Previous research demonstrated that seminal plasma, the fluid that together with sperm composes the semen, modulates the interaction between sperm and polymorphonuclear neutrophils (PMN) within the donkey uterus. This study sought to evaluate whether a specific set of proteins or the entire seminal plasma is involved in that modulation. Seminal plasma was fractioned into six portions: <3, 3–10, 10–30, 30–50, 50–100, and >100 kDa. Sperm incubated with two specific fractions, containing 30–50 kDa and 50–100 kDa proteins, exhibited the highest motility and viability after co-incubation with PMN for 4 h, and the highest sperm–PMN interaction at the beginning of the experiment. These results suggest that proteins of 30–50 and 50–100 kDa, rather than the entire seminal plasma, appear to be involved in the inflammatory reaction within the donkey endometrium.While artificial insemination (AI) with frozen-thawed sperm results in low fertility rates in donkeys, the addition of seminal plasma, removed during cryopreservation, partially counteracts that reduction. Related to this, an apparent inflammatory reaction in jennies is induced following AI with frozen-thawed sperm, as a high amount of polymorphonuclear neutrophils (PMN) are observed within the donkey uterus six hours after AI. While PMN appear to select the sperm that ultimately reach the oviduct, two mechanisms, phagocytosis and NETosis, have been purported to be involved in that clearance. Remarkably, sperm interacts with PMN, but the presence of seminal plasma reduces that binding. As seminal plasma is a complex fluid made up of different molecules, including proteins, this study aimed to evaluate how different seminal plasma fractions, separated by molecular weight , affect sperm–PMN binding. Sperm motility, viability, and sperm–PMN binding were evaluated after 0 h, 1 h, 2 h, 3 h, and 4 h of co-incubation at 38 °C. Two seminal plasma fractions, including 30–50 kDa or 50–100 kDa proteins, showed the highest sperm motility and viability. As viability of sperm not bound to PMN after 3 h of incubation was the highest in the presence of 30–50 and 50–100 kDa proteins, we suggest that both fractions are involved in the control of the jenny’s post-breeding inflammatory response. In conclusion, this study has shown for the first time that specific fractions rather than the entire seminal plasma modulate sperm–PMN binding within the donkey uterus. As several proteins suggested to be involved in the control of post-AI endometritis have a molecular weight between 30 and 100 kDa, further studies aimed at determining the identity of these molecules and evaluating their potential effect in vivo are much warranted. Jackasses show high spermatogenic efficiency and a relatively short spermatogenesis in comparison with stallions . In geneIn mammals, natural breeding or AI induces physiological endometritis. This endometrial inflammatory response is characterized by an apparent infiltration of polymorphonuclear neutrophils (PMN) into the uterus, which is induced by spermatozoa rather than bacteria . The extOn the other hand, seminal plasma, a complex fluid that contains ions, sugars, lipids, hormones, amino acids, and growth factors , is prodWhile, as aforementioned, the modulating role of seminal plasma on sperm–PMN binding is apparent, it is not clear whether this fluid as a whole or specific fractions, are involved in the control of post-AI endometritis in donkeys. Therefore, this study evaluated the effects of different seminal plasma portions on sperm–PMN binding after fractioning this fluid into separate parts based on molecular weight .Five Catalonian jackasses, aged 3–11 years old, and three jennies, aged 5–8 years old, were involved in this study. All animals were in good health and body conditions, and of proven fertility . Animals were housed at the Experimental Farm, Faculty of Veterinary Medicine, Autonomous University of Barcelona . Jackasses were allocated to individual paddocks, whereas jennies were maintained in a big paddock . Animals were fed grain forage, straw, and hay, and water was provided ad libitum. This study was approved by the Ethics Committee, Autonomous University of Barcelona (Code: CEEAH 1424).g and 4 °C for 10 min . Supernatants were transferred into new 50 mL conical tubes and again centrifuged. Following this, supernatants, presumably containing seminal plasma, were examined under a phase-contrast microscope  to ensure that no sperm was present. This action was repeated as many times as needed  until samples were sperm-free. The number of centrifugation cycles ranged between five and seven, depending on the ejaculate. Seminal plasma samples were stored in 15 mL tubes at −80 °C.Three ejaculates per male were collected with an artificial vagina (Hannover model) equipped with an in-line filter . Raw semen was split into 50 mL conical tubes and then centrifuged at 3000× g and 20 °C for 10 min through a specific filter and a programmable centrifuge  to obtain the <3 kDa fraction. The resulting supernatant was used to obtain the next fraction by another specific filter, and this process was repeated for each fraction. Upon completion of the entire process, six fractions were obtained: <3 kDa, 3–10 kDa, 10–30 kDa, 30–50 kDa, 50–100 kDa, and >100 kDa.Seminal plasma samples from three ejaculates collected from five different jackasses were thawed on ice, pooled, and fractioned through ultracentrifugation with filters for protein purification and concentration based on molecular weight , following manufacturer instructions. Seminal plasma was centrifuged at 4000× g and 4 °C for 5 min , and then adjusted to 100×106 PMN/mL through dilution with ice-cold PBS.Peripheral blood was collected from three different jennies via jugular venipuncture with 10 mL BD Vacutainer R tubes containing 18.0 mg of ethylenediaminetetraacetic acid . Eight tubes from each jenny were collected, and PMN were subsequently isolated following the protocol described by Miró et al. [s et al. ). The su6 sperm/mL, whereas the other was centrifuged through a single layer of a silane-coated silica-based colloid  in order to eliminate the seminal plasma proteins attached to the sperm surface. Semen was filtered through Equicoll using the protocol for small tubes described by Morrell et al. [g and 20 °C for 20 min. Supernatants and colloids were discarded, and sperm pellets were diluted with the same skim-milk-based extender and adjusted to a final concentration of 500 × 106 sperm/mL.Semen samples were collected with an artificial vagina (Hannover model) equipped with an in-line filter (Minitüb Ibérica S.L.) to obtain a gel-free semen sample. Semen volume was recorded and sperm concentration was evaluated using a Neubauer chamber . Ejaculates were diluted in a skim-milk-based extender  and divil et al. . Briefly6 PMN/mL + 100 µl 500×106 sperm/mL; A, B, C, D, E, F, and G), were prepared. Six treatments were added with the obtained seminal plasma fractions ; the other one was used as a control and did not contain seminal plasma (G). Detailed description of these seven treatments is as follows: A) 100 µL 100 × 106 PMN/mL + 100 µL 500 × 106 sperm/mL + 100 µL <3 kDa fraction; B) 100 µL 100 × 106 PMN/mL + 100 µL 500 × 106 sperm/mL + 100 µL 3–10 kDa fraction; C) 100 µL 100 × 106 PMN/mL + 100 µL 500 × 106 sperm/mL + 100 µL 10–30 kDa fraction; D) 100 µL 100 × 106 PMN/mL+ 100 µL 500 × 106 sperm/mL + 100 µL 30–50 kDa fraction; E) 100 µL 100 × 106 PMN/mL+ 100 µL 500 × 106 sperm/mL+ 100 µL 50–100 kDa fraction; F) 100 µL 100 × 106 PMN/mL+ 100 µL 500 × 106 sperm/mL+ 100 µL >100 kDa fraction; and G) 100 µL 100 × 106 PMN/mL+ 100 µL 500 × 106 sperm/mL (control). All semen samples were incubated in a water bath at 38 °C, and their viability, motility, and sperm–PMN binding were evaluated at 0, 1, 2, 3, and 4 h. This experiment was repeated 15 times. In each experiment, PMN were obtained from peripheral blood of one jenny (n = 3), semen was obtained from one jackass (n = 5), and seminal plasma consisted of a pool from the five used jackasses.Seven treatments, all containing the same PMN and sperm concentrations (100×10Viability and morphology of the two sperm populations (bound-to-PMN and unbound) were evaluated following eosin-nigrosin staining . Two repMotion characteristics of the unbound sperm population were evaluated through a computer assisted sperm analysis (CASA) system . This system consisted of a negative phase contrast microscope , with a yellow light filter, warm-up plate, and a digital video camera  connected to a personal computer containing the ISAS software. Samples (5 µL) were loaded into a pre-warmed (38 °C) Neubauer chamber. Digital images at 200× magnification were recorded and analyzed to remove wrongly detected particles/artefacts. At least 200 spermatozoa were counted and two technical replicates were evaluated.2/s); absolute mean angular displacement (MADabs), which is the absolute value of the advancing angle of sperm trajectory (units: angular degrees); algebraic mean angular displacement , which is the algebraic value of the advancing angle of sperm trajectory, provided that negative values indicate clockwise displacement (units: angular degree).In each assessment, the following parameters were recorded: curvilinear velocity (VCL), which is the mean path velocity of the sperm head along its actual trajectory (units: µm/s); straight-line velocity (VSL), which is the mean path velocity of the sperm head along a straight line from the first to its least position (units: µm/s); average pathway velocity (VAP), which is the mean velocity of the sperm head along its average trajectory (units: µm/s); linearity coefficient (LIN), which corresponds to VSL/VCL×100 (units: %); straightness coefficient (STR), which results from calculating VSL/VAP×100 (units: %); wobble coefficient (WOB), which is equal to VAP/VCL × 100 (units: %); mean amplitude of lateral head displacement (ALH), which is the mean value of the extreme side-to-side movement of the sperm head in each beat cycle (units: µm); frequency of head displacement (BCF), which is the frequency with which the actual sperm trajectory crosses the average path trajectory (units: Hz); dance, which is equal to VCL/ALH  and plotted with GraphPad Prism 8.0 for Windows . Data were confirmed to be normally distributed (Shapiro–Wilk test) and variances were found to be homogeneous (Levene test). The effects of different seminal plasma fractions  on the viability and motility of unbound and bound-to-PMN sperm populations and sperm–PMN binding  were tested through a linear mixed model followed by post hoc Sidak test for pair-wise comparisons. The seminal plasma fraction was the fixed-effects factor, the animal was the random-effects factor, and the incubation time (4 h at 38 °C) was the intra-subjects factor.Sperm motile subpopulations were set according to the procedure described in Catalán et al. . In briep ≤ 0.05. Data are shown as mean ± standard deviation (SD).In all analyses, the level of statistical significance was set at p < 0.05) higher percentage of spermatozoa bound to PMN at 0 h. At 1 h, 2 h, and 3 h, differences between seminal plasma fractions were less apparent. Interestingly, while no significant differences were observed at 2 h, the fraction containing 50–100 kDa proteins showed significantly (p < 0.05) higher percentage of spermatozoa bound to PMN than the ones containing proteins smaller than 50 kDa  after 4 h of incubation. Moreover, the fraction containing 10–30 kDa proteins showed a significantly (p < 0.05) lower percentage of spermatozoa bound to PMN than the control after 4 h of incubation  higher in the treatments containing seminal plasma proteins bigger than 10 kDa , sperm–PMN ratios  were significantly  lower in the fraction containing proteins of 50–100 kDa than in the fraction containing proteins of 30–50 kDa after 2 h and 3 h of incubation. In addition, viability of bound-to-PMN sperm was the highest in the fraction containing 30–50 kDa proteins at the same specified times.p < 0.05) higher viability than the control after 2 h, 3 h, and 4 h of incubation at 38 °C. In addition, fractions containing small proteins (<3 kDa and 3–10 kDa) showed a significantly lower percentage of viable spermatozoa in the unbound population than the other fractions and the control at 4 h  higher in the presence of 50–100 kDa proteins than in the control, and at 4 h, when the proportions of sperm belonging to SP1 were significantly (p < 0.05) higher in the treatment containing 30–50 kDa proteins than in the control.In general, incubation decreased sperm motility, as the fastest populations  lower than the control at 4 h.In the case of SP2, the most linear one, the fraction containing 50–100 kDa proteins was the one that showed the highest proportion of spermatozoa belonging to that subpopulation. In contrast, the proportion of spermatozoa belonging to SP2 in the treatment containing 50–100 kDa proteins was significantly . Related to this, when they remain in contact with spermatozoa, low molecular proteins  are not beneficial for sperm, but rather they reduce motility and damage plasma membrane integrity in several species ,37,38,39Sperm motile subpopulations with specific patterns have been previously described in donkeys ,34,40 anWhile all the aforementioned findings suggest that not all seminal plasma proteins have the same effect, information about these proteins in donkey seminal plasma is scarce, and needs further research. In addition, not only have a reduced number of proteins been described, but their functions also remain mostly unknown. Based on our results and the available literature, one could suggest that some proteins of 30–50 kDa and 50–100 kDa play that beneficial role. Lactate dehydrogenase (LDH) has a molecular weight of 35 kDa, and its levels are high both in horses and donkeys ,52. In tMoving on to a separate issue, the antioxidant enzyme system involving SOD, CAT, GSR, and GPX has been described in several species ,54,55,56Donkey seminal plasma has been demonstrated to be involved in the modulation of sperm–PMN binding , and hasPrevious studies on sperm–PMN interaction indicate that only a reduced percentage of spermatozoa are phagocytosed by PMN, as most remain attached on the PMN surface or are found within a surrounding halo . BrinkmaFinally, it should be noted that the current study was conducted using fresh, non-contaminated donkey semen. Kotilainen et al.  reportedIn conclusion, this study has reported, for the first time, that specific seminal plasma proteins, notably those of 30–100 kDa, are involved in the modulation of sperm function, including sperm–PMN binding.


Brain radiation exposure, in particular, radiotherapy, can induce cognitive impairment in patients, with significant effects persisting for the rest of their life. However, the main mechanisms leading to this adverse event remain largely unknown. A study of radiation-induced injury to multiple brain regions, focused on the hippocampus, may shed light on neuroanatomic bases of neurocognitive impairments in patients. Hence, we irradiated BALB/c mice  at postnatal day 3 (P3), day 10 (P10), and day 21 (P21) and investigated the long-term radiation effect on brain MRI changes and hippocampal neurogenesis.We found characteristic brain volume reductions in the hippocampus, olfactory bulbs, the cerebellar hemisphere, cerebellar white matter (WM) and cerebellar vermis WM, cingulate, occipital and frontal cortices, cerebellar flocculonodular WM, parietal region, endopiriform claustrum, and entorhinal cortex after irradiation with 5 Gy at P3. Irradiation at P10 induced significant volume reduction in the cerebellum, parietal region, cingulate region, and olfactory bulbs, whereas the reduction of the volume in the entorhinal, parietal, insular, and frontal cortices was demonstrated after irradiation at P21. Immunohistochemical study with cell division marker Ki67 and immature marker doublecortin (DCX) indicated the reduced cell division and genesis of new neurons in the subgranular zone of the dentate gyrus in the hippocampus after irradiation at all three postnatal days, but the reduction of total granule cells in the stratum granulosun was found after irradiation at P3 and P10.The early life radiation exposure during different developmental stages induces varied brain pathophysiological changes which may be related to the development of neurological and neuropsychological disorders later in life. Brain structural changes such as volume reduction, vascular dilatation and permeability, and white matter pathology ranging from demyelination to coagulative necrosis can be detected by magnetic resonance imaging (MRI) . Functiotastasis . At the tastasis . Localiztastasis . Hence, Radio-sensitivity in different brain development stages varies due to differing neurogenesis and gliogenesis; it is, therefore, reasonable to speculate that radiation-induced brain damage varies among animals exposed to different postnatal days. For instance, hippocampal neuronal death peaked at 6 h after X-Ray irradiation at postnatal day 1 with 2 Gy, no cell death was observed at P15 after the irradiation . It has Brain development in rodents from P1 to P3 is comparable to 23–32 weeks of gestation (preterm infant) in the human , 12. At Neonatal specific-pathogen-free BALB/c mice with mother were provided by InVivos, Singapore, and kept in the Comparative Medicine Facility, the National University of Singapore with free access to water and food in a specific pathogen-free (SPF) facility with room temperature ranging from 210 to 240 °C. Animals were provided with toys and checked by vet daily, and were euthanized with CO2 once they suffered pale mucous membranes, hunched posture and ruffled fur, increased respiratory rate/difficulty breathing, ocular/nasal discharge, unresponsive to gentle prodding, and weight loss > 20% compared to age-matched control.A total of 28 mice was used in this study. These animals were divided into four groups based on litters, i.e., the normal control group  without irradiation; The experimental groups were whole-body irradiated with 5 Gy (dose rate 2.48 Gy/min) at postnatal day 3 , 10 , and 21  using the Gamma-Irradiator BIOBEAM8000 . To monitor the animal radiation dose, two nanoDots dosimeters  was attached to the opposite sides of the inner wall of the mouse container for each radiation exposure and was read after irradiation using InLight microStar System  to estimate the actual dose animal received. The actual mean value of nanoDots reading was 0.11 mGy, 4.54 Gy, 4.61 Gy, and 4.77 Gy for the control, P3, P10, and P21 groups respectively. Within thirteen months (13 M) after irradiation, 1, 5, 4, and 2 mice were euthanized in each group of the control, P3, P10, and P21 mice respectively based on the health criteria indicated including (1) weight loss > 20% compared to age-matched control, (2) radiation-induced dermatitis, pale mucous membranes, hunched posture and ruffled fur, increased respiratory rate/difficulty breathing, ocular/nasal discharge, unresponsive to gentle prodding, and (3) skeletal structure extremely prominent, little or no flesh cover and vertebrae distinctly segmented. Euthanization was done by putting animals in a euthanasia chamber, opening the compressed CO2 gas cylinder valve and setting the flow so to displace at least 20% of the chamber volume per minute  to induce rapid unconsciousness with minimal distress to the animals. Gas flow was maintained for at least 1 min after respiration has ceased. We verified that animals were dead before removing it from the chamber by making sure there was no respiratory movement for at least 3 min. If the animal was not dead, cervical dislocation was done following the CO2 administration. Carcass were then stored in −20 °C freezer. MRI scans were therefore performed in 6 , 4 , 3 , and 3  mice in those respective groups at 13 M after radiation exposure. The previous study indicated that the mean longevity or life-span was 27.2 months in BALB/c female and 21.6 months in BALB/c male mice [In vivo imaging was performed at the NUS Comparative Medicine Imaging Facility using a 7 Tesla preclinical animal MRI scanner  equipped with a gradient system reaching a maximum amplitude of 400 mT/m. The animals were positioned head first, prone on a mouse bed and the circularly polarized mouse head coil was positioned and fixed covering the head. The animal bed was then movedinto the center of the magnet bore. Anesthesia was maintained with 1.5–2.5% isoflurane in oxygen (flow rate 1 L/min) during image acquisition. Rectal temperature and respiratory rate and pattern were monitored using an MRI-compatible small animal physiological monitoring system .2-weighted coronal images with a slice thickness of 0.2mm and an in-plane resolution of 0.075 mm × 0.078 mm. Each scan lasted approximately 40 min.For anatomical reference and to check the correct animal alignment in the magnet bore, localizers were acquired in three orthogonal planes: axial, sagittal, and coronal. A 2D Turbo Spin Echo (TSE) protocol  was used to produce T2-weighted sagittal images with a slice thickness of 0.4 mm, and an in-plane resolution of 0.072 mm × 0.072 mm. A 3D TSE sequence  was also used to produce TThree-dimensional (3D) brain images were automatically segmented into 364 labels using an in-house pipeline. This consisted of atlas-based brain extraction  followed3 using an in-house script developed in the programming language R. Percentage differences from the control group were calculated for each region and then converted into a color-coded image  were anesthetized with ketamine (75 mg/kg) + medetomidine (1 mg/kg) at 0.1ml/10 g, perfused with saline and fixed with freshly prepared 4% paraformaldehyde solution. The brain was removed, postfixed overnight, and then transferred to 30% sucrose in 0.1 M PB (pH: 7.4). Sagittal sections were then cut at 40 μm thickness, a serial of alternative 6 sections were collected and put into 6 wells. Three wells were processed for immunohistochemistry to investigate radiation-induced changes of cell proliferation, neurogenesis (immature neurons), and mature neurons using Ki67, doublecortin (DCX), or and neuron-specific nuclear protein (NeuN) respectively.2O2 for 10 min, followed by 1.5% normal goat serum (for Ki67 and NeuN) or 2% horse serum (for DCX) for 2 h at room temperature. The sections were then treated with primary rabbit antibodies for Ki67 (1: 400) , and NeuN (1:1500)  or goat antibodies for DCX  in 0.1 M phosphate buffer saline (PBS) with 0.1% Triton X-100 (PBS-TX) overnight. The sections were then treated with biotinylated goat anti-rabbit or horse anti-goat secondary antibodies for 1 h. The sections were then treated with avidin-biotin complex (ABC) reagent  for 1 h, reacted in DAB Peroxidase Substrate  for 10 min, mounted, and covered with a coverslip.For immunohistochemistry, sections were blocked with 3% H™ software . The experimenter was blind to the group allocation, and only the principal investigator was aware of the group allocation at the different stages of the experiment. “Regional Volume Probe” and “Object Number Probe” were chosen to count positive cell numbers in the entire medial-lateral extent of the dentate gyrus. The section interval was 6. The cell number was indicated as mean ± SEM and was statically analyzed by One-Way ANOVA followed by Tukey’s post hoc test. A p-value < 0.05 was considered as statistical significant.Ki67- or DCX- or NeuN-immunostained cells were analyzed by a stereological method using STEREOLOGERRegional percentage volume changes as characterized by MRI scans were visualized in Fig. 3, t-ratio = 8.759, df = 12, p < 0.0001), P10 + 13 M  and P21 + 13 M .Compared to the control group, the total brain volume was reduced in all three groups of P3 + 13 M (− 51.53 ± 5.88 mmPercentage volume change from control ranged from − 45% (cerebellar vermis WM) to − 2%  at P3 + 13 M, − 28% (cerebellar WM) to − 4%  at P10 + 13 M and − 9%  to − 4%  at P21 + 13 M.3, t-ratio = 15.852, df = 12, p < 0.0001), deep cerebellar nuclei, , cerebellar vermis, , cerebellar white matter, , cerebellar flocculonodular lobe , cerebellar hemisphere , thalamus , cerebellar paravermis WM , cerebellar flocculonodular lobe WM  and olfactory bulbs ,In order, regions with the greatest percentage volume reduction between control and P3 + 13 M groups were cerebellar vermis WM , region and sex  and group and sex . The interaction between region, group and sex was not significant .Statistical testing of the interaction of group, region and sex using a three-way ANOVA showed significant interactions between group and region  or mature (8–12 weeks) mice with high doses of 7.5 or 7 Gy respectively reduced both neurogenesis and volume of the olfactory bulb (OB) , 75. FraThe brain neuropathological changes in different neurological and neuropsychological disorders varied. Delli Pizzi et al.  reportedIn patients with epilepsy and intellectual disability, cortical dysplasia has been reported, it may be closely linked to clinical manifestation . The atrIn the present study, both male and female mice were used as we kept litters with dams. While different brain regions are sexually dimorphic in terms of volume , volume,Both epidemiological and animal experimental studies have suggested the link between prenatal or postnatal radiation exposure and different neurological and neuropsychological disorders. In the present MRI study, characteristic regional volume changes were observed at 13 months after irradiation at P3, P10, and P21, suggesting different brain disorders may be developed at later stages of animal life. These characteristic regional volume change patterns may be used as biomarkers to retrospectively calculate the radiation doses animals are exposed at the early stages of their life. From this point of view, non-invasive MRI scanning may provide morphological biomarkers for retrospective estimation of the radiation doses animals may be exposed in addition to prospective monitoring of radiation-induced brain damage from both radiotherapy and accidental radiation exposure. Further study in patients with radiotherapy or radiodiagnostic exposure may provide more evidence for the development of MRI biomarkers for radiation exposure detection.


Honey bees are eusocial insects that live in colonies comprised of ~30,000 individuals. They are the primary pollinators of plants that produce fruits, nuts, and vegetables. High annual losses of honey bee colonies have made it challenging for beekeepers to provide enough colonies to meet the demand for pollination services. Multiple stressors, including viruses, contribute to colony deaths. To better understand viral pathogenesis and bee antiviral defense mechanisms, we obtained primary cells from honey bee larvae and pupae, maintained them in culture, and infected them with a panel of viruses. We determined that larval hemocytes, which are primary immune cells, support replication of a honey bee virus (sacbrood virus) and a model virus (Flock House virus). Similarly, we determined that mixed cell populations derived from honey bee pupae support replication of sacbrood virus, Flock House virus, and another honey bee virus (deformed wing virus). Evaluation of the host cellular level response to infections with each of these viruses revealed unique expression profiles of three immune genes. In summary, this study demonstrates the utility of honey bee primary cell cultures to investigate the impacts of virus infection on honey bees at the cellular level, which in turn affects individual bee and colony health.Apis mellifera) health is impacted by viral infections at the colony, individual bee, and cellular levels. To investigate honey bee antiviral defense mechanisms at the cellular level we further developed the use of cultured primary cells, derived from either larvae or pupae, and demonstrated that these cells could be infected with a panel of viruses, including common honey bee infecting viruses  and deformed wing virus (DWV)) and an insect model virus, Flock House virus (FHV). Virus abundances were quantified over the course of infection. The production of infectious virions in cultured honey bee pupal cells was demonstrated by determining that naïve cells became infected after the transfer of deformed wing virus or Flock House virus from infected cell cultures. Initial characterization of the honey bee antiviral immune responses at the cellular level indicated that there were virus-specific responses, which included increased expression of bee antiviral protein-1 (GenBank: MF116383) in SBV-infected pupal cells and increased expression of argonaute-2 and dicer-like in FHV-infected hemocytes and pupal cells. Additional studies are required to further elucidate virus-specific honey bee antiviral defense mechanisms. The continued use of cultured primary honey bee cells for studies that involve multiple viruses will address this knowledge gap.Honey bee ( Apis mellifera). While approximately 1000 cell lines have been derived from insect tissues, including 19 hymenopteran cell lines , only one immortalized honey bee cell line, AmE-711, has been established [All organisms are infected by viruses, which are obligate intracellular pathogens that rely on host cell machinery to replicate. In turn, hosts evolve mechanisms to prevent and/or limit virus infections. To investigate host–virus interactions at the cellular and molecular levels, scientists rely extensively on cell cultures, including immortalized cell lines and primary cells ,2,3. Culablished ,9,10.Varroa destructor mite, and pathogens including bacteria, fungi, trypanosomatids, and viruses [The development of additional tools to understand honey bee host–virus interactions at the cellular level is critical for determining the impact of virus infections at the individual bee and colony levels and may ultimately result in the development of strategies that reduce the number of virus-associated honey bee colony deaths. Honey bee colony losses averaged 38% annually from 2010 to 2018, in part due to viral infections ,17,18,19 viruses ,29,30,31 viruses ,32). The viruses ,40,41 . The viruses , black q viruses , chronic viruses ,45, defo viruses ,47, Isra viruses , Kashmir viruses , sacbroo viruses , and the viruses . Viral i viruses ,53,54.Iflaviridae family comprises several arthropod-associated viruses, including two honey-bee-infecting viruses  [Varroa destructor mite [The g virus) ,56,57,58g virus) ,60. Whilg virus) ,62,63,64g virus) ,66,67,68g virus) ,70,71,72g virus) ,74,75. Dtor mite ,79,80,81tor mite ). Varroator mite ,85,86,87tor mite ,91,92,93tor mite ,96,97,98tor mite ,78,99. Itor mite ,103,104.Nodaviridae family. FHV is genetically and structurally well-characterized and has been extensively utilized in studies aimed at understanding basic viral processes, including genome replication and virion assembly [Costelytra Zealandica), it infects a range of insects, including mosquitoes, fruit flies, tsetse flies, and honey bees [Model viruses, including Sindbis virus and Flock House virus, have also been utilized to investigate honey bee antiviral defense mechanisms ,108,109.assembly ,119,120.ney bees ,121,122.ney bees ). Suppreney bees ,125,126)ney bees ,127,128.Similar to other insects, honey bee antiviral defense involves numerous processes and pathways, including autophagy, endocytosis, apoptosis, eicosanoid signaling, melanization, and the Toll and Imd (Immune Deficiency) pathways involving NF-κB (Nuclear Factor κB) homologues Dorsal and Relish, JAK/STAT , JNK , MAPK (Mitogen-Activated Protein Kinases), and RNA interference (RNAi) pathways There is only one immortalized honey bee cell line, AmE-711, which was established from honey bee embryonic tissue  . There oid wasp . In addioid wasp . While rot known ,170,171.bee antiviral protein-1 (GenBank: MF116383) in SBV-infected pupal cells and increased expression of argonaute-2 and dicer-like in FHV-infected hemocytes and pupal cells [To investigate honey bee antiviral defense mechanisms at the cellular level, we further developed the use of cultured primary cells, derived from either larvae or pupae, and demonstrated that these cells could be infected with a panel of viruses, including common honey bee infecting viruses  and a model virus (FHV). Specifically, hemocytes obtained from stage L4-L5 honey bee larvae supported infection and replication of SBV and FHV. Virus replication was verified via detection of the replicative intermediate of both viruses using negative-strand-specific reverse transcription (RT), followed by polymerase chain reaction (PCR), and using quantitative PCR (qPCR) to demonstrate increased virus abundance over an infection time course. Mixed cell populations derived from purple-eyed honey bee pupae supported replication of SBV, DWV, and FHV. Virus replication was verified via negative-strand-specific RT-PCR and qPCR. Peak virus abundance was observed at 96 h post-infection (hpi) for SBV and DWV, while virus abundance peaked at 72 hpi for FHV. Furthermore, the production of infectious virions in cultured honey bee pupal cells was demonstrated by determining that naïve cells became infected after the transfer DWV or FHV infected cell culture material. Initial characterization of the honey bee antiviral immune responses mounted against SBV, DWV, and FHV at the cellular level indicate varying virus-specific responses, including increased expression of al cells ,172. AddApis mellifera carnica) were purchased from a commercial producer in Montana and installed in Langstroth hives at Montana State University, Bozeman, MT, USA. Colonies were maintained using standard apicultural practices, including bi-monthly Varroa destructor mite checks by the sugar roll method and treatment for mites with Mite Away Quick Strips  when mite infestation was evaluated to be greater than 3% (3 mites per 100 bees [Packages (~1.5 kg of worker bees and a mated queen) of Carniolan honey bees .5 cells per well) for five wells on a 48-well plate.Three days before experiments using primary honey bee hemocytes were performed, frames of brood with a lot of 4th and 5th instar larvae (L4–L5) were brought to the laboratory and maintained in a humidified incubator at 28 °C. Larvae that fell out of their cells were caught in a glass dish and collected in batch the next morning. After collection, larvae were surface-sterilized for 3 min in 0.6% hypochlorite (dilute bleach), followed by 3 min in 70% ethanol, and then briefly washed in sterile water for injection (Gibco) in a biological safety cabinet . Larvae were placed into 200 μL of WH2 media , supplemented with 1× penicillin/streptomycin , 10 at a time ,175. Sten = 37) on the lateral cuticle of the abdomen. Each larva was wounded one on each side and exsanguinated. Hemolymph was directly collected by pipetting (approximately 40 μL). Hemolymph was diluted with 100 μL WH2 media; then, hemocytes were pelleted by centrifuging at 1000× g for 10 min. Supernatant was removed, and hemocytes were suspended in 100 μL WH2 media. Then, 800 μL of TRIzol® reagent (Invitrogen) was added to the hemocytes, and they were stored at −80 °C until RNA extraction.To test for virus association with carcasses versus hemocytes, an independent set of larvae was obtained from two different colonies, and sterile 18-guage needles were used to wound individual larva (6 cells) were pooled. Cell suspensions with approximately 106 cells per 300 μL were plated into each well of a 48-well plate and incubated at room temperature overnight before infection.Two days prior to honey bee pupal cell experiments, a brood frame containing purple-eyed pupae was brought into the lab. Fine-point, curved-tip forceps (Bioquip) were used to carefully remove the wax capping from pupae. Wide-tip, featherweight forceps (Bioquip) were used to gently grasp pupae between the eyes to remove them from the wax comb cells in which they develop. Pupae were incubated at 28 °C overnight in 12-well plates, and wounded  pupae were discarded. Primary cells were harvested from surface-sterilized honey bee pupae in a biosafety cabinet . Surface sterilization was carried out in a sterile polystyrene petri dish , in which pupae were swirled in 0.6% hypochlorite solution (diluted bleach) for 3 min, 70% ethanol for 3 min, and briefly in sterile water for injection (Gibco). In groups of two, pupae were dissected into head, thorax, and abdomen segments in 2 mL WH2 medium in a 47 mm dish using sterile 18-gauge needles to vigorously disturb tissues and release cells into the medium . The medg for 10 min at 4 °C, and the clarified lysate was sequentially filtered through a 0.45 μm filter and then a 0.2 μm filter to remove small debris and microbes. To characterize this virus preparation, RNA was isolated from 100 μL using TRIzol® reagent (as described below), and cDNA template was used for pathogen-specific PCR and qPCR. PCR confirmed the presence of SBV and the absence of other common honey bee viruses, including ABPV, BQCV, CBPV, DWV, IAPV, KBV, and LSVs  using a sterile steel ball (5 mm) in a 2 mL microcentrifuge tube, using a Tissue Lyser II (Qiagen) at 30 Hz for 2 min. Cell debris was pelleted by centrifugation at 10,000× and LSVs . Quantit7 DWV RNA copies in 2 μL) between the 2nd and 3rd integuments of the abdomen using the borosilicate glass needle and a Harbo syringe (Honey bee Insemination Service). Pupae were incubated at 30 °C in a humid incubator over the course of infection and examined daily to ensure they were alive. Live pupae were harvested at 10 days post-injection, and individual pupae were homogenized in 1 mL PBS (pH 7.4) using a sterile steel ball (5 mm) and Tissue Lyser II (Qiagen) at 30 Hz for 2 min. Cell debris was pelleted by centrifugation at 10,000× g for 10 min at 4 °C. To characterize this virus preparation, RNA was isolated from 100 μL using TRIzol® reagent (Invitrogen) (as described below), and cDNA template was used for pathogen-specific PCR and qPCR. PCR confirmed the presence of DWV and the absence of other common honey bee viruses, including ABPV, BQCV, CBPV, IAPV, KBV, LSVs, and SBV  were inoculated with 4.3 × 106 DWV RNA copies.DWV inoculum was prepared according to standard methods . In brie and SBV . QuantitDrosophila melanogaster Schneider 2 (S2) cells (Invitrogen), an immortalized cell line originally derived from D. melanogaster embryos. S2 cells were grown in Schneider’s Drosophila medium supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies) and 1% penicillin-streptomycin. A sterile T75 flask was seeded with 4 × 107 S2 cells/mL and infected with FHV at a multiplicity of infection of 1 pfu/cell, as provided by Dr. Anette Schneemann . The virus stock was quantified via plaque assay by the Schneemann lab using previously described protocols [v/v) Nonidet P-40, and incubated on ice for 10 min with periodic swirling [g for 10 min at 4 °C, and the clarified supernatant was transferred to a fresh tube. Using an ultracentrifuge (Beckman Coulter), virus was pelleted through 1 mL volume of 30% (wt/wt) sucrose in 50 mM HEPES (pH 7) at 40,000 rpm for 2.5 h at 11 °C in a SW41 Ti swinging-bucket rotor. The resulting pellets were resuspended in 0.5 mL buffer , and any remaining insoluble material was removed by centrifugation at 10,000 rpm for 10 min. The clarified supernatant was layered on a continuous sucrose gradient ) and centrifuged at 40,000 rpm for 1.5 h at 11 °C to sediment the virus between 25–35% sucrose gradients. RNA was isolated from 100 µL virus preparations (as described below), and FHV abundance was quantified by qPCR, with the copy number based on a standard curve. For the infection experiments described herein, hemocytes were infected with 1 × 106 FHV RNA copies, and pupal cells were infected with 2 × 108 FHV RNA copies. FHV infection experiments were monitored over a time course from 0 h to 96 h post-infection.FHV was propagated in rotocols . The S2 swirling . Cell de6 cells per 300 μL media and incubated at room temperature overnight before infection. Each well was infected with 10 µL of mock- or virus-infected pupal cell culture. The resulting infected cells were incubated at room temperature for 0 h, 48 h, and 72 h post-infection.Small amounts of virus-infected cell culture, including cells and culture supernatant, from initial infections were transferred to naïve pupal cells, seeded in 48-well plates as described above. Primary purple-eyed honey bee pupal cells were seeded at approximately 10® reagent (Invitrogen) according to manufacturer’s instructions. In brief, cell culture medium from each well (300 µL) was collected into a microcentrifuge tube. Then, 300 μL of TRIzol® reagent (Invitrogen) was added to each well, incubated for 1 min, and then combined with the corresponding cell culture media sample. Samples were stored at −80 °C until RNA extraction. To complete RNA isolation, an additional 450 μL of TRIzol was added to each sample, which was vortexed and incubated at room temperature for 5 min. Then, 160 μL of chloroform was added, and samples were shaken by hand (15 s) and incubated at room temperature (2 min). Samples were centrifuged at 12,000× g for 15 min, and the aqueous phase was transferred to a clean microcentrifuge tube. An equal volume of isopropanol (~550 μL) and 20 μg of glycogen (Thermo Scientific) were added to the aqueous phase, which was then mixed by inverting. RNA was precipitated after overnight incubation (~18 h) at −20 °C by centrifugation at 4 °C for 15 min at 14,000× g. Supernatants were carefully removed by pipetting, and each pellet was washed twice with 75% ethanol. Final pellets were air-dried at room temperature until visually dry (~20 min). The pellet was suspended in 30 μL nuclease-free water; then, 3 μL 3M sodium acetate (pH 5.5) and 120 μL ethanol were added, and RNA was precipitated overnight at −20 °C. Next, samples were centrifuged at 14,000× g for 15 min, and supernatants were carefully removed by pipetting. Each pellet was washed twice with 75% ethanol. Finally, RNA pellets were air-dried at room temperature until visually dry (~20 min) and then dissolved in 20 μL nuclease-free water. RNA concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher).RNA was isolated from cultured adherent hemocytes or non-adherent pupal cells/cell culture media at indicated time points using TRIzol2O was added to the exsanguinated carcass. The carcass was homogenized with a sterile steel BB using a TissueLyzer (Qiagen) at 30 Hz for 2 min. Then, 800 μL TRIzol® reagent (Invitrogen) was added to the lysate and vortexed and incubated at room temperature for 5 min; 200 μL of chloroform was then added. The tubes were shaken by hand for 15 s and incubated at room temperature for 2 min. The samples were then centrifuged for 15 min at 4 °C at 12,000× g. The aqueous phase was transferred to a clean microcentrifuge tube (approximately 800 μL), one volume of isopropanol was added, and tubes were mixed by inverting. Samples were incubated at −20 °C for one hour and then centrifuged for 10 min at 4 °C at 12,000× g. The supernatant was decanted, and the pellet was washed by adding 500 μL 75% ethanol and centrifuging for 5 min (7500× g) at 4 °C. The supernatant was decanted, and pellets were air-dried at room temperature. Pellets were suspended in 100 μL of dH2O.To extract RNA from larval carcasses, 300 μL of deionized HReverse transcription (RT) reactions to produce complementary DNA (cDNA) were performed by incubating 100–250 ng total RNA for cultured cell experiments, 200 units M-MLV reverse-transcriptase (Promega), and 500 ng random hexamer primers (IDT) for 2 h at 37 °C, according to the manufacturer’s instructions. For larvae/hemocyte assays, 2000 and 1000 ng total RNA, respectively, were used for RT reactions.Virus-treated samples  were analyzed for the presence of the negative-strand RNA of the viral genome  using strand-specific PCR ,179,180.PCR was performed according to standard methods ,63,64,97rpl8), and three immune genes  using the primer sets listed in 2. Reactions were carried out in 96-well plates using a CFX Connect Real-Time instrument using Maestro software (Bio-Rad) with the following thermo-profile: initial denaturation at 95 °C for one minute; followed by 40 cycles of 95 °C for 10 s, 58 °C for 20 s, and 72 °C for 15 s, with a final melt curve analysis at 65 °C for 5 s to 95 °C. Positive and negative control reactions were included for all qPCR analyses and exhibited the expected results. Viral RNA copies were estimated using a plasmid DNA standard for SBV, DWV, and FHV, with a detection range of 103 to 109 to create a linear standard curve used for copy number interpolation. The linear equation for the plasmid standard for FHV was Ct = −3.240x + 40.138 , determined by a previously described method [2 = 0.994, efficiency = 97.2%). The relative expression of host genes was determined by a ranked ΔΔCt method in which the ΔCt was calculated by subtracting the rpl8 Ct value from the Ct of the gene of interest. Then, the within-group ΔCt values were ranked from highest to lowest, and the relevant corresponding control ΔCt value was subtracted from the treatment group ΔCt to obtain the ΔΔCt. The fold-change in cDNA abundance was calculated by the equation 2−ΔΔCt. See Quantitative PCR (qPCR) was used to quantify the viral RNA  abundance in the samples and assess the relative abundances of honey bee encoded transcripts  in WH2 media. Live-cell and fixed-cell images were taken on a Nikon Ti-Eclipse (Nikon Instruments) inverted microscope equipped with a SpectraX LED (Lumencor) excitation module and fast-switching emission filter wheels (Prior Scientific). Brightfield images were captured using a Plan Fluor 20 phase contrast (Ph) objective and an iXon 896 EM-CCD (Andor Technology Ltd.) camera using NIS Elements software. To visualize cells with an intact nucleus, the cell culture media was removed, and the cells were washed with PBS and then incubated in 4% paraformaldehyde in PBS for 10 min at room temperature. The paraformaldehyde containing PBS was removed, and cells were washed two times with PBS. Hoechst stain  prepared to 30 mg/mL in PBS was further diluted 1:5000 in PBS to create a working solution, which was then overlaid on the cells. Then, fluorescent images were captured using paired excitation/emission filters and dichroic mirrors for DAPI (Chroma Technology Corp).DunnettTest function in the DescTools R stats package to perform the post hoc pairwise multiple comparisons to a control [p < 0.05.All data were analyzed using R v4.0.2 in RStudio V1.3.1073. Unless otherwise stated, comparisons for virus abundance were evaluated using the  control . Relativ control . For eac5 larval (L4–L5) hemocytes were seeded into wells of a 48-well tissue culture plate with relatively few contaminating adipocytes and treated with either phosphate buffered saline (PBS) or SBV  (p = 0.042) (p < 0.001), and it rose to 69.2× by 48 hpi (p < 0.001), to 147.9× by 72 hpi (p < 0.0001), and peaked at 354.8× at 96 hpi (p < 0.0001). In a second biological replicate of this experiment, confounding SBV infection was not detected (p = 0.0001), and by 48 hpi SBV had increased 182× (p < 0.0001) and reached peak infection. There was a decrease in SBV relative to 48 hpi at 72 hpi , with a subsequent rise at 96 hpi . Although it is unclear why there was a reduction in viral abundance at 72 hpi, it could have been due to a decrease in the number of permissive hemocytes in the culture; by 96 hpi, cell division and/or differentiation may have occurred and resulted in additional susceptible host cells.To determine whether sacbrood virus (SBV) infects hemocytes, the primary immune cell in honey bees, approximately 1 × 10 copies) . Cells w= 0.042) A. This rdetected B. In inoCrassostrea virginica) [Drosophila melanogaster remain uninfected [To further validate that the increase in SBV over the infection time course was due to active virus replication in honey bee hemocytes, we carried out strand-specific RT-PCR. The SBV genome is positive-sense single-stranded RNA (+ssRNA). During virus replication, a negative strand template is produced ,180,184.rginica) . In addirginica) . Resultsinfected . As descAmel\102, a gene involved in melanization and encapsulation [To our knowledge, this is the first quantitative evidence that SBV productively infects honey bee hemocytes. A previous study used transmission electron microscopy to visualize SBV virions within larval and adult hemocytes . While tsulation .Drosophila melanogaster. Direct evidence for an antiviral role for hemocytes in fruit flies includes the results of in vivo experiments in which hemocytes were either saturated via injection of latex beads or genetically depleted [In addition to hosting viral infection, honey bee hemocytes are likely instrumental in antiviral immune responses, as they are in depleted . Flies wdepleted . These rdepleted ,171. ThiWhile the function of hemocytes in honey bee antiviral response requires further investigation, there is limited indirect evidence of their antiviral role. Specifically, genes involved in migration, phagocytosis, and expression immunoglobulin domain encoding, were expressed at greater levels in honey bees infected with a model virus, SINV, relative to mock-infected bees . Additio6 cells were treated with either PBS or SBV . Cells were harvested at distinct time points post-infection, and virus abundance was assessed by qPCR. Three independent experiments were performed (p < 0.0001). SBV abundance remained high at 72 hpi  and 96 hpi  (p < 0.0001), remained high at 72 hpi , and peaked at 96 hpi  (p < 0.0001). Like the other two replicates, SBV levels increased by 48 hpi , remained high at 72 hpi , and peaked at 96 hpi  (p < 0.0001)), which was similar to replicate two, which had an overall 1047× increase, whereas the replication and relative increase in abundance for replicate one was lower   . Likewis 0.0001) . While S 0.0001) . The ovenly 72×) . To furtnly 72×) B. Togethprophenoloxidase activating enzyme (PPAE) and increased expression of a putative serpin protein encoding gene (GB48820) [Varroa destructor mites. It is not known whether SBV-infected pupae can clear infections prior to eclosion, and in turn if SBV-infected pupae can develop into healthy adults. Therefore, tools to study the impact of SBV and the molecular mechanisms of antiviral defense at all life stages are needed and include the SBV-permissive cultured primary cells described herein.While the primary concern of SBV infection of honey bee colonies is larval mortality, SBV also reduces adult lifespan. SBV infections in pupae may inhibit the melanization pathway by causing reduced expression of GB48820) ,145. Pup6 purple-eyed pupal cells were incubated with 4.3 × 106 DWV RNA copies. Cells were harvested at distinct time points post-infection , and virus abundance, using RNA copy number as a proxy, was determined by qPCR. Data from two independent experiments demonstrated that DWV abundance increased from 0 hpi to 96 hpi (p = 0.002). DWV abundance increased 3.5× and 4× at 72 hpi (p < 0.001) and 96 hpi (p < 0.001), respectively, with peak DWV levels at 96 hpi relative to 0 hpi (p = 0.001) and 6.6× higher at 96 hpi, relative to 0 hpi (p = 0.003)  . To veri= 0.003) B. TogethThe ability to infect primary honey bee cells with DWV opens new avenues for studying a pathogen that has co-evolved with honey bees. Primary cell cultures may also facilitate the propagation of virus stocks that are free of confounding viruses. Experiments in cultured honey bee cells also provide an opportunity to study the mechanistic details of DWV infections and virus–host dynamics in a system that complements experiments carried out in larvae, pupae, and adult honey bees. Recent efforts have focused on exploring the ability of alternative host cell lines to support honey bee virus infections; for example, the Lepidopteran hemocyte-derived cell line (P1) has been shown to support DWV infection . While e5 larval-derived hemocytes maintained in culture were incubated with PBS or FHV (1 × 106 FHV RNA copies). Cells were harvested at distinct time points post-infection , and virus abundance was determined by qPCR. Data from two independent experiments demonstrated that FHV abundance increased from 0 hpi to 72 hpi and decreased from 72 hpi to 96 hpi (p = 0.026) and 10.7× by 48 hpi, relative to 0 hpi (p < 0.001) (p < 0.001) the initial FHV level  (p = 0.034) but remained significantly higher relative to 0 hpi (p < 0.001) (p < 0.01) relative to 0 hpi. At 72 hpi, FHV levels peaked, with 39.8× higher FHV levels compared to 0 hpi (p < 0.001). FHV abundance was reduced at 96 hpi relative to 72 hpi but was still 12.6× higher that levels at 0 hpi (p < 0.001)  A. FHV abo 0 hpi) A. By 96 < 0.001) A. One po< 0.001) . To furt< 0.001) B. TogethThe ability to infect hemocytes with FHV provides an additional tool for investigating virus–honey bee host interactions at the cellular level. This model virus may prove particularly useful in investigating RNAi in the context of viral suppressors, since FHV-B2 protein suppresses RNAi, as confirmed by studies that utilized deletion mutants  ,191. To 6 primary cells in culture were incubated with FHV (2 × 108 FHV RNA copies), and virus abundance was assessed at distinct time points post-infection. FHV abundance increased from 0 hpi to 72 hpi and was reduced or remained constant at 96 hpi in two experimental replicates. In replicate one, FHV levels increased by 1.7× at 24 hpi (p = 0.024) and by 3.4× at 48 hpi, relative to initial levels at 0 hpi (p < 0.001). The peak of infection was reached at 72 hpi, with 5× greater FHV levels relative to 0 hpi (p < 0.001). FHV abundance in pupal cells did not increase from 72 hpi to 96 hpi but remained 4× higher than at 0 hpi (p < 0.001) (p = 0.013) and 7.5× higher at 48 hpi (p < 0.001), relative to levels at 0 hpi. FHV abundance increased 20× (p < 0.001) at 72 hpi relative to 0 hpi, which was the peak of virus infection. Similar to FHV infection in hemocytes, FHV levels in pupal cells was lower at 96 hpi relative to 72 hpi (p < 0.01) but was still 15.8× higher at 96 hpi compared to 0 hpi (p < 0.001)  A. Simila< 0.001) . To vali< 0.001) B. TogethThe ability to infect mixed cell types derived from honey bee pupae with a model virus helps address some challenges in studying honey bee host–virus interactions using naturally infecting honey bee viruses. FHV can be easily propagated and purified in the lab, which helps circumvent the problem of mixed virus inoculum produced from infected bees, since many honey bee viruses co-purify due to their similar size and the high prevalence of mixed infections in colonies ,63,64,978 FHV RNA copies) was transferred to freshly seeded pupal cells (p = 0.003) and at 72 hpi 28× greater than levels at 0 hpi (p < 0.001). In the second replicate, FHV-infected pupal cells  from the first round of infection were transferred to naïve pupal cells, and FHV abundance remained constant from 0 hpi to 48 hpi but was 10.4× higher at 72 hpi (p < 0.001)  was transferred to naïve pupal cells. Virus abundance in the secondary host cells was monitored over time. In replicate one, a small volume of FHV-infected pupal cell culture  . As expe< 0.001) . Togethe< 0.001) A.5 DWV RNA copies. By 48 hpi, DWV abundance increased 2.7× relative to 0 hpi (p < 0.001). DWV levels continued to increase over time, with 4.1× increase at 72 hpi (p < 0.001) (4 DWV RNA copies). DWV abundance increased by 2.3× at 48 hpi (p < 0.01) and was 10.4× greater at 72 hpi, relative to 0 hpi (p < 0.001)  from either mock-infected or DWV-infected cultures, which contained an equivalent of 1.04 × 10< 0.001) B. In rep< 0.001) . As expedicer-like (dcr-like), argonaute-2 (ago2), and bee antiviral protein-1 (bap1), which was formerly designated as GenBank MF116383, were analyzed by qPCR in either two or three experimental replicates  ,146,190.plicates .bap1 (GenBank: MF116383) was greater in SBV-infected cells than mock-infected pupal cells at 72 hpi. Specifically, in first biological replicate, bap1 expression was 0.67 log2-fold greater in SBV-infected pupal cells relative to the expression level in mock-infected cells      and unchanged in the second replicate, compared to mock-infected cells    and dcr-like expression  than mock-infected hemocytes in both biological replicates  . Likewis= 0.032) . Express= 0.032) . Similar= 0.032) . In DWV-eplicate  and sligal cells . Specified cells . Interes= 0.032) . Similar= 0.032) . The tre= 0.032) . Specifiplicates .Together these experiments indicate that studies in cultured honey bee primary cells recapitulate data obtained from transcriptome level studies in individual honey bees, including those carried out in pupae and adult bees , though dcr-like, ago2, and bap1). These cultures can be established using rather simple techniques that are amenable to higher sample size studies. Future work to optimize transfection in these different cell culture types will be paramount for performing more advanced mechanistic studies. This includes utilizing RNAi-mediated gene knockdown, which facilitates the study of honey bee host–virus interactions by directly demonstrating the importance of specific genes in limiting particular viruses. For example, this study indicates that hemocytes are a natural site of infection for SBV and that SBV results in higher bap1 expression in infected pupal cells. By reducing the expression of bap1 in cell cultures that are infected with SBV, one could test the hypothesis that this gene is important for cells to combat SBV infection. Additionally, the use of cell culture will facilitate the study of basic virological questions, including virus cellular tropism, entry, and uncoating mechanisms. Additional efforts to establish virus-free cell lines from explanted honey bee tissues will prove useful for the production of pure virus stocks, especially in concert with the development of cDNA clones capable of producing infectious virus. The data presented in this study represent one of many steps toward the development of sophisticated cell culture techniques for the study of honey bee host–virus interactions.In summary, these data indicate that primary honey bee cell cultures of larval hemocytes and explanted pupal tissue serve as a useful tool for studying honey bee–host interactions. In addition to demonstrating that a panel of viruses  replicates in hemocytes and/or pupal cells, pupal cell cultures showed virus-specific transcriptional regulation of three immune genes (


Chloride nutrition at macronutrient levels reduces stress symptoms and enhances plant growth during water deficit, promoting water deficit avoidance (water-use efficiency) and tolerance (osmoregulation) mechanisms that improve drought resistance. −), traditionally considered harmful for agriculture, has recently been defined as a beneficial macronutrient with specific roles that result in more efficient use of water (WUE), nitrogen (NUE), and CO2 in well-watered plants. When supplied in a beneficial range of 1–5 mM, Cl− increases leaf cell size, improves leaf osmoregulation, and reduces water consumption without impairing photosynthetic efficiency, resulting in overall higher WUE. Thus, adequate management of Cl− nutrition arises as a potential strategy to increase the ability of plants to withstand water deficit. To study the relationship between Cl− nutrition and drought resistance, tobacco plants treated with 0.5–5 mM Cl− salts were subjected to sustained water deficit  and water deprivation/rehydration treatments, in comparison with plants treated with equivalent concentrations of nitrate, sulfate, and phosphate salts. The results showed that Cl− application reduced stress symptoms and improved plant growth during water deficit. Drought resistance promoted by Cl− nutrition resulted from the simultaneous occurrence of water deficit avoidance and tolerance mechanisms, which improved leaf turgor, water balance, photosynthesis performance, and WUE. Thus, it is proposed that beneficial Cl− levels increase the ability of crops to withstand drought, promoting a more sustainable and resilient agriculture.Chloride (Cl The resulting enlargement of leaf cell size reduces the stomatal density, which in turn lowers stomatal conductance (gs) and water consumption. Secondly, Cl− also increases mesophyll diffusion conductance to CO2 (gm), which makes it possible to maintain the plant photosynthetic capacity despite the reduction of gs, resulting in overall higher WUE in well-watered plants  quantifying the degree of WD resistance of Cl−-treated plants compared with plants treated with equivalent concentrations of anionic macronutrients such us NO3−, phosphate, and sulfate; and (ii) identifying relevant physiological mechanisms regulated by Cl− nutrition that improve WD resistance in plants.Cl closure . TherefoNicotiana tabacum L. var. Havana) plants were grown under greenhouse experimental conditions . Plants were grown in 7.5 liter pots (20×17×25 cm) containing a mix of perlite:vermiculite (4:6), and watered with a basal nutrient solution supplemented with three nutritional treatments: 5 mM Cl− salts (CL), 5 mM NO3− salts (N), and a mix of sulfate+phosphate (SO42−+PO43−) salts (SP), as previously reported in −: 2.5 mM KCl, 0.625 mM MgCl2, and 0.625 mM CaCl2. To evaluate the specificity of Cl− in the studied phenomena, two additional treatments were used: N treatment containing 2.5 mM KNO3, 0.625 mM Mg(NO3)2, and 0.625 mM Ca(NO3)2; and SP treatment containing 1.25 mM KH2PO4, 0.625 mM K2SO4, 0.625 mM MgSO4, and 0.625 mM CaSO4. All treatments  contained the same cationic balance as shown in 3, 0.625 mM KH2PO4, 0.053 mM K2HPO4, 2 mM Ca(NO3)2, 1 mM MgSO4, 0.1 mM FeNa-EDTA, 0.1 mM H3BO3, 0.1 mM MnSO4, 29 μM ZnSO4, 0.11 μM CoCl2, 53 μM KCl, 0.1 μM CuSO4, 1 μM Na2MoO4, and 5 μM KI. All experimental solutions were adjusted to pH 5.7 with KOH.Tobacco (−1 substrate) throughout the experiment, and WD, with pots irrigated every 2–3 d up to 60% of field capacity (2.1 ml g−1 substrate) for 20 d (65 DAS). During the WD treatment, the resulting average soil water content ranged between 60% and 10% of field capacity , in addition to the three nutritional treatments, plants were subjected to two irrigation treatments: optimal irrigation , in which pots containing tobacco plants were irrigated up to 100% field capacity  as explained above, was performed for 26 d under similar experimental conditions to those previously described  was calculated as follows  and the concentration of Clπ), leaf Ψ w, and leaf turgor (or pressure) potential (Ψ p) were determined as previously described in Leaf water content, RWC, succulence, leaf osmotic potential (Ψ i) was calculated as the increase of plant DW over time related to the accumulated water consumption (g DW ml−1 H2O), as well as the DW obtained throughout the experiment and after harvesting related to total water consumption (g DW ml−1 H2O)  were maintained under CTR conditions up to 73 DAS, when water deprivation was applied for 4 d, and, at 77 DAS, plants were rehydrated at 100% of field capacity and monitored until 80 DAS. Three plants from each nutritional treatment were monitored every day by gravimetric methods to verify the water content in the soil, and PSII quantum yield (Qy) measurements were performed. For Qy determination, chlorophyll fluorescence in light-adapted plants was measured using a portable fluorometer , as described in Pp) is recorded in a leaf that is patched between a metallic sensing chip and a magnetic pad. Pp is inversely correlated with the leaf turgor pressure , a non-invasive technique that records leaf turgor pressure in real-time  and stomatal conductance (gs) were measured between 12.00 h and 14.00 h using an open gas exchange system  equipped with a 2×3 cm LED chamber (LI-6400-02B) as described in i was calculated as the ratio between the rate of photosynthesis and stomatal conductance (AN/gs).Net photosynthetic rate  differences were indicated when P was >0.05. Multiple comparisons of means were determined by the Tukey’s HSD (honestly significant difference) and MRT (multiple range test) statistical tests included in the mentioned software. Analysis of covariance (ANCOVA) was performed with R software (https://www.r-project.org/) to compare the slopes of the relationship of total biomass with water consumption between CL and SP treatments. Values represent the mean of at least six tobacco plants in each treatment, which were reproduced in at least three independent experiments . Shapiro–Wilk (W) test was used to verify the normality of the data sets. One-way ANOVA and multivariate analysis of variance (MANOVA) were performed to determine significant differences between groups of samples, and levels of significance were described by asterisks: *eriments .− nutrition participates in plant adaptive responses to drought stress, greenhouse experiments were performed under two irrigation regimes: optimal irrigation (CTR) and sustained water deficit (WD). For the WD treatment, plants were watered every 2–3 d with the three nutritional treatments  until the substrate reached 2.1 ml g−1 (60% of field capacity). The WD treatment was maintained for 20 d, whereas watering up to 100% of field capacity was established for the CTR treatment (3.5 ml g−1). The time course of the substrate water loss throughout a representative experiment is presented in To study whether Cl− treatment (CL) on plant growth were consistent with those previously obtained in − (SP and N treatments). Consistently, leaf anion contents  were differentially accumulated in plants according to the respective nutritional treatments  under both irrigation regimes (− and NO3− contents in tobacco leaves (− concentration in CL-treated leaves was 106.5 mM (i.e. 55.7 mg g−1 DW), reaching typical macronutrient levels. In SP and N plants, Cl− content was 100 times lower, although far exceeding the critical levels of deficiency required to fulfil essential micronutrient functions , whereas no changes were observed in N plants. In addition, the NO3− concentration was strongly decreased by WD in SP and N plants , whereas no relevant changes in SO42− and PO43− contents were observed  than SP and N treatments  during WD . Under control conditions, N plants showed the significantly highest leaf area due to the occurrence of larger and more numerous leaves, while CL plants presented higher leaf area than SP plants  gradually increased during the day, indicating turgor loss after sunrise, and abruptly decreased during sunset, indicating leaf turgor recovery during the night. Although some differences in amplitude were found between treatments, the kinetics of the Pp curves from different plants showed the same circadian trends. Irrigation with the three nutritional treatments  was withheld for 4 d until the water content of drought-stressed pots reached between 10% and 20% of the water content measured in well-watered pots. Subsequently, irrigation was restored to control water levels. After WD imposition, strong loss of turgor  was observed in plants subjected to SP and N treatments. However, turgor values were not significantly altered by WD in CL plants, which maintained a Pp pattern similar to that of well-watered plants  and were compared with equivalent gradients of SO42− and PO43− (SP) salts. With this aim, we normalized each measurement obtained under WD conditions with respect to the CTR in each nutritional treatment and, in turn, CL was also normalized to SP plants to show the percentage of improvement induced by Cl− during WD. In − treatments, beyond 1 mM Cl−. In this work, after 26 d under sustained WD, both 2 mM and 5 mM Cl− treatments significantly induced plant growth and leaf area when compared with SP plants  could stimulate crop yield  compared with SP and CL treatments , non-protein antioxidants (e.g. polyamines), and drought-induced proteins ; and (iii) the stored NO3− being used in maintaining the plant growth  increased sensitivity to WD, with a strong reduction of growth and WUE compared with CL and SP plants , which implies strong dynamic interactions between the two monovalent anions in plants , nitrogen (NUE), and CO2. Thus, our results showed that Cl− treatments (2–5 mM) reduced stress symptoms and allowed continued growth in tobacco plants during WD as a result of two main mechanisms  impThe following supplementary data are available at Fig. S1. Time course of substrate water loss.− nutrition and WD on plant growth.Fig. S2. Images showing effects of Cl− nutrition and sustained WD on plant and leaf growth.Fig. S3. Effect of ClFig. S4. Relationship between total biomass and water consumption.AN and RWC.Fig. S5. Relationship between − nutrition and sustained WD on leaf biomass, RWC, and WUEi in tomato plants.Fig. S6. Effect of Cl− nutrition in different physiological processes.Table S1. List of experiments conducted between 2010 and 2018 to characterize the role of ClTable S2. Anion content in leaves subjected to different nutritional and irrigation treatments.Table S3. Osmotic potential calculated from ion concentration measured in mature leaves.erab143_suppl_Supplementary_Figures_and_TablesClick here for additional data file.


Panax notoginseng. Despite the NGR1 having been used as a traditional medicine, little is known about the neuroprotective effects. In this study, we investigate the protective effects of NGR1 against glutamate-induced cytotoxicity in HT22 cells and its possible molecular mechanism. We assessed the toxicity of NGR1 and the protective activity by MTT assay. The levels of oxidative stress indices superoxide dismutase (SOD), glutathione (GSH), and mitochondrial membrane potential (MMP) were measured by the kits. The levels of reactive oxygen species (ROS) and Ca2+ concentration were measured by flow cytometry. Furthermore, we determined the expression of mitochondrial dysfunction related protein PINK1, Parkin, silent mating type information regulation 2 homolog-1 , and Wnt/β-catenin by Western blotting. Here, we discovered that glutamate treatment led to cell viability loss, apoptosis facilitation, Ca2+ upregulation, MMP fluorescence intensity downregulation, and ROS generation of HT22 cells. In parallel, expression of Parkin was declined by glutamate. While, NGR1 treatment alleviated all the above phenomena. We further clarified that NGR1 alleviated glutamate-induced oxidative stress, apoptosis, and mitochondrial dysfunction by upregulating SIRT1 to activate Wnt/β-catenin pathways. These findings demonstrate that NGR1 alleviated glutamate-induced cell damage, and NGR1 may play a protective role in neurological complications.Notoginsenoside R1 (NGR1) is an active compound isolated from  Oxidative stress caused by the accumulation of reactive oxygen species (ROS) is related to the development of many neurological complications , 2. NormPanax notoginseng [Notoginsenoside R1 (NGR1) is an active compound isolated from oginseng , 6. It hoginseng . Previouoginseng . In diaboginseng . Howeverβ-catenin pathway.Glutamate is the main excitatory neurotransmitter in the brain and is considered to be one of the initiating factors of neuronal damage –12. In oβ-catenin agonist SKL2001 (S8302) was brought from Selleck Chemicals . Superoxide dismutase (SOD), lactate dehydrogenase (LDH), and glutathione (GSH) assay kits were brought from Nanjing Jiancheng Bioengineering Institute . Primary antibodies against Parkin, PINK1, SIRT1, Wnt1, β-catenin, cyclin D1, GAPDH, and secondary antibodies were all brought from Cell Signaling Technology .L-glutamic acid, notoginsenoside R1 (NGR1), 98% (HPLC), 2′, 7′dichlorodihydrofluorescein diacetate (H2DCFDA), MTT, and nicotinamide were brought from Sigma-Aldrich . Wnt/2 in a humidified incubator. When cell confluency reached 80%, cells were passaged. All experiments were performed on third passage cells and next, treated with 5 mM glutamate and different concentrations of NGR1.Mouse hippocampal HT22 cells were bought from the Procell Cell Bank . HT22 cells were cultured in the DMEM with 10% fetal bovine serum , supplemented with penicillin-streptomycin  at 37°C with 5% CO4 cells/cm2, according to the manufacturer's instructions. Cells were treated with 5 mM glutamate and different concentrations of NGR1 for 24 h. After treatment, MTT solution (50 μL) was added to the cell culture medium at 37°C for 4 h and added DMSO to plates. Absorbance was measured at 570 nm using a microplate reader. The experiment was repeated three times.Cell viability was detected by the MTT assay. We followed the methods of Wang et al. . Brieflyβ-catenin (1 : 1000), and GAPDH (1 : 2000). Then, they were incubated with diluted secondary antibodies (1 : 3000). followed by immunodetection using an enhanced chemiluminescence kit  and analyzed with Image J software .In the HT22 cells after treatment, add RIPA buffer  to collect total protein, and the concentration of protein was determined by a bicinchoninic acid protein assay kit . An equivalent of total protein was resolved on SDS-PAGE and transferred to the polyvinylidene fluoride membrane. Next, the membranes were blocked with 5% nonfat milk and probed with the following primary antibodies overnight at 4°C : SIRT1 (1 : 1000), PINK1(1 : 1000), Parkin (1 : 1000), Wnt1 (1 : 1000), LDH activity was detected by LDH assay kit. Briefly, cells were collected after glutamate and NGR1 treatment, and separate the supernatant; next, cells' supernatant was incubated with 2,4-dinitrophenylhydrazine; the release of LDH was examined by measurement of the absorbance at 450 nm. The experiment was repeated three times.4 cells/well) were treated by NGR1, glutamate, or both. 10 μM H2DCFDA was added into cells and reacted for 20 min at 37°C in the dark. HT22 cells were washed with the serum-free medium for three times. The ROS generation was examined by the flow cytometer .HT22 cells in 6-well plates  working solution for 15 min (in dark). Finally, the fluorescence intensity was measured by flow cytometry .HT22 cells were treated by NGR1, glutamate, or both. Collect cells and incubate with 5 We followed the methods of Parinee et al. . MMP wast-test. Statistical analysis was performed using GraphPad Prism 7.0 software. A value of P < 0.05 was considered as statistically significant .In our study, all experimental data are presented as mean ± standard error of mean. The difference between two groups was compared by using the μM NGR1 as optimum dosage for the following experiments . Simultaneously, treatment with 50 μM NGR1 significantly inhibited Glu-induced LDH leakage  to treat cells. The result showed that viability of HT22 cells was unchanged by nicotinamide (20 μM) treatment alone. However, treatment with nicotinamide (20 μM) significantly reduced the cell viability of HT22 cells treated with NGR1  inhibited the effect of nicotinamide  in nerve cells is associated with neurological complications, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and neuropathic pain –17. The n nerves , there is system –23. The s system , 24. Ears system . In the s system . In addi2+ concentration, reduced mitochondrial membrane potential, and ultimately leads to cellular apoptosis [2+ concentration in HT22 cells [2+ concentration. When mitochondria are damaged, activated PINK1 aggregates on the outer mitochondrial membrane and induces mitochondrial autophagy through direct/indirect activated Parkin [2+ concentration and increases the level of mitochondrial membrane potential in HT22 cells.Glutamate induces the further development of oxidative stress in HT22 cells, leads to cell mitochondrial dysfunction, increased Capoptosis . Mitochopoptosis . Plant e22 cells . In thisd Parkin , therebyd Parkin . In thisβ-catenin signaling system is an important pathway in the development of the CNS [β-catenin signaling pathway, can increase the transcriptional activity of Wnt1 and β-catenin by affecting the stability of c-myc, promote the nuclear transfer of β-catenin, and activates the Wnt/β-catenin signaling pathway [β-catenin signaling pathway participates in the development process of neurological diseases [β-catenin, and cyclin D1. NGR1 treatment upregulated the expression of SIRT1, Wnt1, β-catenin, and cyclin D1. Furthermore, the effects of SIRT1 inhibitor nicotinamide and Wnt/β-catenin inhibitor SKL2001 both affect the regulation of SIRT1/Wnt/β-catenin by NGR1.As the main regulator of neurological diseases, SIRT1 plays a protective role in neurological diseases by regulating inflammation, oxidative stress, and cell apoptosis . Previou the CNS . Previou pathway , 34. Mordiseases , 36. In β-catenin pathway. However, one limitation should not be ignored in this study; it is necessary to further verify the mechanism of NGR1 regulation in animal models of neurological diseases.In summary, this study demonstrated that NGR1 plays a neuroprotective role in glutamate-induced cytotoxicity. To the best of our knowledge, this is the first report to show NGR1-protecting HT22 cells against glutamate-induced cytotoxicity by upregulating SIRT1 to activate the Wnt/


Neisseria meningitidis is a gram-negative bacterium that often asymptomatically colonizes the human nasopharyngeal tract. These bacteria cross the epithelial barrier can cause life-threatening sepsis and/or meningitis. Antimicrobial peptides are one of the first lines of defense against invading bacterial pathogens. Human beta-defensin 2 (hBD2) is an antimicrobial peptide with broad antibacterial activity, although its mechanism of action is poorly understood. Here, we investigated the effect of hBD2 on N. meningitidis. We showed that hBD2 binds to and kills actively growing meningococcal cells. The lethal effect was evident after 2 h incubation with the peptide, which suggests a slow killing mechanism. Further, the membrane integrity was not changed during hBD2 treatment. Incubation with lethal doses of hBD2 decreased the presence of diplococci; the number and size of bacterial microcolonies/aggregates remained constant, indicating that planktonic bacteria may be more susceptible to the peptide. Meningococcal DNA bound hBD2 in mobility shift assays and inhibited the lethal effect of hBD2 in a dose-dependent manner both in suspension and biofilms, supporting the interaction between hBD2 and DNA. Taken together, the ability of meningococcal DNA to bind hBD2 opens the possibility that extracellular DNA due to bacterial lysis may be a means of N. meningitidis to evade immune defenses. Neisseria meningitidis is a strictly human pathogen that inhabits the nasopharynx asymptomatically in approximately 10% of the population  are highly conserved molecules that are found in mammals, insects and plants. They are relatively small peptides that are often positively charged and amphipathic in nature. They constitute the first line of defense against invading pathogens and contribute to eliminating infection by direct killing of microbes . BacteriEscherichia coli, Porphyromonas gingivalis, and Pseudomonas aeruginosa (Human beta-defensin 2 (hBD2) is an AMP belonging to the defensin family. Defensins are characterized by a beta sheet structure stabilized by three disulfide bridges  and are ruginosa . While truginosa , the comN. meningitidis. We found that hBD2 binds to and kills N. meningitidis in a time-dependent manner. Planktonic bacteria disappeared upon hBD2 treatment, suggesting that bacterial aggregates are better protected against hBD2 killing. Furthermore, we found that meningococcal DNA binds to hBD2 and that the presence of DNA protects against the peptide, suggesting a potential evasion strategy employed by N. meningitidis against hBD2-mediated killing both in suspension and in biofilms.In this work, we investigated the effect of hBD2 on Neisseria meningitidis serogroup C strain FAM20 and its pilE mutant (ΔpilE) have been previously described (2 for 16-18 h before experiments. Lactobacillus reuteri ATCC PTA5289 (2 on Rogosa (Oxoid) agar for 30-72 h and then in MRS broth for 16-18 h. Escherichia coli DH5α was grown on LA-plates and then in LB broth (Acumedia) at 37°C 5% CO2 at 200 rpm shaking.escribed . The strescribed  at 37°C  PTA5289  was grow2 in a humidified environment.The human pharyngeal epithelial cell line FaDu (ATCC-HBT43) was cultured in Dulbecco’s modified Eagle’s medium (DMEM)  and supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich) at 37°C and 5% COSynthetic hBD2 peptide (GIG DPV TCL KSG AIC HPV FCP RRY KQI GTC GLP GTK CCK KP) was synthesized by Innovagen, Lund, Sweden. hBD2 was dissolved in sterile water at a concentration of 0.4 mg/ml and stored at −80°C.2 for 2 h under shaking conditions. Log phase bacteria were suspended to 106 CFU/ml in modified Dulbecco’s Modified Eagle’s Medium , i.e., DMEM diluted 4× in sterile water, and then mixed with hBD2. It is known that the killing capacity of hBD2 is reduced in the presence of serum and high salt concentration  and incubated in polypropylene tubes for 1, 2, 3, or 6 h at 37°C and 5% CO2 under shaking conditions. Sterile water instead of hBD2 served as the control. Bacterial viability was assessed by serial dilutions, which were plated for viable counts and enumerated the next day. Samples were carefully vortexed and pipetted up and down and examined by microscopy before plating to check for absence of aggregates.Bacteria from plates were grown to log phase in GC broth supplemented with 1% Kellogg’s at 37°C and 5% COntration ; for thi6 CFU/ml, and incubated as in the killing assays above. For the binding assay, bacteria were incubated in either 0.1 μM or 1 μM hBD2 for 3 h. Samples were washed once in PBS and resuspended in PBS. Bacteria-bound hBD2 was detected using an hBD2 ELISA development kit (Peprotech) according to the manufacturer’s instructions.Bacteria from plates were grown to log phase, diluted to 10g for 3 min and resuspended in 5 mM HEPES buffer to 107 CFU/ml, and 50 μl of the bacterial suspension was added to the microplate. The plate was incubated at 37°C, and the fluorescence was immediately measured every minute for 3 h using a Spectramax i3x microplate reader (Molecular Devices) with an excitation wavelength of 485 nm and emission wavelength of 535 nm.To measure hBD2-induced permeabilization of the outer membrane, we used a previously described protocol . This me6 CFU/ml, and incubated as in the killing assays above. For flow cytometry, bacteria were incubated in either 0.1 μM hBD2, 1 μM hBD2, 2 μg/ml tetracycline or 10 μg/ml cephalexin for 2 h. Samples were washed once in PBS before fixation in 4% formaldehyde solution. Cells were permeabilized with 0.1% Triton X-100 and resuspended in 50 mM sodium citrate buffer. Samples were stained with 2.5 μM SYTOX Green (Thermo Fisher Scientific) before being analyzed by flow cytometry using an LSRFortessa flow cytometer (BD Biosciences). For each sample, 30 000 cells were counted. Data were analyzed with FlowJo software.Bacteria from plates were grown to log phase, diluted to 106 CFU/ml, and incubated as in the killing assays above. For microscopy, bacteria were incubated in 0.1 μM hBD2, 1 μM hBD2, or 10 μg/ml cephalexin for 2 h. Samples were washed once in PBS before fixing in 4% formaldehyde solution. Cells were permeabilized with 0.1% Triton X-100 and resuspended in PBS supplemented with 0.2% Tween-20. Cells were stained with 20 nM DAPI for 30 min and resuspended in PBS. A small volume of cells was mounted on 1% agarose pads and sealed under a coverslip. Images were obtained using a Ti eclipse inverted research microscope with a 100×/1.45 NA objective (Nikon). Fluorescence images were captured using excitation and emission filter cubes for Hoechst 33258. Image preparation was performed using Fiji software. The assay was performed two times with at least 12 images captured per sample. Aggregates were enumerated by counting and were defined as a cluster consisting of more than 4 cells to exclude the possibility of diplococci undergoing division. The relative number of aggregates was calculated as the number of aggregates divided by the number of diplococci.Bacteria from plates were grown to log phase, diluted to 10600 of 0.1 and incubated for 3 h at 37°C and 5% CO2 under shaking conditions. Bacterial cultures were centrifuged at 2500 × g for 5 min, and the pellet was resuspended in an equal volume of the modified DMEM used for the killing assays. Bacterial suspensions were incubated at room temperature under static conditions, and the OD600 of the supernatant was measured at 20 min intervals. Absorbance values were normalized against the first time point (0 min).To quantify aggregation of the bacterial strains, we used a previously described sedimentation assay  with sliN. meningitidis FAM20, a 1786 bp non-coding DNA fragment was amplified using the primers: F20_non-coding_fwd, 5′-GGAACCGGTTTGATGTCCATTT-3′, and F20_non-coding_rev, 5′-GCTATTGCATGCGCTTAATGA-3′. For E. coli, a 1702 bp fragment was amplified using the primers: 5′-AGCCTTATTCTTCATCGTTTTT-3′ and 5′-CATCATTGAGTGCGGCATTTTC-3′. For L. reuteri, fragments of 1610 bp (5′-GTTAGGTTGAGGGCCAGGTT-3′ and 5′-GTTGGGTTGGAGGGGTAGTT-3′) and 1791 bp (5′-AATGGACGCTAAAATGGAAGT-3′ and 5′-AGGGGAAAAGAATATGAGAAAT-3′) was amplified. For preparation of eukaryotic DNA, human pharyngeal epithelial FaDu cells were trypsinized, centrifuged at 8000 g for 10 min, washed with PBS and resuspended in TE buffer with 0.1% Triton x100 (Sigma-Aldrich). The cell suspension was boiled at 100°C for 5 min and then centrifuged at 13000 g for 10 min. The supernatant was collected and stored at −20°C or used immediately for amplification. A 1735 bp fragment was amplified using primers (5′-CAGTTGGTGCGGGAAGGAGT-3′ and 5′-GGAAGAGAAGGAAAGGAGGGCT-3′).Bacterial genomic DNA (gDNA) was isolated using the Wizard Genomic DNA Purification kit (Promega) according to the manufacturer’s instructions. PCR fragments were amplified with Phusion DNA polymerase (Thermo Fisher Scientific). For Two hundred ng of purified DNA fragments was incubated with hBD2  or BSA  in binding buffer , 1 mM EDTA, 1 mM dithiothreitol, 20 mM KCl, 50 μg/ml BSA). The reaction mixture was incubated at room temperature for 1 h and subjected to 1% agarose gel electrophoresis. Gels were stained with ethidium bromide, and images were captured using a Gel Doc 2000 Imaging System (Bio-Rad).N. meningitidis, we used a previously described protocol  at a 1:1 ratio in black-walled, clear bottom microplates. Fluorescence was measured using a Spectramax i3x microplate reader with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. DNA was quantified based on a Lambda DNA standard.Log-phase bacteria were prepared as performed in the killing assay above, but without peptide. After 3 h of incubation, samples were centrifuged at 5000 × 2 under static conditions, washed in PBS, and incubated for 1 h or 3 h with 100 μl of either 5 μM BSA, 5 μM hBD2, 200 μg/ml DNase or 0.5% Triton X-100.To measure the stability of meningococcal biofilm upon treatment with hBD2, a static biofilm assay previously described was used with some modifications . Bacteri600 and viable counts were assessed after the treatments. For viable counts bacterial suspensions were serially diluted, plated and counted the next day. Before plating for viable counts, bacteria were suspended and vortexed to dissolve aggregates, and then examined by microscopy to verify the absence of any aggregates.For measurement of the total biofilm mass clear plates were washed in PBS, stained with 0.3% crystal violet for 2 min, washed twice with PBS, and solubilized in 30% acetic acid. A crystal violet standard was included with known concentration and the absorbance was measured at 630 nm. The eDNA content was measured in the black-walled plates using Quant-iT PicoGreen dsDNA dye. 100 μl TE buffer and PicoGreen solution (1 μl:199 μl) was added into the biofilm and pipetted 10 times for it to dissolve. eDNA was quantified with a lambda DNA standard and fluorescence was measured at excitation wavelength 485 nm and emission wavelength 535 nm. Measurement of both plates was performed in a Spectramax i3x microplate reader. ODt-tests were used to compare differences between two groups. Analysis of variance (ANOVA) with Bonferroni’s post hoc test was used to compare differences between multiple groups. P-values below 0.05 were considered statistically significant.Testing of statistical significance was performed in Graph Pad Prism (version 5). Two-tailed and unpaired Student’s N. meningitidis to hBD2, killing assays were performed using a range of 0.1 μM to 15 μM hBD2. It has been demonstrated that basal levels of hBD2 in lung fluid are 0.5 μM  or 1 μM hBD2  for 3 h; then, the level of bound hBD2 was assessed by ELISA. Bacteria incubated with 1 μM hBD2 showed strong and significant hBD2 binding, while treatment with 0.1 μM resulted in a signal similar to the untreated control . These deability . Compareeability , we did eability . We furtngitidis , stronglngitidis .These data indicate that hBD2 may induce bacterial killing by non-membrane disruptive mechanisms.N. meningitidis. We used flow cytometry of SYTOX green-stained bacteria and compared hBD2 treatment to tetracycline and cephalexin treatment. Tetracycline inhibits the initiation of DNA replication but allows the ongoing round of replication to finish, producing fully replicated chromosomes that are visualized as an integer number of peaks, while cephalexin inhibits cell division and will result in cells with increased DNA content  increased during incubation   , could bin vivo relevance of hBD2 has been challenged. It is noteworthy that the local environment can greatly alter AMP activity by influencing peptide conformation and secondary structure (It is known that beta-defensins are affected by physiological concentrations of salt and serum . For thitructure . For exatructure . Furthertructure , which ctructure . It is pN. meningitidis. The lethal effect was not immediate, as is known for membrane-disruptive AMPs, but appeared at 2 h postincubation. Bacteria in aggregates survive treatment with lethal concentrations of hBD2 more often. Finally, the binding of hBD2 to extracellular DNA due to bacterial lysis is a possible evasion mechanism against hBD2. In this study, we used a N. meningitidis serogroup C strain, in the future it would be interesting to assess whether other serogroups behave in a similar way. With the emerging rise in antibiotic resistance, AMPs have increasingly become the focus as a novel antimicrobial treatment strategy. Although antibiotic resistance to N. meningitidis does not yet pose a problem, the widespread antimicrobial resistance exhibited by N. gonorrhoeae, a closely related strain, may increase the likelihood of the emergence of meningococcal resistant clones (In summary, hBD2 binds to and kills actively growing t clones . Here, wThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.GW, KL, SS, JF, KS, A-CL, KJ, and A-BJ conceived and designed the experiments. GW, KL, SS, JF, KS, and NI performed the experiments. GW, KL, SS, JF, A-CL, KJ, and A-BJ analyzed the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We determined if meeting the 2018 physical activity guidelines was associated with better ambulatory function, health-related quality of life, and inflammation than failing to meet the guidelines in patients with peripheral artery disease and claudication. Secondly, we determined the optimal number of total daily steps that are needed to meet the physical activity guidelines. Five hundred seventy-two patients were assessed on their daily ambulatory activity for one week with a step activity monitor, and were grouped according to whether they achieved less than 150 minutes of moderate intensity physical activity per week , or whether they were above this threshold . Treadmill peak walking time (mean±SD) was significantly higher (p<0.001) in Group 2 (709±359 sec) than in Group 1 (427±281 sec). The health-related quality of life score for physical function was significantly higher (p<0.001) in Group 2 (61±22%) than in Group 1 (44±21%). High sensitivity C-reactive protein was significantly lower (p<0.001) in Group 2 (3.6±4.5 mg/L) than in Group 1 (5.9±6.1 mg/L). Finally, 7,675 daily steps was the optimal threshold associated with meeting the physical activity guidelines, with a sensitivity of 82.9% and a specificity of 88.4%. In conclusion, patients with claudication who meet the 2018 physical activity guidelines for US adults had better ambulation, HRQoL, and inflammation outcomes than those who failed to meet the guidelines. From a practical standpoint, patients with claudication best achieved the physical activity guidelines by taking a total of 7,675 daily steps.