microbes to improve the solubility of nutrients (indirect) [+ and augment the concentration of osmolytes such as K+ within leaves [Phosphogypsum is a new alternative approach to inorganic chemicals. Phosphogypsum (PG) is a by-product of the phosphate fertilizer industry, due to the manufacturing of phosphoric acid from rock phosphate (fluorapatite). Globally, around 160 Mt of phosphogypsum are manufactured yearly and it is mainly removed in big stocks or discharged into waterways . It comp2+) . Because2+) . Worldwirtilizer , which hndirect)  owing ton leaves . Variousn leaves , therebyn leaves .Inoculation of crop plants with beneficial microbes (plant growth-promoting rhizobacteria and PGPR) has the potential to enhance soil–water–plant relations ,29 and aThe aim of the study was to include phosphogypsum by-product combined with plant growth-promoting rhizobacteria under water stress in salt-affected soil and enhance soil characteristics; this may, in turn, be beneficial as an environmentally friendly approach to increase quinoa productivity and ameliorate the soil properties.−1 soil d−1) and dehydrogenase (mg NH4+-N g−1 soil d−1) enzymes was significantly (p < 0.05) reduced upon irrigating quinoa plants with deficit irrigation in saline soil during 2019–2020 and 2020–2021 (The activity of soil urease (mg TPF g020–2021 . However020–2021 . Similar020–2021 . Moreove+ content and a decrease in K+ content in quinoa leaves relative to the plants grown under the untreated plots (control)  . Deficitrigation .p < 0.05) the levels of chlorophyll a, chlorophyll b, and total carotenoids (p < 0.05) declined the levels of chlorophyll a, chlorophyll b, and total carotenoids during 2019–2020 and 2020–2021 seasons. Quinoa plants had the highest levels of photosynthetic pigments when irrigated well. Under regular irrigation, the combined application of phosphogypsum and PGPR together had the highest levels of chlorophyll a, chlorophyll b, and total carotenoids during 2019–2020 and 2020–2021 seasons, followed by phosphogypsum alone, then PGPR. Although all photosynthetic pigments were significantly (p < 0.05) reduced under deficit irrigation treatment, the coupled application of phosphogypsum and PGPR had the highest levels of chlorophyll a, chlorophyll b, and total carotenoids during 2019–2020 and 2020–2021 seasons.Under saline soil, deficit irrigation treatment declined the levels of photosynthetic pigments, and soil application using phosphogypsum and PGPR improved significantly , catalase (CAT), and peroxidase (POD) have been colorimetrically assessed during two growing seasons (2019–2020 and 2020–2021), as shown in 2O2 and MDA in 2019–2020 and 2020–2021 seasons (p < 0.05) reduced the accumulation of both molecules. Concisely, MDA levels significantly (p < 0.05) declined, owing to the soil application by phosphogypsum, PGPR, or both, during 2019–2020 and 2020–2021 seasons. Similarly, H2O2 levels were declined in 2019–2020 and 2020–2021 seasons (p < 0.05) in both growing seasons.Deficient irrigation noticeably increased the stress biochemical indicators as confirmed by H seasons . Coupled seasons ; however−1), foliage yield (5.08 ± 0.090 and 5.37 ± 0.015 t ha−1), biological yield (8.67 ± 0.050 and 7.96 ± 0.072 t ha−1), and harvest index (41.3 ± 0.33 and 40.6 ± 0.09%) in the 2019–2020 and 2020–2021 seasons, respectively. Similarly, the combined application of phosphogypsum and PGPR to deficiently irrigated soils produced the second highest plant height , 1000-seed weight (2.50 ± 0.012 and 2.35 ± 0.035 g), seed yield (2.65 ± 0.049 and 2.73 ± 0.085 t ha−1), foliage yield (4.15 ± 0.049 and 4.23 ± 0.085 t ha−1), biological yield (6.79 ± 0.099 and 6.95 ± 0.170 t ha−1), and harvest index (39.0 ± 0.16 and 39.2 ± 0.24%)) in the 2019–2020 and 2020–2021 seasons, respectively. Control quinoa plants had the lowest plant height  compared to unamended treatment (control) [Under the same abiotic stress, high He damage . From oucontrol) . Howevercontrol) . This is+ uptake, augment K+ uptake, thereby keeping healthy flag leaf, increase photosynthesis with high net assimilation rate, translocate from sources to sink alongside starch accumulation in the chloroplast, decline oxidative damage, delay senescence, improve water uptake, and augment the antioxidative capacity [Grain yield is the most important variable, considering its use in the market. The decrease in plant height (cm), 1000-grain weight (gm), grain yield, foliage yields, biological yield, and harvest index as well as qualities such as grain protein content and saponin content have been adversely influenced under water stress and soil salinity due to the decline in the incorporation and transfer of the nutrition materials through grain maturity and their filling periods. In addition, soil salinity and water stress can prompt severe damage to the ovary, and consequently may result in a decline in productivity . Howevercapacity . Howevercapacity . In thiscapacity ,57. Soilcapacity . All of 2+ (27.43 meq L−1), P (26.43 g kg−1), and S (14.33%) as major elements. Moreover, the value of Mg2+ was (3.79 meq L−1), O.M was (1.01%), and CEC was (61.36 cmol kg−1). PG had a pH of 3.25, which was measured by pH–meter and EC 3.45 dS m−1 (1:5 soil/water ratio). Total Cd concentration was not detected in PG samples. In addition, the concentrations of P, K, and Al accounted for 2.45%, 0.08%, and 0.11%, respectively, which were determined by coupled plasma-optical emission spectrometry (ICP-OES) (PerkinElmer Optima 4300 DV). The application rate of PG was 9 ton ha−1.Phosphogypsum was obtained from a fertilizer industry factory in El-Sharkia Governorate, Egypt. Some samples were taken in the lab to be ground and passed through a 2-mm mesh and then the samples were digested using nitric acid in a microwave to measure the physical and chemical characteristics of PG. Phosphogypsum consists of gypsum as the key compound, which has higher concentrations of Ca−1 dry soil, Bacillus); . Total counts were performed by pasteurization in a water bath at 70 °C for 10 min to kill the vegetative cells. The inoculation treatments were prepared as peat-based inoculums, with 30 mL of 109 CFU mL−1 from each culture per 60 g of sterilized carrier, which were mixed carefully with the quinoa seeds, and seed planting was performed directly after seed inoculation.Bacterial inoculants of plant growth-promoting rhizobacteria (PGPR), namely Azospirillum lipoferum SP2, Bacillus coagulans NCAIM B.01123, Bacillus circulance NCAIM B.02324, and Bacillus subtilis MF497446 were attained from Bacteriology Laboratory, Sakha Agricultural Research Station, Kafr El-Sheikh, Egypt. The standard culture circumstances were set with semi-solid malate medium for A. lipoferum , and witChenopodium quinoa) under water stress in salt-affected soil. Quinoa seeds  were attained from Plant Breeding Unit, Desert Research Center. Quinoa seeds were planted on 15th and 18th of November for both years of study, respectively. Soil samples were collected at depth (0–30 cm) before sowing to measure the physical and chemical characteristics in both seasons. The experimental soil was analyzed and its physical and chemical properties were as follows; soil texture was clay loam and the chemical properties in both seasons were pH (8.35 and 8.58), EC (11.14 and 12.09 dS m−1), O.M. (1.51 and 1.48%), field capacity (29.4 and 31.1%), Na+ (17.78 and 18.65 meq L−1), K+ (10.73 and 11.86 meq L−1), Mg2+ (14.76 and 16.23 meq L−1), Ca2+ (6.54 and 18.29 meq L−1), HCO3− (17.22 and 19.67 meq L−1), and SO42− (19.03 and 21.15 meq L−1), respectively. Meteorological data of the experimental site are also presented in We performed the experiments at the Elamaar village in the region of Sidi Salem , Kafr El-sheik Governorate, Egypt during the 2019–2020 and 2020–2021 cropping seasons, to investigate the application of phosphogypsum as soil amendment coupled with PGPR inoculation on soil, growth, physiological, and quality properties as well as yield traits and productivity of quinoa (2 (7 rows of 50 cm width and 3 m length) with plant spacing 20 cm (maximum 210 plants/each subplot). The research experiments were conducted in accordance with the most important technical recommendations contained in the Bulletin of the Ministry of Agriculture and Land Reclamation. Soil of experimental plot was prepared in the same manner as the nursery area. Prior to quinoa planting, ploughing was performed at 30 cm depth and hoeing at 15 cm. During soil preparation, the experimental soil was fertilized with phosphorus fertilizer in the form of calcium superphosphate (15.5% P2O5) at the rate of 107 kg P2O5 ha−1 during land preparation and potassium (K) at the rate of 120 kg ha−1 in the form of potassium sulfate (48% K2O) during seedbed preparation and before ridging, and nitrogen fertilizer was applied at a rate of 286 kg ha−1as ammonium nitrate (33.5%) was side dressed at two equal doses, then nitrogen fertilizer was added into two portions, half being applied before the first irrigation, while the remaining portion was applied before the second irrigation.The field experiment design was arranged in a split-plot at three replicates comprising of eight treatment combinations, i.e., two water irrigation regimes as follows: regular irrigation (four irrigations) and deficit irrigation (two irrigations) in the main plots and four soil treatments in subplots. There was a total of four treatments, comprised from (1) non-treated control, without the application of phosphogypsum or PGPR; (2) soil application of phosphogypsum; (3) inoculation with PGPR; and (4) combined application of phosphogypsum and PGPR. The subplot net area was 10.50 m+, Ca2+, and Mg2+, which are soluble cations in soil solution (meq L–1) in paste extract by Atomic Absorption Spectrophotometer  to assess soil sodium adsorption ratio (SAR). Then ESP was computed by Arshad et al. [A total of ten soil samples were selected at random from different plots of the experimental site at harvest by an auger and oven-dried to determine soil Nad et al. :ESP = 1.Soil samples were collected at 60 days after seed sowing, to determine the activity of soil enzymes: dehydrogenases (Deh) as described by Öhlinger  by addin3 was applied for cation extraction. The leaf Na+ and K+ contents were assessed as mg g−1 dry weight by an ion chromatograph based on the method of Jackson [At 75 days after seed sowing, milled five leaf samples (>1.0 g) were weighed and converted to ash with an electric oven. Then, 100 μL of HNO Jackson .−1 FW) = 12.7 (A663) − 2.69 (A645); Chl b (mg g−1 FW) = 25.8 (A645) − 4.68 (A663); Carotenoids (mg g−1 FW) = (1000 (A470) − 2.27 (Chl a) − 81.4 (Chl b))/227.At 75 days after seed sowing, milled five leaf samples (>1.0 g) were weighed and used to determine photosynthetic pigment contents . Pigment contents in green leaves were estimated in 80% acetone for 48 h at 4 °C. The absorbance of the filtrates was assessed at different wavelengths  by a UV-1900 BMS  spectrophotometer. Pigment contents were computed based on method of Anaraki . EquatioAt 75 days after seed sowing, five green leaves were selected from the mid-section and weighed . The fresh weight attained from each sample was >0.5 g, as recommended by Weatherly . So as t−1 FW.Proline content was assessed by the method of Bates et al. . At 75 d15 − EC0))/((leaf fresh weight)) × 100, where EC0 represents conductivity at the initial time and EC15 represents conductivity after 15 min.At 75 days after seed sowing, green leaves were selected from the mid-section and cut into segments (ca. 1 cm) and weighed. The segments were saved into 20 mL of distilled water in the test tube. The leaf fragments were washed gradually then put into a shaker (100 rpm) at room temperature and the rate of leakage was read at 15 min intervals for 60 min via a LF 92 conductivity meter. The leakage rate was measured as the slope of the line to the leaf dry weight by the method of Bajji et al. . The eleg, 4 °C for 30 min.At 75 days after seed sowing, green leaves were selected from the mid-section (250 mg each) and were cut into segments and homogenized in 5 mL of cold phosphate buffer  to utilize as an enzyme extract. Then, the mixed sample was centrifuged at 15,000× (SOD: 1.15.1.1) was assessed by 50% NBT reduction assay at 560 nm, as found by Beauchamp and Fridovich .2O2 in company with 50 mM K-phosphate buffer (pH 7.0). The reaction started by applying H2O2 and the absorbance was examined at 240 nm for 60 s [(CAT: 1.11.1.6) was assessed respecting the reaction between 50 µL enzyme extract and 12.5 mm Hfor 60 s .2O2 and enzyme extract at 417 nm, as found by Vetter et al. [−1 protein.(POX: 1.11.1.7) was assessed by o-phenylenediamine as a chromogenic indicator in company with Hr et al. . The act2 and tri chloroacetic acid (TCA: 0.1%) was performed, then the samples were centrifuged (3000 rpm) for 20 min to determine H2O2, as found by Velikova et al. [2O2 was computed as µmol g−1 FW by a standard curve.At 75 days after seed sowing, green leaves selected from the mid-section (250 mg each) were cut into segments, and homogenization of samples (0.5 g) by liquid Na et al. . The reav/v) was performed. A total of 0.65% thiobarbituric acid (TBA) and 0.01% Butyl hydroxyl toluene (BHT) were used to determine thiobarbituric acid reactive substances (TBARS), following the method found by Du and Bramlage [g ) for 15 min. The absorbance was examined at 532 and 600 nm by a spectrophotometer. MDA was computed as µmol g−1 FW by a standard curve.At 75 days after seed sowing, green leaves selected from the mid-section (250 mg each) were cut into segments and homogenization of sample (0.5 g) by liquid N2 and hydro-acetone buffer  was measured from a 6-m2 area in each experimental unit except the outer border, and the standard grain moisture content of 14% was added to yield computation. Harvest index  was computed as the ratio between grain and biological yields and expressed as %. HI (%) was calculated using Equation:After maturity, canopy biomass was collected and estimated after oven drying at 70 °C until constant weight. Plant height (cm), 1000-grain weight (g), grain yield, foliage yields, and harvest index were measured from the remaining plants in each replicate. The biological yield  according to the Association of Official Analytical Chemists (AOAC) methods. The extracts obtained were subjected to preliminary phytochemical screening as follows. Grains protein (%) was estimated by multiplying the total nitrogen by 6.25 using Kjeldahl method according to .Saponin content was estimated using reverse phase based on the grains for extraction (10 g), which were thoroughly ground and then extracted with water at 60 °C for 3 h. The ratio of water to grains was 15 to 1 (by weight). The extract was centrifuged and the supernatant filtered. The Saponin content was calculated as following formula:p < 0.05, 0.01, and 0.001. The data were presented as mean ± standard deviation. Spearman coefficient was run to investigate the non-parametric correlation between water regimes and treatments.Normality and homoscedasticity of dependent variables were checked. Data analysis was performed using the SPSS 13.0 software package . The analysis of variance using two-way ANOVA was performed between water regimes and treatments ecotypes. One-way ANOVA was applied to assess the differences either among water regimes or treatments. Separation of means was performed by post-hoc test (Tukey’s test), and significant differences were accepted at the levels Our results investigate that coupled phosphogypsum soil amendment and seed inoculation using PGPR can be an effective, sustainable, and ecological strategy to enhance the resistance of quinoa plants grown under deficiently irrigation in salt-affected soils, particularly in arid and semi-arid zones. Coupled application of phosphogypsum soil amendment and seed inoculation using PGPR significantly improved the growth, physiology, productivity, seed quality, and salt stress tolerance of quinoa plants, along with soil characterization and nutrient absorption in 2019–2020 and 2020–2021 seasons under field conditions. Our results showed that the useful role of phosphogypsum soil amendment and seed inoculation using PGPR might be owing to the activation of the enzymatic antioxidant defense machinery to scavenge reactive oxygen species (ROS) homeostasis within stressed plants. However, itis required in the future to investigate the long-term studies on the impact(s) of phosphogypsum soil amendment and seed inoculation using PGPR on both plant and soil ecosystems.


This is the first clinical study that wants to investigate the treatment patterns, clinical outcomes, and prognostic factors of regorafenib plus PD-1 inhibitors therapy in Chinese elderly patients with advanced colorectal cancer.A cohort of metastatic colorectal cancer patients 60 years or older who received treatment with regorafenib combined with PD-1 inhibitors was included in our analysis. The endpoints included overall survival (OS), progression-free survival (PFS), and prognostic factors.In total, 24 patients were enrolled with the median age of 68 years, and 62.5% were female. The median OS and PFS were 15.03 months (95% CI 7.0–23.0) and 4.0 months (95% CI 1.8–6.2), respectively. The objective response rate was 8.3%, and the disease control rate was 70.8%. Patients previously treated with regorafenib had a longer median PFS than those without (6.3 versus 2.8 months). In terms of final daily doses, it showed a trend toward better PFS (median PFS was 10.0 months) in high-dose group (daily dose above 80 mg of regorafenib) compared to low-dose group (daily dose no more than 80 mg of regorafenib) (median PFS was 3.5 months).This real-world evidence confirms that Chinese elderly patients with advanced colorectal cancer may benefit from the treatment of regorafenib combined with PD-1 inhibitors, similarly with this combination therapy strategies in all age patients. Colorectal cancer (CRC), as one of the most prevalent types of cancer in China, is still a leading cause of cancer-related mortality . Based oRegorafenib, an oral small-molecule multi-kinase inhibitor, has demonstrated significantly improved survival in two randomized, double-blind, placebo-controlled phase 3 trials (CORRECT and CONCUR) , 5. TherRecent data suggest a possible synergic effect between regorafenib and immune checkpoint inhibitors, as has been shown in the REGONIVO trial, which reported a response rate of 33% and median progression-free survival of up to 7.9 months in a cohort of 24 Japanese patients with MSS treatment-refractory metastatic colorectal cancers . In the We retrospectively reviewed the patients treated with regorafenib plus PD-1 inhibitors for unresectable mCRC in Henan Cancer Hospital (China) from January 2019 to July 31, 2021.Patients were eligible for participation if they were 60 years of age or older and had histologically or cytologically confirmed mCRC. Patients were required to have failed to respond to all the available systemic agents, including fluoropyrimidine, oxaliplatin, irinotecan, bevacizumab, and cetuximab when applicable, and received at least one cycle of regorafenib plus PD-1 inhibitors. Besides, the medical record should be complete and legible. Patients who met any of the following criteria at the time of screening will be excluded: other histological types instead of adenocarcinoma of the colon or rectum; at stage I ~ III according to TNM staging system; Eastern Cooperative Oncology Group (ECOG) performance status of 3 or more. This work was approved by the ethics committee of Henan Cancer Hospital . Patients were followed up by telephone questionnaires every three months until death or this study cutoff date .Regorafenib was prescribed at 40, 80, 120, or 160 mg daily from day 1 to day 21 of each 28-day cycle. Depending on the patient’s tolerability, the daily dose was allowed at the discretion of treating physicians. All patients received immune checkpoint inhibitors on the first day of regorafenib treatment, PD-1 inhibitors were used according to the recommended doses: nivolumab 240 mg every two weeks, camrelizumab 200 mg every two weeks, sintilimab 200 mg every three weeks, toripalimab 240 mg every three weeks, pembrolizumab 200 mg every three weeks, tislelizumab 200 mg every three weeks.Demographics, disease features, and therapy information of patients were documented at baseline examinations. Tumors response was evaluated every 2 or 3-cycle treatment of PD-1 inhibitors, according to the Response Evaluation Criteria in Solid Tumors (RECIST) Version 1.1.The primary endpoint was overall survival (OS), which was defined as the duration from treatment to death as a result of any cause. Other endpoints included progression-free survival (PFS), disease-control rate (DCR), and objective response rate (ORR). Progression-free survival (PFS) was defined as the duration from treatment to the first documented disease progression or death. The objective response rate (ORR) was defined as complete response (CR) plus partial response (PR), and the disease-control rate (DCR) was defined as CR plus PR together with stable disease (SD). Adverse Events were graded and analyzed according to National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE), Version 4.03.P value < 0.05 was considered statistically significant. All statistical analysis was performed using IBM SPSS Statistics for Windows Version 25.0 . Quantitative data were displayed as mean with standard deviation (SD), and qualitative data were expressed as a number with percentage [No. (%)]. Survival data were analyzed using the Kaplan–Meier method and compared by log-rank test. n = 8) of patients, the left-side colon in 29.2% (n = 7), and the rectum in 37.5% (n = 9). The main metastatic sites were the liver and/or lung (91.7%), only the lung (25.0%), only the liver (29.2%), distal lymph nodes (45.8%), and peritoneum (16.7%). Other baseline characteristics of the patients are presented in Table At a median follow-up of 16.2 months, a total of 24 patients were enrolled. Nine of these patients (37.5%) were male; the median age was 68.0 years. The primary tumor site was the right-side colon in 33.3% (Regorafenib plus PD-1 inhibitors were given to 13 patients (54.2%) in third-line treatment and 11 patients (45.8%) in fourth-line treatment and beyond. The most common PD-1 inhibitors were sintilimab (50.0%), followed by carrelizumab (25.0%), nivolumab (8.3%), toripalimab (8.3%), pembrolizumab (4.2%) and tislelizumab (4.2%). There were eight patients (33.3%) who previously received regorafenib therapy, and no one had taken PD-1 inhibitors before entering this study. MSI/MMR status data were available from 20 patients (83.3%). Of these, only one patient (4.2%) had an MSI-high tumor, and the remaining 19 patients (79.2%) were MSS or MMR proficient.The median OS and median PFS were 15.0 months and 4.0 months , respectively , K-RAS status (P = 0.150), tumor sites (P = 0.321), and any kind of PD-1 inhibitors (P = 0.477).Specifically, patients previously treated with regorafenib had longer median PFS than those without it. Median PFS was 6.3 months  in the former and 2.8 months 95% CI, 1.9–3.7) in the latter (.9–3.7 inn = 4) started regorafenib at daily doses of 120 mg, 79.2% of patients (n = 19) started at 80 mg, and 4.2% of patients (n = 1) started at 40 mg. There were 12.5%, 70.8%, 4.2%, and 12.5% of patients who received the final daily doses of 40, 80, 120, and 160 mg, respectively. Dose modifications were performed in 9 overall patients (37.5%), including dose reduction in 5 patients (20.8%) and dose escalation in 4 patients (16.7%) . The most common grade 3 to 4 events were hand-foot skin reaction, hypertension, and oral mucositis.As the majority of CRC cases display a molecular MSS/pMMR profile, it is particularly meaningful to investigate the clinical applications of adaptive immune or combination regimens in MSS CRC patients. Several studies have shown that the combination regimen using regorafenib combined with PD-1 inhibitors has promising efficacy for those patients –18. The In this single-center, retrospective study involving Chinese elderly patients with refractory advanced colorectal cancer, the median age of patients enrolled in our study is 68 years, which is older than that in most studies conducted with regorafenib or regorafenib plus PD-1 inhibitors (53.0 to 61.3 years) , 14, 15.P > 0.05). In addition, PFS was similar for patients with KRAS wild-type versus mutant, left- versus right-sided tumors, and among various types of PD-1 inhibitors. However, patients with previous treatment with regorafenib had longer PFS than those without it . Several reports showed that regorafenib modulated immuno-suppressive tumor microenvironment by blocking VEGFRs, TIE2, and CSF-1R, RET/Src axis signal pathways, and increased intratumoral CD45+ leukocytes, CD8+ T cells, which would enhance anti-tumor immunity when using regorafenib alone and plus various immunotherapies [Further analyses of effectiveness in our study showed that there was no obvious correlation for survival between patients who only had liver metastasis and lung metastasis  raised the doses up to 120 mg or 160 mg. When compared with PFS and OS in different initial daily dose groups of regorafenib (≤ 80 versus > 80 mg), we concluded that there was no significant difference between these groups. And similar results were found in the final daily doses groups. However, it showed a trend toward better PFS in patients with final daily doses > 80 mg group compared to the low-dose group (median PFS was 10.0 months in the high-dose group versus 3.5 months in ≤ 80 mg). Considering the safety and tolerability profile of regorafenib, we argue that it should be started with a lower dose for regorafenib and adjusted until the maximum tolerated dose was reached by periodic follow-up and communication in time between nurses and patients. This strategy could contribute to enhancing tolerability and improving adherence, and reducing the risk of adverse events from regorafenib and/or PD-1 inhibitors.The limitations of our study include its retrospective nature, the inadequate data on toxicity evaluation, the small sample size, and the short median follow-up. Besides, there were 4 PD-1 inhibitors just approved in China but no other countries, which means these therapeutic drugs are not available in other countries. However, the results may provide a clearer picture of the efficacy and safety of regorafenib plus PD-1 inhibitors in elderly Chinese patients with mCRC.To our knowledge, this is the first paper describing the detailed information on regorafenib plus PD-1 inhibitors in elderly Chinese patients with mCRC in real-world settings. This real-world evidence confirms that Chinese elderly patients with advanced colorectal cancer can benefit from the treatment of regorafenib combined with PD-1 inhibitors, similarly with this combination therapy strategies in all age patients. However, further larger cohorts research should investigate whether the PFS advantage in the high-dose group could eventually lead to improved OS outcomes. Besides, considering the quality of life, close monitoring and management for adverse drug events are needed.


L. infantum infection using canine (n = 140) and human (n = 145) sera samples. Results: in the trials, rMELEISH was able to discriminate between VL cases and cross-reactive diseases and healthy samples, with sensitivity and specificity values of 100%, as compared to the use of a soluble Leishmania antigenic extract (SLA). Conclusions: the preliminary data suggest that rMELEISH has the potential to be tested in future studies against a larger serological panel and in field conditions for the diagnosis of canine and human VL. Background: visceral leishmaniasis (VL) is a critical public health problem in over ninety countries. The control measures adopted in Brazil have been insufficient when it comes to preventing the spread of this overlooked disease. In this context, a precise diagnosis of VL in dogs and humans could help to reduce the number of cases of this disease. Distinct studies for the diagnosis of VL have used single recombinant proteins in serological assays; however, the results have been variable, mainly in relation to the sensitivity of the antigens. In this context, the development of multiepitope-based proteins could be relevant to solving such problem. Methods: a chimeric protein (rMELEISH) was constructed based on amino acid sequences from kinesin 39 (k39), alpha-tubulin, and heat-shock proteins HSP70 and HSP 83.1, and tested in enzyme-linked immunosorbent (ELISA) for the detection of  Leishmaniasis is a neglected disease caused by protozoan parasites of the genus Leishmania; the disease is endemic in 99 countries . There aL. infantum infections to prevent VL from spreading [The strategy adopted by the Brazilian Ministry of Health to control VL is based on early diagnosis, the treatment of human cases, the monitoring of seroreactive dogs, and vector control. However, such measures have been shown to have little effect ,6,7. Thepreading ,9,10. ThA variety of laboratory methods have been used to diagnose VL in humans and dogs, such as indirect fluorescence antibody tests, enzyme-linked immunosorbent (ELISA), dot-ELISA, the direct agglutination test, Western blotting, and immunochromatographic assays ,12,13. TThe ELISA is the candidate of choice for a rapid and reliable diagnostic test for Leishmania infection because it is practical, standardizable, and suitable for mass screening ,21. HoweSuch an experimental strategy has been shown to be a valuable basis for the serological diagnosis of diseases ,26,27,28L. infantum infection in humans and dogs. The preliminary results described here showed that the recombinant protein was identified by antibodies in sera from canine and human VL, but not by samples from cross-reactive diseases or those from endemic controls.In this study, a recombinant multiepitope-based protein  was constructed based on amino acid sequences from kinesin 39 (k39), alpha-tubulin, and heat-shock proteins (HSP70 and HSP 83.1); the resultant chimeric protein was used for the detection of L. infantum (MHOM/BR/1970/BH46) strain was used. Stationary promastigotes were grown at 24 °C in Schneider’s medium , which was supplemented with 20% inactivated fetal bovine serum , 20 mM L-glutamine, 200 U/mL penicillin, and 100 µg/mL streptomycin, at pH 7.4. The soluble Leishmania antigen extract (SLA) was prepared as described Coelho et al. 2003 [9 stationary-phase promastigotes per mL of L. infantum were washed 3 times in 5 mL of cold sterile phosphate-buffered saline (PBS). After 7 cycles of freezing (−196 °C) and thawing (+37 °C), the suspension was centrifuged at 8000× g for 20 min at 4 °C. The supernatant containing SLA was collected in 500 µL aliquots and then stored at −80 °C until use. The protein concentration was estimated using the Bradford method [An al. 2003 . Brieflyd method .g for 10 min at room temperature); then, the sera were recovered from the supernatant, aliquoted, and stored at −70 °C, until use. Sera were collected from asymptomatic (n = 20) or symptomatic (n = 25) VL dogs, who were diagnosed by means of clinical evaluation and by a PCR technique using spleen and/or bone marrow aspirates to identify L. infantum kinetoplast DNA (kDNA). In addition, all VL animals presented positive serological results when evaluated using the EIE-CVL Bio Manguinhos® kit . Symptomatic animals presented three or more of the following clinical signs: emaciation, alopecia, anemia, conjunctivitis, dehydration, dermatitis, erosion, ulcerations, lymphadenopathy, and onychogryphosis. Otherwise, asymptomatic dogs were free of any sign of disease. Sera were also collected from healthy dogs living in endemic  and non-endemic  areas of VL. These animals showed no clinical sign of leishmaniasis and presented negative serological results when evaluated with the EIE-CVL BioManguinhos® kit . Sera from Leish-Tec®-vaccinated dogs (n = 20) and from those experimentally infected with Ehrlichia canis (n = 15) or Babesia canis (n = 10), all of whom were kept in kennels to prevent their contact with transmitting vectors of leishmaniasis, were used.The study was approved by the Ethical Committee on the Use of Animals in Research of the Federal University of Minas Gerais (UFMG), with the protocol number 244/2018. In total, 140 canine samples were used in this study. Blood samples were collected in vacutainer tubes without anticoagulant , and processed by centrifugation (5000× g for 10 min at room temperature); then, sera were recovered from the supernatant, aliquoted, and stored at −70 °C until use. Samples were collected from VL patients (n = 35), who were diagnosed by means of clinical evaluation and PCR to identify L. infantum kDNA in organic aspirates. In addition, all patients presented positive serological results when evaluated with the Kalazar Detect™ Rapid Test kit . Sera were also obtained from healthy subjects living in areas where VL is endemic (n = 30); these subjects did not present any clinical sign of disease and showed negatve serological results. Samples from patients infected with Chagas Disease (n = 25), leprosy (n = 10), tuberculosis (n = 10), malaria (n = 10), and HIV (n = 25) were employed in the serological assays.The study was approved by the Human Research Ethics Committee of UFMG, with protocol number CAAE–32343114.9.0000.5149. In total, 145 human sera samples were used in the experiments. Blood samples were collected in vacutainer tubes without anticoagulant (BD Biosciences), and processed by centrifugation  plysS competent cells, and selection was performed on LB agar plates containing 100 µg/mL ampicillin. The DNA and amino acid sequences for the entire synthetic gene construct are proprietary (under Brazilian patent No. BR1020140313311) and cannot be shared at this stage. An individual colony was inoculated in 5 mL LB  containing 100 µg/mL ampicillin and allowed to grow overnight at 37 °C under agitation (200 rpm). Then, 1.25 mL of the pre-culture was transferred to 25 mL LB in a 250 mL Erlenmeyer flask. The culture was grown in the aforementioned conditions until an OD600 of 0.6, at which point 1 mM IPTG was added. Aliquots were withdrawn 0.5, 1.5, and 2.5 h after induction. The induced culture was harvested by centrifugation at 6000× g for 15 min at 4 °C and the pellet was stored at −80 °C. The amino acid sequence of the rMELEISH protein was submitted to the Protein Homology Recognition Engine V 2.0 server (Phyre2)  for mode2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) following incubation at 4 °C for 16 h. Cell suspension was then sonicated  using a Vibra Cell sonicator  and incubated on ice for 2 h following centrifugation at 6000× g for 15 min at 4 °C. The supernatant was added to 0.5 mL Ni-Sepharose 6 Fast Flow resin  (resuspended in lysis buffer), which was then incubated at 4 °C for 90 min on a vertical disc rotator. Next, the resin was sedimented and washed 4 times with 1 mL washing buffer . The protein was eluted in 3 fractions using 0.5 mL elution buffer .The frozen pellet was resuspended in 1 mL lysis buffer , for 2 h at room temperature. It was then washed 3 times with TBS-T and incubated with monoclonal mouse anti-His AP , diluted 1:1000 in TBS for 2 h at room temperature. Following 3 washes with TBS-T, the specific protein band was visualized using the nitroblue tetrazoli-um/5-bromo-4chloro-3′-indolylphosphate (NBT/BCIP) detection method.Protein integrity and molecular mass calculation were evaluated by running sam-ples on 12% SDS-PAGE. Proteins were stained with Coomassie Brilliant Blue R-250 (Sigma-Aldrich). Following electrophoresis, the proteins were transferred electrophoretically to a Polyvinylidene difluoride (PVDF) membrane for Western blotting. The membrane was blocked with 5% skim milk powder in Tris-buffered saline with 0.1% Tweenw/v) dried skim milk powder and washed again with PBST. Subsequently, 100 µL of a dilution  dried skim milk powder, and 5 µL serum) was placed into the wells, resulting in a final dilution of approximately 1/20. After incubation for 1 h at 37 °C, the wells were washed with PBST and 100 µL of goat anti-dog IgG conjugate peroxidase-labeled diluted at 1:25,000 in PBS buffer (pH 7.2), containing 5% (w/v) dried skim milk powder, was added following incubation for 1 h at 37 °C. The wells were again washed with 100 µL TMB by incubating for 30 min at room temperature. The optical density (OD) values were read at 450 nm.Titration curves were plotted to determine the most appropriate antigen concentration and antibody dilution to be used. The wells of polystyrene plates (Sarsted) were sensitized with 35 ng purified rMELEISH protein, which was diluted in 100 µL 0.1 M sodium carbonate-bicarbonate buffer (pH 9.6). After incubation at 4 °C for 16 h, the coated wells were washed with PBST  and blocked for 2 h at 37 °C with PBS containing 5% (θ) based on the molecular mass of 115 Da per residue [Conformational changes in the chimeric protein were evaluated by fluorescence spectroscopy using a Jasco FP-6500 Spectrofluorimeter  coupled to a Jasco ETC-273T Peltier system  with water circulation. The rMELEISH protein (0.05 mg/mL) was diluted in 10 mM sodium acetate buffer at pH 4.0 and 10 mM Tris-HCl, pH 7.0 and 9.0, at 25 °C. The excitation and emission slits were fitted at 5.0 and 10.0 nm, respectively. The excitation wavelength was 295 nm and emission spectra were recorded from 300–400 nm. The circular dichroism (CD) assays were performed using a Jasco J-815 spectropolarimeter  equipped with a Peltier-type temperature controller and a thermostatized cuvette cell linked to a thermostatic bath. The far-ultraviolet (UV) CD spectra of the rMELEISH (0.1 mg/mL) in 2 mM acetate, pH 4.0, and 2 mM Tris HCl, pH 7.0 and 9.0, were recorded using a 0.1 cm-path-length quartz cuvette. After five consecutive measurements, the mean spectrum for each pH was recorded and the buffer contribution spectrum was subtracted. The ellipticities were converted into molar ellipticity ( residue . TM (version 6.0 for Windows). Receiver–operator characteristic (ROC) curves were constructed using the OD values from VL sera versus those from negative or cross-reactive samples. The diagnostic performance of the antigens was evaluated by calculating the sensitivity (Se), specificity (Sp), area under the curve (AUC), and Youden index (J). Confidence intervals (CI) were defined using a 95% confidence level (95%CI). Differences were considered significant with p < 0.05.Results were entered into Microsoft Excel (version 10.0) spreadsheets and analyzed using GraphPad PrismIn order to design a multiepitope protein that could be of diagnostic use, linear and conserved B-cell epitopes, which were shown to be antigenic for VL and present specific antibodies against them, were selected, grouped in tandem, and connected by flexible glycine–serine linkers. This allowed the epitopes to be freely available for interaction with their cognate antibodies, thus contributing to the overall sensitivity and specificity of the diagnostic test. Some molecules were randomly repeated to increase epitope density, resulting in a chimeric protein called rMELEISH. After purification, the recombinant protein had a molecular weight of ~25.0 kDa C. A WestFluorescence spectra of rMELEISH showed typical emission bands of solvent exposed tryptophan residues A. The inL. infantum SLA was used as comparative antigen. The results showed that the chimeric protein was recognized by antibodies in VL patients’ sera, presenting a low reaction in healthy subjects and in cross-reactive patient sera : Brazilian patent No. BR1020140313311.