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The rise of antibiotic resistance has increased the need for alternative ways of preventing and treating enteropathogenic bacterial infection. Various probiotic bacteria have been used in animal and human. However, Saccharomyces boulardii is the only yeast currently used in humans as probiotic. There is scarce research conducted on yeast species commonly found in kefir despite its claimed potential preventative and curative effects. This work focused on adhesion properties, and antibacterial metabolites produced by Kluyveromyces lactis and Saccharomyces unisporus isolated from traditional kefir grains compared to Saccharomyces boulardii strains. Adhesion and sedimentation assay, slide agglutination, microscopy and turbidimetry assay were used to analyze adhesion of Salmonella Arizonae and Salmonella Typhimurium onto yeast cells. Salmonella growth inhibition due to the antimicrobial metabolites produced by yeasts in killer toxin medium was analyzed by slab on the lawn, turbidimetry, tube dilution and solid agar plating assays. Alcohol and antimicrobial proteins production by yeasts in killer toxin medium were analyzed using gas chromatography and shotgun proteomics, respectively. Salmonella adhered onto viable and non-viable yeast isolates cell wall. Adhesion was visualized using scanning electron microscope. Yeasts-fermented killer toxin medium showed Salmonella growth inhibition. The highest alcohol concentration detected was 1.55%, and proteins with known antimicrobial properties including cathelicidin, xanthine dehydrogenase, mucin-1, lactadherin, lactoperoxidase, serum amyloid A protein and lactotransferrin were detected in yeasts fermented killer medium. These proteins are suggested to be responsible for the observed growth inhibition effect of yeasts-fermented killer toxin medium. Kluyveromyces lactis and Saccharomyces unisporus have anti-salmonella effect comparable to Saccharomyces boulardii strains, and therefore have potential to control Salmonella infection.

Introduction Currently, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can be induced to differentiate at the cellular level but not to form mature tissues or organs suitable for transplantation. ESCs/iPSCs form immature teratomas after injection into immunodeficient mice. In humans, immature teratomas often transform into fully differentiated mature teratomas after administration of anticancer agents. Methods We first investigated the ability of cisplatin to induce changes in mouse ESCs/iPSCs in vitro. Next, we designed experiments to analyze ESC/iPSC-derived immature teratoma tissue in vivo after treatment of cisplatin. Groups of six mice carrying ESC- or iPSC-derived teratomas were given either low or high dose intraperitoneal injection of cisplatin, while the control group received saline for 4 weeks. Results Treatment of ESC/iPSC cultures with cisplatin for 3 days caused a dose-related decrease in cell numbers without inducing any morphological changes to the cells. ESC/iPSC-derived teratomas showed lower growth rates with a significantly higher mature components ratio in a concentration dependent manner after cisplatin treatment (P, Highlights • Transformation of immature to mature teratoma after chemotherapy was verified. • Mice bearing ESC/iPSC-derived immature teratomas were used. • Mice were treated with intraperitoneal injection of cisplatin for 4 weeks. • Newly differentiated structures were found only in the tumors of treated mice. • Cisplatin can induce differentiation in ESC/iPSC-derived immature teratomas.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection resulting in the coronavirus disease 2019 (COVID-19) has afflicted tens of millions of people in a worldwide pandemic. A recently developed recombinant Plant-Derived Virus-Like Particle Vaccine candidate for COVID-19 (CoVLP) formulated with AS03 has been shown to be well-tolerated and highly immunogenic in healthy adults. Since the target population for the vaccine includes women of childbearing potential, the objective of the study was to evaluate any untoward prenatal and postnatal effects of AS03-adjuvanted CoVLP administered intramuscularly to Sprague-Dawley female rats before cohabitation for mating (22 and 8 days prior) and during gestation (Gestation Days [GD] 6 and 19). The embryo-fetal development (EFD) cohort was subjected to cesarean on GD 21 and the pre/post-natal (PPN) cohort was allowed to naturally deliver. Effects of AS03-adjuvanted CoVLP was evaluated on pregnant rats, embryo-fetal development (EFD), during parturition, lactation and the development of the F1 offspring up to weaning Vaccination with AS03-adjuvanted CoVLP induced an antibody response in F0 females and anti-SARS-CoV-2 spike-specific maternal antibodies were detected in the offspring at the end of the gestation and lactation periods. Overall, there was no evidence of untoward effects of AS03-adjuvanted CoVLP on the fertility or reproductive performance of the vaccinated F0 females. There was no evidence of untoward effects on embryo-fetal development (including teratogenicity), or early (pre-weaning) development of the F1 offspring. These results support the acceptable safety profile of the AS03-adjuvanted CoVLP vaccine for administration to women of childbearing potential.

Introduction Regenerative solutions of the skin represent a hope for burn victims with extensive skin loss and chronic wound patients. The development of xeno-free workflow is crucial for clinical application in compliance with the directives of the European Medicines Agency. This study aimed at evaluating the outcome of the xeno-free isolation workflow of keratinocytes from human skin biopsy. Methods Skin biopsies were obtained from volunteers. The epidermis was digested with TrypLE™ Select, which was deactivated by dilution or with trypsin, deactivated by media with fetal bovine serum. Freshly isolated cells were compared for total cell number, viability, activity of caspase 3, gene expression and the presence of the keratinocyte surface markers cytokeratin 14. The cells were cultured in xeno-free conditions for one week and characterized regarding the number and viability as well as the metalloproteinase secretion. Results The number of obtained cells was similar in both workflows. The cell viability was less in the TrypLE group, with slight reduction of the cell surface marker cytokeratin 14. Caspase 3 activity was comparable as well as the gene expression of the apoptotic markers BAX, BCL2 and SLUG, as well as the keratinocyte markers cytokeratin 14, stratifin and filaggrin. Upon culture, the number of keratinocytes, their viability and secretion of matrix metalloproteinases 1 and 10 were equal in both groups. Conclusion This study reports the possibility of isolating functioning and viable keratinocytes through a xeno-free workflow for clinical application., Highlights • The xenofree workflows are essential for clinical application of regenerative medicine. • TrypLE based digestion of skin biopsies can be deactivated by dilution in a xenofree workflow to isolate keratinocytes. • Although the xenofree workflow was efficient, keratinocytes viability was better with the classical trypsin-based workflow. • The cell characteristics were equivalent upon culturing the cells.

Anti-tumour efficacy of doxorubicin is hindered by the cumulative dose-dependent cardiotoxicity induced by reactive oxygen species during its metabolism. As Cinnamomum zeylanicum has proven antioxidant potential, objective of this study was to investigate the cardioprotective activity of Cinnamomum bark extract against doxorubicin induced cardiotoxicity in Wistar rats. Physicochemical and phytochemical analysis was carried out and dose response effect and the cardioprotective activity of Cinnamomum were determined in vivo. 180 mg/kg dexrazoxane was used as the positive control. Plant extracts were free of heavy metals and toxic phytoconstituents. In vivo study carried out in Wistar rats revealed a significant increase (p

Highlights • Ravulizumab and eculizumab differ by 4 amino acids in their heavy chains. • Validation of ravulizumab LDT with quantitation by intact light chain. • TOF-MS allows for detection by heavy chain., Introduction Ravulizumab (RAVUL) is a new complement inhibitor, with a difference of 4 amino acids in the heavy chain from a predecessor compound, eculizumab (ECUL). Objectives First, to utilize mass spectrometry (MS) to characterize RAVUL and verify differences from its predecessor and, second, to validate and implement a lab developed test (LDT) for RAVUL that will allow for quantitative therapeutic monitoring. Methods A time-of-flight mass spectrometer (TOF-MS) was used to characterize and differentiate the molecular weight differences between RAVUL and ECUL by both digest and reduction experiments. In parallel, an LDT for RAVUL was validated and implemented utilizing IgG4 enrichment with light chain detection and quantitation on a high throughput orbitrap MS platform. Results The TOF-MS platform allowed for the mass difference between RAVUL and ECUL to be verified along with providing a proof of concept for a new intact protein quantitation software. An LDT on an orbitrap MS was validated and implemented using intact light chain quantitation, with the limitation that it cannot differentiate between ECUL and RAVUL. The LDT has an analytical measuring range from 5 to 600 mcg/mL, inter-assay imprecision of ≤13% CV (n = 13) and accuracy with

Highlights • Obesity contributes to male infertility. • Can lycopene or moringa improve male infertility? • Tested in a rodent model of diet-induced obesity. • Lycopene was superior to Moringa in improving male fertility parameters., Obesity is a condition of chronic tissue inflammation and oxidative stress that poses as a risk factor for male infertility. Moringa oleifera oil extract is known to have cholesterol-lowering properties and a potential to treat obesity, while lycopene is a potent antioxidant. We hypothesize that Moringa or lycopene may improve male fertility markers in an animal model of diet-induced obesity. Male Albino rats (n = 60) were randomized to receive regular chow (RC) or high-fat diet (HFD) for 12 weeks (n = 30 each). Animals in each arm were further randomized to receive gavage treatment with corn oil (vehicle), lycopene (10 mg/kg), or Moringa (400 mg/kg) for four weeks starting on week 9 (n = 10 each). Animals were sacrificed at 12 weeks, and blood was collected to assess lipid profile, serum testosterone, and gonadotropin levels. The testes and epididymides were removed for sperm analysis, oxidative stress and inflammatory markers, and histopathological assessment. In comparison to their RC littermates, animals on HFD showed an increase in body weights, serum lipids, testosterone and gonadotrophin levels, testicular oxidative stress and inflammatory markers, as well as sperm abnormalities and disrupted testicular histology. Moringa or lycopene reduced body weight, improved oxidative stress, and male fertility markers in HFD-fed animals with lycopene exhibiting better anti-antioxidant and anti-lipidemic effects. Lycopene is superior to Moringa in improving male fertility parameters, possibly by attenuating oxidative stress.

Graphical abstract, For the whole GFP family, a few cases, when a single mutation in the chromophore environment strongly inhibits maturation, were described. Here we study EYFP-F165G – a variant of the enhanced yellow fluorescent protein – obtained by a single F165G replacement, and demonstrated multiple fluorescent states represented by the minor emission peaks in blue and yellow ranges (~470 and ~530 nm), and the major peak at ~330 nm. The latter has been assigned to tryptophan fluorescence, quenched due to excitation energy transfer to the mature chromophore in the parental EYFP protein. EYFP-F165G crystal structure revealed two general independent routes of post-translational chemistry, resulting in two main states of the polypeptide chain with the intact chromophore forming triad (~85%) and mature chromophore (~15%). Our experiments thus highlighted important stereochemical role of the 165th position strongly affecting spectral characteristics of the protein. On the basis of the determined EYFP-F165G three-dimensional structure, new variants with ~ 2-fold improved brightness were engineered.

Trichomonas vaginalis is a neglected urogenital parasitic protist that causes 170 million cases of trichomoniasis annually, making it the most prevalent non-viral, sexually transmitted disease. Trichomoniasis treatment relies on nitroheterocyclics, such as metronidazole. However, with increasing drug-resistance, there is an urgent need for novel anti-trichomonals. Little progress has been made to translate anti-trichomonal research into commercialised therapeutics, and the absence of a standardised compound-screening platform is the immediate stumbling block for drug-discovery. Herein, we describe a simple, cost-effective growth assay for T. vaginalis and the related Tritrichomonas foetus. Tracking changes in pH were a valid indicator of trichomonad growth (T. vaginalis and T. foetus), allowing development of a miniaturised, chromogenic growth assay based on the phenol red indicator in 96- and 384-well microtiter plate formats. The outputs of this assay can be quantitatively and qualitatively assessed, with consistent dynamic ranges based on Z′ values of 0.741 and 0.870 across medium- and high-throughput formats, respectively. We applied this high-throughput format within the largest pure-compound microbial metabolite screen (812 compounds) for T. vaginalis and identified 43 hit compounds. We compared these identified compounds to mammalian cell lines, and highlighted extensive overlaps between anti-trichomonal and anti-tumour activity. Lastly, observing nanomolar inhibition of T. vaginalis by fumagillin, and noting this compound has reported activity in other protists, we performed in silico analyses of the interaction of fumagillin with its molecular target methionine aminopeptidase 2 for T. vaginalis, Giardia lamblia and Entamoeba histolytica, highlighting potential for fumagillin as a broad-spectrum anti-protistal against microaerophilic protists. Together, this new platform will accelerate drug-discovery efforts, underpin drug-resistance screening in trichomonads, and contributing to a growing body of evidence highlighting the potential of microbial natural products as novel anti-protistals., Graphical abstract Image 1

Triple negative breast cancer (TNBC) is an aggressive disease with a 5-y relative survival rate of 11% after distant metastasis. To survive the metastatic cascade, tumor cells remodel their signaling pathways by regulating energy production and upregulating survival pathways. AMP-activated protein kinase (AMPK) and Akt regulate energy homeostasis and survival, however, the individual or synergistic role of AMPK and Akt isoforms during lung colonization by TNBC cells is unknown. The purpose of this study was to establish whether targeting Akt, AMPKα or both Akt and AMPKα isoforms in circulating cancer cells can suppress TNBC lung colonization. Transient silencing of Akt1 or Akt2 dramatically decreased metastatic colonization of lungs by inducing apoptosis or inhibiting invasion, respectively. Importantly, transient pharmacologic inhibition of Akt activity with MK-2206 or AZD5363 inhibitors did not prevent colonization of lung tissue by TNBC cells. Knockdown of AMPKα1, AMPKα2, or AMPKα1/2 also had no effect on metastatic colonization of lungs. Taken together, these findings demonstrate that transient decrease in AMPK isoforms expression alone or in combination with Akt1 in circulating tumor cells does not synergistically reduce TNBC metastatic lung colonization. Our results also provide evidence that Akt1 and Akt2 expression serve as a bottleneck that can challenge colonization of lungs by TNBC cells.

Morphological and functional characterizations of cultured microglia are essential for the improved understanding of their roles in neuronal health and disease. Although some studies (phenotype analysis, phagocytosis) can be carried out in mixed or microglia-enriched cultures, in others (gene expression) pure microglia must be used. If the use of genetically modified microglial cells is not feasible, isolation of resident microglia from nervous tissue must be carried out. In this study, mixed primary cultures were established from the forebrains of newborn rats. Secondary microglia-enriched cultures were then prepared by shaking off these cells from the primary cultures, which were subsequently used to establish tertiary cultures by further shaking off the easily detachable microglia. The composition of these cultures was quantitatively analyzed by immunocytochemistry of microglia-, astrocyte-, oligodendrocyte- and neuron-specific markers to determine yield and purity. Microglia were quantitatively characterized regarding morphological and proliferation aspects. Secondary and tertiary cultures typically exhibited 73.3% ± 17.8% and 93.1% ± 6.0% purity for microglia, respectively, although the total number of microglia in the latter was much smaller. One in seven attempts of culturing the tertiary cultures had ~99% purity for microglia. The overall yield from the number of cells plated at DIV0 to the Iba1-positive microglia in tertiary cultures was ~1%. Astrocytic and neuronal contamination progressively decreased during subcloning, while oligodendrocytes were found sporadically throughout culturing. Although the tertiary microglia cultures had a low yield, they produced consistently high purity for microglia; after validation, such cultures are suitable for purity-sensitive functional screenings (gene/protein expression)., Highlights • Microglia can be preferentially shaken off from primary mixed and secondary cultures. • Secondary cultures contaminated mainly by astrocytes (12–19%) and neurons (2–3%). • Tertiary cultures had low cell yields (x̄ = 5.1% for T1, x̄ = 2.4% for T2). • Tertiary cultures had consistently high purity (x̄ = 93.1%; 99% in 1 of 7 attempts). • Tertiary cultures are suitable for gene or protein expression analyses.

Background The absolute number of new stroke patients is annually increasing and there still remains only a few Food and Drug Administration (FDA) approved treatments with significant limitations available to patients. Tanshinone IIA (Tan IIA) is a promising potential therapeutic for ischemic stroke that has shown success in pre-clinical rodent studies but lead to inconsistent efficacy results in human patients. The physical properties of Tan-IIA, including short half-life and low solubility, suggests that Poly (lactic-co-glycolic acid) (PLGA) nanoparticle-assisted delivery may lead to improve bioavailability and therapeutic efficacy. The objective of this study was to develop Tan IIA-loaded nanoparticles (Tan IIA-NPs) and to evaluate their therapeutic effects on cerebral pathological changes and consequent motor function deficits in a pig ischemic stroke model. Results Tan IIA-NP treated neural stem cells showed a reduction in SOD activity in in vitro assays demonstrating antioxidative effects. Ischemic stroke pigs treated with Tan IIA-NPs showed reduced hemispheric swelling when compared to vehicle only treated pigs (7.85 ± 1.41 vs. 16.83 ± 0.62%), consequent midline shift (MLS) (1.72 ± 0.07 vs. 2.91 ± 0.36 mm), and ischemic lesion volumes (9.54 ± 5.06 vs. 12.01 ± 0.17 cm3) when compared to vehicle-only treated pigs. Treatment also lead to lower reductions in diffusivity (−37.30 ± 3.67 vs. −46.33 ± 0.73%) and white matter integrity (−19.66 ± 5.58 vs. −30.11 ± 1.19%) as well as reduced hemorrhage (0.85 ± 0.15 vs 2.91 ± 0.84 cm3) 24 h post-ischemic stroke. In addition, Tan IIA-NPs led to a reduced percentage of circulating band neutrophils at 12 (7.75 ± 1.93 vs. 14.00 ± 1.73%) and 24 (4.25 ± 0.48 vs 5.75 ± 0.85%) hours post-stroke suggesting a mitigated inflammatory response. Moreover, spatiotemporal gait deficits including cadence, cycle time, step time, swing percent of cycle, stride length, and changes in relative mean pressure were less severe post-stroke in Tan IIA-NP treated pigs relative to control pigs. Conclusion The findings of this proof of concept study strongly suggest that administration of Tan IIA-NPs in the acute phase post-stroke mitigates neural injury likely through limiting free radical formation, thus leading to less severe gait deficits in a translational pig ischemic stroke model. With stroke as one of the leading causes of functional disability in the United States, and gait deficits being a major component, these promising results suggest that acute Tan IIA-NP administration may improve functional outcomes and the quality of life of many future stroke patients., Highlights • Tanshinone IIA loaded nanoparticles demonstrate antioxidative capabilities in neural stem cell cultures. • Tanshinone IIA loaded nanoparticles leads to reduced lesion volume, midline shift, and white matter damage post-stroke. • Tanshinone IIA loaded nanoparticle treatment leads to marked improvements in spatiotemporal and kinetic gait parameters.

Urea transporters (UT) play a vital role in the mechanism of urine concentration and are recognized as novel targets for the development of salt-sparing diuretics. Thus, UT inhibitors are promising for development as novel diuretics. In the present study, a novel UT inhibitor with a diarylamide scaffold was discovered by high-throughput screening. Optimization of the inhibitor led to the identification of a promising preclinical candidate, N-[4-(acetylamino)phenyl]-5-nitrofuran-2-carboxamide (1H), with excellent in vitro UT inhibitory activity at the submicromolar level. The half maximal inhibitory concentrations of 1H against UT-B in mouse, rat, and human erythrocyte were 1.60, 0.64, and 0.13 μmol/L, respectively. Further investigation suggested that 8 μmol/L 1H more powerfully inhibited UT-A1 at a rate of 86.8% than UT-B at a rate of 73.9% in MDCK cell models. Most interestingly, we found for the first time that oral administration of 1H at a dose of 100 mg/kg showed superior diuretic effect in vivo without causing electrolyte imbalance in rats. Additionally, 1H did not exhibit apparent toxicity in vivo and in vitro, and possessed favorable pharmacokinetic characteristics. 1H shows promise as a novel diuretic to treat hyponatremia accompanied with volume expansion and may cause few side effects., Graphical abstract In the present study, a novel urea transporters (UT) inhibitor with a diarylamide scaffold was discovered by high throughput screening. Optimization of the hit compound E04 led to the identification of 1H, with orally diuretic activity, more powerful inhibition activity on UT-A1, favorable pharmacokinetic characteristics and without electrolyte disturbance.Image 1

Graphical abstract, Highlights • Solid Lipid Nanoparticles, Nanostructured Lipid Carriers, and Nanoemulsion are lipid nanocarriers with different matrix fluidity. • Lipid nanocarriers with different matrix fluidity may contribute differently to the activity of the incorporated curcumin. • Curcumin may be protected against photodegradation by incorporation in nanocarrier of any fluidity. • Matrix fluidity regulates the occlusive index, film forming capacity, curcumin release and wound healing. • Incorporation of curcumin in nanocarriers may achieve faster wound healing at lower dose compared to free molecule., Curcumin (CUR) has a long history of use as an antimicrobial, anti-inflammatory and wound healing agent, for the treatment of various skin conditions. Encapsulation in nanocarriers may overcome the administration limitations of CUR, such as lipophilicity and photodegradation. Lipid nanocarriers with different matrix fluidity (Solid Lipid Nanoparticles; SLN, Nanostructured Lipid Carriers; NLC, and Nanoemulsion; NE) were prepared for the topical delivery of curcumin (CUR). The occlusive properties and film forming capacity, as well as the release profile of incorporated CUR, its protection against photodegradation and wound healing were studied in vitro, using empty nanocarriers or free CUR as control. The results suggest that incorporation of CUR in nanocarriers offers a significant protection against photodegradation that is not influenced by the matrix fluidity. However, this characteristic regulates properties such as the occlusion, the release rate and wound healing ability of CUR. Nanoparticles of low fluidity provided better surface occlusion, film forming capacity and retention of the incorporated CUR. All nanocarriers but especially NLC, achieved faster wound healing at lower dose of incorporated CUR. In conclusion, nanotechnology may enhance the action of CUR against skin conditions. Important characteristics of the nanocarrier such as matrix fluidity should be taken into consideration in the design of CUR nanosystems of optimal efficiency.

Introduction Basic fibroblast growth factor (bFGF) is a promising cytokine in regenerative therapy for spinal cord injury. In this study, recombinant canine bFGF (rc-bFGF) was synthesized for clinical use in dogs, and the ability of rc-bFGF to differentiate canine bone marrow mesenchymal stem cells (BMSCs) into functional neurons was investigated. Methods The rc-bFGF was synthesized using a wheat germ cell-free protein synthesis system. The expression of rc-bFGF mRNA in the purification process was confirmed using a reverse transcription-polymerase chain reaction (RT-PCR). Western blotting was performed to confirm the antigenic property of the purified protein. To verify function of the purified protein, phosphorylation of extracellular signal-regulated kinase (ERK) was examined by in vitro assay using HEK293 cells. To compare the neuronal differentiation capacity of canine BMSCs in response to treatment with rc-bFGF, the cells were divided into the following four groups: control, undifferentiated, rh-bFGF, and rc-bFGF groups. After neuronal induction, the percentage of cells that had changed to a neuron-like morphology and the mRNA expression of neuronal markers were evaluated. Furthermore, to assess the function of the canine BMSCs after neuronal induction, changes in the intracellular Ca2+ concentrations after stimulation with KCl and l-glutamate were examined. Results The protein synthesized in this study was rc-bFGF and functioned as bFGF, from the results of RT-PCR, western blotting, and the expression of pERK in HEK293 cells. Canine BMSCs acquired a neuron-like morphology and expressed mRNAs of neuronal markers after neuronal induction in the rh-bFGF and the rc-bFGF groups. These results were more marked in the rc-bFGF group than in the other groups. Furthermore, an increase in intracellular Ca2+ concentrations was observed after the stimulation of KCl and l-glutamate in the rc-bFGF group, same as in the rh-bFGF group. Conclusions A functional rc-bFGF was successfully synthesized, and rc-bFGF induced the differentiation of canine BMSCs into voltage- and glutamate-responsive neuron-like cells. Our purified rc-bFGF may contribute, on its own, or in combination with canine BMSCs, to regenerative therapy for spinal cord injury in dogs., Highlights • Functional rc-bFGF was successfully synthesized. • rc-bFGF induced the differentiation of canine BMSCs into neuron-like cells. • rc-bFGF may aid in regenerative therapy of spinal cord injury in dogs.

Photodynamic therapy (PDT) is a well-known cancer therapy that utilizes light to excite a photosensitizer and generate cytotoxic reactive oxygen species (ROS). The efficacy of PDT primarily depends on the photosensitizer and oxygen concentration in the tumor. Hypoxia in solid tumors promotes treatment resistance, resulting in poor PDT outcomes. Hence, there is a need to combat hypoxia while delivering sufficient photosensitizer to the tumor for ROS generation. Here we showcase our unique theranostic perfluorocarbon nanodroplets as a triple agent carrier for oxygen, photosensitizer, and indocyanine green that enables light triggered spatiotemporal delivery of oxygen to the tumors. We evaluated the characteristics of the nanodroplets and validated their ability to deliver oxygen via photoacoustic monitoring of blood oxygen saturation and subsequent PDT efficacy in a murine subcutaneous tumor model. The imaging results were validated with an oxygen sensing probe, which showed a 9.1 fold increase in oxygen content inside the tumor, following systemic administration of the nanodroplets. These results were also confirmed with immunofluorescence. In vivo studies showed that nanodroplets held higher rates of treatment efficacy than a clinically available benzoporphyrin derivative formulation. Histological analysis showed higher necrotic area within the tumor with perfluoropentane nanodroplets. Overall, the photoacoustic nanodroplets can significantly enhance image-guided PDT and has demonstrated substantial potential as a valid theranostic option for patient-specific photodynamic therapy-based treatments., Graphical Abstract ga1

Graphical abstract The map illustrating mechanisms associated with lncRNA HIF1A-AS2-mediated inflammation in the atherosclerosis. LncRNA HIF1A-AS2 forms a complex with USF1, which is recruited into the ATF2 promoter to elevate ATF2 expression, thus promoting the development of atherosclerotic inflammation. Meanwhile, downregulation of lncRNA HIF1A-AS2 decreases the expression of ATF2 by reducing the binding of USF1 to the ATF2 promoter regions, thereby inhibiting atherosclerotic inflammation, as reflected by decreased inflammatory factors TNF-α, IL-1β and IL-6 in serum and protein levels of adhesion molecules VCAM-1, ICAM-1, and MCP-1, as well as increased cell viability and reduced apoptosis in ox-LDL-induced inflammation in ECs, SMCs, and HCAECs., Introduction In atherosclerotic lesions, extensive inflammation of the vessel wall contributes to plaque instability. Long noncoding RNAs (lncRNAs) play important roles in diverse biological processes in atherosclerosis. Objectives Here, we aim to identify the functional role and regulatory mechanisms of lncRNA hypoxia-inducible factor 1 alpha-antisense RNA 2 (HIF1A-AS2) in atherosclerotic inflammation. Methods An atherosclerotic mouse model was induced in ApoE-/- mice by high fat diet (HFD). Endothelial cells (ECs), human aortic smooth muscle cells (SMCs) or human coronary artery endothelial cells (HCAECs) were exposed to ox-LDL to develop the in vitro model. The effects of lncRNA HIF1A-AS2 on inflammation were evaluated by determining levels of inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) and levels of adhesion molecules vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and macrophage cationic peptide 1 (MCP-1). Results It was established that lncRNA HIF1A-AS2 and ATF2 were highly expressed in atherosclerotic ApoE-/- mice. Downregulating lncRNA HIF1A-AS2 in ox-LDL-exposed ECs, SMCs and HCAECs inhibited inflammation by reducing levels of pro-inflammatory factors and adhesion molecules. LncRNA HIF1A-AS2 bound to the transcription factor USF1 to elevate ATF2 expression. USF1 overexpression counteracted the suppressive effect of lncRNA HIF1A-AS2 silencing on ox-LDL-induced inflammation. Knockdown of lncRNA HIF1A-AS2 or ATF2 could also attenuate inflammation in atherosclerotic mice. Collectively, the present study demonstrates that downregulation of lncRNA HIF1A-AS2 represses the binding of USF1 to the ATF2 promoter region and then inhibits ATF2 expression, thereby suppressing atherosclerotic inflammation. Conclusion This study suggests lncRNA HIF1A-AS2 as an promising therapeutic target for atherosclerosis.

Background Liver cirrhosis is a chronic disease in which normal liver tissue is replaced by fibrous tissue, leads to liver malfunction. Although transplantation is the most certain cure, stem cell therapies are shedding light on efforts to regenerate cirrhotic liver. The purpose of this study was to evaluate the regenerative potential of mesenteric fat stem cells in CCL4-induced liver cirrhosis in an animal model. Methods Thirty rats were treated with the mixture of CCL4 and olive oil intraperitoneally by a dose of 0.2 ml (0.1 ml CCL4 and 0.1 ml olive oil) every other day for 16 weeks till cirrhosis signs appeared. Fifteen rats were randomly selected as control group. Others treated by mesenteric fat derived mesenchymal stem cells transferred into the liver parenchyma. Results After 5 weeks, rats received stem cells had improved clinically by increased movements, appetite, improvement in overall behavior and decreased abdomen size. Histopathologically, liver cells showed state of regeneration and forming new colonies. Conclusion Liver cirrhosis was induced. The mesenchymal stem cells derived from mesenteric adipose tissue could improve hepatic status of the rats, as cirrhotic livers were regenerated back into normal appearing parenchyma. Rats’ clinical behavior also reached healthy status., Highlights • The study evaluated the regenerative potential of mesenteric fat stem cells in cirrhotic livers. • All aspects of a patient (in this study animals) including clinical behavior, paraclinical investigation, serum parameters and histopathologic views were analysed before and after the surgical treatment. • Both intravenous and intraparenchymal administration of stem cells was the new method used in this study.

Interferon Regulatory Factors (IRFs) are key regulators of immunity, cell survival and apoptosis. IRF transcriptional activity and subcellular localization are tightly regulated by posttranscriptional modifications including phosphorylation. The IκB kinase family member IKK-ε is essential in regulating antiviral innate immunity mediated by IRFs but is now also recognized as an oncoprotein amplified and overexpressed in breast cancer cell lines and patient-derived tumors. In the present study, we report that the tumor suppressor IRF-1 is a specific target of IKK-ε in breast cancer cells. IKK-ε-mediated phosphorylation of IRF-1 dramatically decreases IRF-1 protein stability, accelerating IRF-1 degradation and quenching IRF-1 transcriptional activity. Chemical inhibition of IKK-ε activity, fully restores IRF-1 levels and function and positively correlates with inhibition of cell growth and proliferation of breast cancer cells. By using a breast cancer cell line stably expressing a dominant negative version of IRF-1 we were able to demonstrate that IKK-ε preferentially exerts its oncogenic potential in breast cancer through the regulation of IRF-1 and point to the IKK-ε-mediated phosphorylation of IRF-1 as a therapeutic target to overcome IKK-ε-mediated tumorigenesis.

Graphical abstract, Highlights • Corneal stromal dystrophies are a group of hereditary disorders caused by mutations in the TGFBI gene and affect the corneal stroma and epithelium. • The disease is characterized by the accumulation of insoluble deposits of the mutant TGFBIp leading to poor visual acuity in patients. • Mutations are hypothesized to disrupt the protein folding and stability, leading oligomerization of the mutant protein. • Current treatment relies on surgical intervention, either tissue removal or substitution, both of which are associated with disease recurrence. • The lead compounds reported here prevent/delay the atypical proteolysis of the mutant protein and the generation of amyloidogenic fragments., Corneal dystrophies are a group of genetically inherited disorders with mutations in the TGFBI gene affecting the Bowman’s membrane and the corneal stroma. The mutant TGFBIp is highly aggregation-prone and is deposited in the cornea. Depending on the type of mutation the protein deposits may vary (amyloid, amorphous powdery aggregate or a mixed form of both), making the cornea opaque and thereby decreases visual acuity. The aggregation of the mutant protein is found to be specific with a unique aggregation mechanism distinct to the cornea. The proteolytic processing of the mutant protein is reported to be different compared to the WT protein. The proteolytic processing of mutant protein gives rise to highly amyloidogenic peptide fragments. The current treatment option, available for patients, is tissue replacement surgery that is associated with high recurrence rates. The clinical need for a simple treatment option for corneal dystrophy patients has become highly essential either to prevent the protein aggregation or to dissolve the preformed aggregates. Here, we report the screening of 2500 compounds from the Maybridge RO3 fragment library using weak affinity chromatography (WAC). The primary hits from WAC were validated by 15N-HSQC NMR assays and specific regions of binding were identified. The recombinant mutant proteins (4th FAS-1 domain of R555W and H572R) were subjected to limited proteolysis by trypsin together with the lead compounds identified by NMR assays. The lead compounds (MO07617, RJF00203 and, BTB05094) were effective to delay/prevent the generation of amyloidogenic peptides in the R555W mutant and compounds (RJF00203 and BTB05094) were effective to delay/prevent the generation of amyloidogenic peptides in the H572R mutant. Thus the lead compounds reported here upon further validation and/or modification might be proposed as a potential treatment option to prevent/delay aggregation by inhibiting the formation of amyloidogenic peptides in TGFBI-corneal dystrophy.

Graphical abstract, Highlights • Rhodomyrtone, a bioactive acylphloroglucinol compound isolated from the leaves of Rhodomyrtus tomentosa, has been scientifically evidenced as a potential antibacterial agent. • Rhodomyrtone did not cause any signs of toxicity in invertebrate (Galleria mellonella) and vertebrate (zebrafish, murine) models. • Rhodomyrtone at 256 μg/mL did not cause any observable human erythrocyte haemolysis. • As the minimal inhibitory concentration of rhodomyrtone against most Gram-positive pathogens is 0.5-1 μg/mL, the results suggest that it should produce no toxic effects at concentrations used in human., Background Rhodomyrtus tomentosa (Aiton) Hassk. has been traditionally used to relieve various diseases. Rhodomyrtone, a bioactive acylphloroglucinol compound isolated from the leaves of Rhodomyrtus tomentosa, has been scientifically evidenced as a potential antibacterial agent. This study aimed to assess safety of rhodomyrtone in both invertebrate and vertebrate models. Material and Methods Safety of rhodomyrtone was determined in an invertebrate model, Galleria mellonella as well as vertebrate models including zebrafish (Danio rerio) and murine. In addition, toxicity to human erythrocytes was also measured. Results Treatment of Galleria mellonella with rhodomyrtone at 100 mg/kg body weight up to four days showed no visible toxic effects (100 % survival). In zebrafish embryo model, at least 80 % survival of embryos was demonstrated when treated with rhodomyrtone at 0.5 μg/mL for three days. Prior to clinical trial, it is a prerequisite that rhodomyrtone has to be evaluated for its biocompatibility with human blood components. The results displayed that rhodomyrtone at 256 μg/mL did not cause any observable human erythrocyte haemolysis. Furthermore, preclinical assessment of rhodomyrtone formulation justified potential applications of rhodomyrtone in humans. Oral toxicity testing in a mouse model indicated the absence of systemic toxicity when the animals received up to 5000 mg/kg body weight of rhodomyrtone formulation for a period of fourteen days. Conclusions As the minimal inhibitory concentration of rhodomyrtone against most Gram-positive pathogens is 0.5−1 μg/mL, the results suggest that it should produce no toxic effects at concentrations used in human, thus support further development in pharmaceutical industries and public health applications.

Introduction In the present study, we examined the effect of oriented collagen tube (OCT) implantation on the recovery of sensory function of the resected rat sciatic nerve. Materials and methods After a 10-mm long portion of the sciatic nerve of a rat was resected, an OCT was placed in the site of nerve defect. Recovery of the sensory function was evaluated using Von Frey test every 3 days after surgery. The regenerated tissue were histologically and ultrastructurally analyzed 2 and 4 weeks after the surgery. Results The sensory reflexes of the OCT group were restored to the level of that of the intact group after 15 days. Hematoxylin and eosin staining revealed the cross-linking between the proximal and distal stumps after 2 weeks. After 4 weeks, Luxol Fast Blue and immunohistochemical staining revealed the presence of myelin sheath from the proximal to distal region of the regenerated tissue and S100B staining confirmed the presence of Schwann cells. Interestingly, no myelin sheath was ultrastructurally observed around the regenerated axons at the central region after 2 weeks. Conclusions These results suggest that OCTs facilitate the recovery of sensory function. Additionally, the non-myelinated axons contributed to the recovery of the sensory function., Highlights • Von Frey test results in the OCT group on POD 15 were comparable at the sham group. • OCT group showed regeneration of unmyelinated axons in 2 weeks. • Myelination was observed from proximal to distal after 4 weeks OCT implantation. • In the OCT group, a large number of blood vessels were observed in nerve in 2 weeks.

Introduction Regenerative therapy using chondrocyte sheets is effective for osteoarthritis. The clinical application of chondrocyte sheet therapy is expected to be further advanced by the use of a feasible cryopreservation technique. Previously, we developed a chondrocyte sheet vitrification method; however, it was too complex to be used for routine clinical application. Here, we aimed to develop a prototype method for vitrifying chondrocyte sheets for clinical practice. Methods We developed a “circulating vitrification bag” as a container to process cell sheets for vitrification in an efficient and sanitary fashion. Moreover, we invented the “vitrification storage box”, which is useful for the vitrification of cell sheets, long-term preservation, and transportation. These devices were used to vitrify rabbit chondrocyte sheets, which were then assessed for their structural characteristics and the viability of the component cells after rewarming. Results In all cell sheet samples (n = 7) vitrified by the circulating vitrification bag method, the integrity of the sheet structure was maintained, and the cell survival rate was similar to that of non-vitrified samples (91.0 ± 2.9% vs. 90.0 ± 3.0%). Proteoglycan and type II collagen, which are major components of cartilage, were densely and evenly distributed throughout the chondrocyte sheet subjected to vitrification similarly to that observed in the non-vitrified sheet. After long-term storage using the vitrification storage box, the cell sheets maintained normal structure and cell viability (survival rate: 81.2 ± 1.0% vs. 84.3 ± 1.8%) compared to the non-vitrified sheet. Conclusion Our results indicate that the circulating vitrification bag method is an effective approach for realizing the clinical application of vitrified chondrocyte sheets. The vitrification storage box is also useful for the long-term preservation of vitrified cell sheets, further enhancing the feasibility of the clinical application of cryopreserved chondrocyte sheets., Highlights • We developed a new device and method for the cryopreservation of cell sheets. • The method and device efficacy were evaluated using vitrified rabbit chondrocyte sheets. • Our approach allows efficient clinical application of vitrified cell sheets.

In vitro models of the human lung play an essential role in evaluating the toxicity of inhaled compounds and understanding the development of respiratory diseases. Three-dimensional (3D) organotypic models derived from lung basal epithelial cells and grown at the air–liquid interface resemble human airway epithelium in multiple aspects, including morphology, cell composition, transcriptional profile, and xenobiotic metabolism. Whether the different characteristics of basal cell donors have an impact on model characteristics and responses remains unknown. In addition, studies are often conducted with 3D cultures from one donor, assuming a representative response on the population level. Whether this assumption is correct requires further investigation. In this study, we compared the morphology and functionality of 3D organotypic bronchial and small airway cultures from different donors at different weeks after air-lift to assess the interdonor variability in these parameters. The thickness, cell type composition, and transepithelial electrical resistance varied among the donors and over time after air-lift. Cilia beating frequency increased in response to isoproterenol treatment in both culture types, independent of the donor. The cultures presented low basal cytochrome P450 (CYP) 1A1/1B1 activity, but 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment induced CYP1A1/1B1 activity regardless of the donor. In conclusion, lung epithelial cultures prepared from different donors present diverse morphology but similar functionality and metabolic activity, with certain variability in their response to stimulation., Graphical abstract Unlabelled Image, Highlights • 3D lung cultures derived from various donors differed mostly at the morphological level. • Epithelial thickness, presence of cysts, ciliation, and goblet cell number are donor dependent. • Cilia beating frequency varied across donors but the response to isoproterenol was similar. • CYP450 activity in response to xenobiotics was preserved across donors.

Cholinergic agonists, like levamisole, are a major class of anthelmintic drugs that are known to act selectively on nicotinic acetylcholine receptors (nAChRs) on the somatic muscle and nerves of nematode parasites to produce their contraction and spastic paralysis. Previous studies have suggested that in addition to the nAChRs found on muscle and nerves, there are nAChRs on non-excitable tissues of nematode parasites. We looked for evidence of nAChRs expression in the cells of the intestine of the large pig nematode, Ascaris suum, using RT-PCR and RNAscope in situ hybridization and detected mRNA of nAChR subunits in the cells. These subunits include components of the putative levamisole receptor in A. suum muscle: Asu-unc-38, Asu-unc-29, Asu-unc-63 and Asu-acr-8. Relative expression of these mRNAs in A. suum intestine was quantified by qPCR. We also looked for and found expression of G protein-linked acetylcholine receptors (Asu-gar-1). We used Fluo-3 AM to detect intracellular calcium changes in response to receptor activation by acetylcholine (as a non-selective agonist) and levamisole (as an L-type nAChR agonist) to look for evidence of functioning nAChRs in the intestine. We found that both acetylcholine and levamisole elicited increases in intracellular calcium but their signal profiles in isolated intestinal tissues were different, suggesting activation of different receptor sets. The levamisole responses were blocked by mecamylamine, a nicotinic receptor antagonist in A. suum, indicating the activation of intestinal nAChRs rather than G protein-linked acetylcholine receptors (GARs) by levamisole. The detection of nAChRs in cells of the intestine, in addition to those on muscles and nerves, reveals another site of action of the cholinergic anthelmintics and a site that may contribute to the synergistic interactions of cholinergic anthelmintics with other anthelmintics that affect the intestine (Cry5B)., Graphical abstract Image 1, Highlights • Novel levamisole anthelmintic receptors identified in a nematode parasite intestine. • Molecular techniques and RNAscope show dissimilar distributions of nAChR subunits. • Fluo-3 calcium sensitive dye detects two types of response to agonists. • Evidence of heterogeneous nAChRs present in the intestine. • Intestinal levamisole receptors can explain synergistic anthelmintic interactions.

Liposomal formulations for the treatment of cancer and other diseases are the most common form of nanotechnology enabled pharmaceuticals (NEPs) submitted for market approval and in clinical application today. The accurate characterization of their physical-chemical properties is a key requirement; in particular, size, size distribution, shape, and physical-chemical stability are key among properties that regulators identify as critical quality attributes. Here we develop and validate an optimized method, based on multi-detector asymmetrical-flow field flow fractionation (MD-AF4) to accurately and reproducibly separate liposomal drug formulations into their component populations and to characterize their associated size and size distribution, whether monomodal or polymodal in nature. In addition, the results show that the method is suitable to measure liposomes in the presence of serum proteins and can yield information on the shape and physical stability of the structures. The optimized MD-AF4 based method has been validated across different instrument platforms, three laboratories, and multiple drug formulations following a comprehensive analysis of factors that influence the fractionation process and subsequent physical characterization. Interlaboratory reproducibility and intra-laboratory precision were evaluated, identifying sources of bias and establishing criteria for the acceptance of results. This method meets a documented high priority need in regulatory science as applied to NEPs such as Doxil and can be adapted to the measurement of other NEP forms (such as lipid nanoparticle therapeutics) with some modifications. Overall, this method will help speed up development of NEPS, and facilitate their regulatory review, ultimately leading to faster translation of innovative concepts from the bench to the clinic. Additionally, the approach used in this work (based on international collaboration between leading non-regulatory institutions) can be replicated to address other identified gaps in the analytical characterization of various classes of NEPs. Finally, a plan exists to pursue more extended interlaboratory validation studies to advance this method to a consensus international standard., Graphical abstract Unlabelled Image

The broadly neutralizing anti-HIV antibody, 10-1074, is a highly somatically hypermutated IgG1 being developed for prophylaxis in sub-Saharan Africa. A series of algorithms were applied to identify potentially destabilizing residues in the framework of the Fv region. Of 17 residues defined, a variant was identified encompassing 1 light and 3 heavy chain residues, with significantly increased conformational stability while maintaining full neutralization activity. Central to the stabilization was the replacement of the heavy chain residue T108 with R108 at the base of the CDR3 loop which allowed for the formation of a nascent salt bridge with heavy chain residue D137. Three additional mutations were necessary to confer increased conformational stability as evidenced by differential scanning fluorimetry and isothermal chemical unfolding. In addition, we observed increased stability during low pH incubation in which 40% of the parental monomer aggregated while the combinatorial variant showed no increase in aggregation. Incubation of the variant at 100 mg/mL for 6 weeks at 40°C showed a 9-fold decrease in subvisible particles ≥2 μm relative to the parental molecule. Stability-based designs have also translated to improved pharmacokinetics. Together, these data show that increasing conformational stability of the Fab can have profound effects on the manufacturability and long-term stability of a monoclonal antibody.

Graphical abstract, Highlights • Phenylhydrazine induces structural and functional changes in erythrocytes. • Oleic acid ameliorates these changes possibly through its antioxidant mechanisms. • Oleic acid can be a benefitting molecule in combating phenylhydrazine toxicity., Phenylhydrazine (PHZ), an intermediate in the synthesis of fine chemicals is toxic for human health and environment. Despite of having severe detrimental effects on different physiological systems, exposure of erythrocytes to PHZ cause destruction of haemoglobin and membrane proteins leading to iron release and complete haemolysis of red blood cells (RBC). Involvement of oxidative stress behind such action triggers the urge for searching a potent antioxidant. The benefits of consuming olive oil is attributed to its 75% oleic acid (OA) content in average. Olive oil is the basic component of Mediterranean diet. Hence, OA has been chosen in our present in vitro study to explore its efficacy against PHZ (1 mM) induced alterations in erythrocytes. Four different concentrations of OA (0.01 nM, 0.02 nM, 0.04 nM and 0.06 nM) were primarily experimented with, among which 0.06 nM OA has shown to give maximal protection. This study demonstrates the capability of OA in preserving the morphology, intracellular antioxidant status and the activities of metabolic enzymes of RBCs that have been diminished by PHZ, through its antioxidant mechanisms. The results of the present study firmly establish OA as a promising antioxidant for conserving the health of erythrocyte from PHZ toxicity which indicate toward future possible use of OA either singly or in combination with other dietary components for protection of erythrocytes against PHZ induced toxic cellular changes.

Graphical abstract, Highlights • The pattern of the effect on Ea.hy926 redox status differs among flavored e-liquids. • Tobacco flavored e-liquids increase ROS generation with concomitant increase in TBARS. • Vanilla flavored e-liquids profile depends on the nicotine content. • Apple/mint flavored e-liquids activate the cellular antioxidant defense. • Flavorings and not the nicotine content play a key role in free radical generation., Electronic cigarettes are constantly gaining ground as they are considered less harmful than conventional cigarettes, and there is also the perception that they may serve as a potential smoking cessation tool. Although the acute effects of electronic cigarette use have been extensively studied, the long-term potential adverse effects on human health remain largely unknown. It has been well-established that oxidative stress is involved in the development of various pathological conditions. So far, most studies on e-cigarettes concern the effects on the respiratory system while fewer have focused on the vascular system. In the present study, we attempted to reveal the effects of electronic cigarette refill liquids on the redox state of human endothelial cells (EA.hy926 cell line). For this purpose, the cytotoxic effect of three e-liquids with different flavors (tobacco, vanilla, apple/mint) and nicotine concentrations (0, 6, 12, 18 mg/ml) were initially examined for their impact on cell viability of EA.hy926 cells. Then, five redox biomarkers [reduced form of glutathione (GSH), reactive oxygen species (ROS), total antioxidant capacity (TAC), thiobarbituric acid reactive substances (TBARS) and protein carbonyls (CARBS)] were measured. The results showed a disturbance in the redox balance in favor of free radicals in tobacco flavored e-liquids while vanilla flavored e-liquids exhibited a more complex profile depending on the nicotine content. The most interesting finding of the present study concerns the apple/mint flavored e-liquids that seemed to activate the cellular antioxidant defense and, thus, to protect the cells from the adverse effects of free radicals. Conclusively, it appears that the flavorings and not the nicotine content play a key role in the oxidative stress-induced toxicity of the e-liquids.

The airborne transmission of SARS-CoV-2 remains surprisingly controversial; indeed, health and regulatory authorities still require direct proof of this mode of transmission. To close this gap, we measured the viral load of SARS-CoV-2 of an infected subject in a hospital room (through an oral and nasopharyngeal swab), as well as the airborne SARS-CoV-2 concentration in the room resulting from the person breathing and speaking. Moreover, we simulated the same scenarios to estimate the concentration of RNA copies in the air through a novel theoretical approach and conducted a comparative analysis between experimental and theoretical results. Results showed that for an infected subject's viral load ranging between 2.4 × 106 and 5.5 × 106 RNA copies mL-1, the corresponding airborne SARS-CoV-2 concentration was below the minimum detection threshold when the person was breathing, and 16.1 (expanded uncertainty of 32.8) RNA copies m-3 when speaking. The application of the predictive approach provided concentrations metrologically compatible with the available experimental data (i.e. for speaking activity). Thus, the study presented significant evidence to close the gap in understanding airborne transmission, given that the airborne SARS-CoV-2 concentration was shown to be directly related to the SARS-CoV-2 emitted. Moreover, the theoretical analysis was shown to be able to quantitatively link the airborne concentration to the emission., Graphical Abstract ga1

Background: BM32, a grass pollen allergy vaccine containing four recombinant fusion proteins consisting of hepatitis B-derived PreS and hypoallergenic peptides from the major timothy grass pollen allergens adsorbed on aluminium hydroxide has been shown to be safe and to improve clinical symptoms of grass pollen allergy upon allergen-specific immunotherapy (AIT). We have investigated the immune responses in patients from a two years double-blind, placebo-controlled AIT field trial with BM32. Methods: Blood samples from patients treated with BM32 (n = 27) or placebo (Aluminium hydroxide) (n = 13) were obtained to study the effects of vaccination and natural allergen exposure on allergen-specific antibody, T cell and cytokine responses. Allergen-specific IgE, IgG, IgG1 and IgG4 levels were determined by ImmunoCAP and ELISA, respectively. Allergen-specific lymphocyte proliferation by 3H thymidine incorporation and multiple cytokine responses with a human 17-plex cytokine assay were studied in cultured peripheral blood mononuclear cells (PBMCs). Findings: Two years AIT comprising two courses of 3 pre-seasonal injections of BM32 and a single booster after the first pollen season induced a continuously increasing (year 2 > year 1) allergen-specific IgG4 response without boosting allergen-specific IgE responses. Specific IgG4 responses were accompanied by low stimulation of allergen-specific PBMC responses. Increases of allergen-specific pro-inflammatory cytokine responses were absent. The rise of allergen-specific IgE induced by seasonal grass pollen exposure was partially blunted in BM32-treated patients. Interpretation: AIT with BM32 is characterised by the induction of a non-inflammatory, continuously increasing allergen-specific IgG4 response (year 2 > year1) which may explain that clinical efficacy was higher in year 2 than in year 1. The good safety profile of BM32 may be explained by lack of IgE reactivity and low stimulation of allergen-specific T cell and cytokine responses. Fundings: Grants F4605, F4613 and DK 1248-B13 of the Austrian Science Fund (FWF). Keywords: Allergy, Allergen, Allergen-specific immunotherapy (AIT), Recombinant allergy vaccine, BM32, IgE, IgG subclass, T cell response, Cytokine response

We report clinical, serologic, and immunogenetic studies of a set of monozygotic male twin patients who develop autoimmune thyroiditis and vitiligo associated with the HLA-DRB1*04-DQB1*03:02 and HLA-DRB1*03-DQB1*0201 haplotypes. The patients had detectable anti-thyroid and anti-melanocyte autoantibodies. A critical review is presented regarding the role of MHC II molecules linked to clinical manifestations of various autoimmune diseases displayed in a single patient, as is the case in the twin patients reported here., Highlights • Multiple autoimmunity is a clinicopathological issue that is not well understood. • Monozygotic twins with thyroiditis, vitiligo, HLA-DRB1*04-DQB1*03:02 and HLA-DRB1*03-DQBI*0201 haplotypes. • Multiple autoantibodies related with intermolecular epitope spreading. Epitope handling by MHC proteins probably related with multiple autoimmunity.

Highlights • Spontaneous dog cancers closely resemble human cancer. • Dogs with EGFR associated tumors were immunized with an EGFR/HER2 peptide vaccine. • EGFR peptide vaccinated dogs developed anti-EGFR/HER2 antibodies. • Vaccinated dogs have anti-EGFR antibody and T cells infiltrating tumors. • Vaccinated dogs with osteosarcoma had tumor regression and increased survival., Epidermal Growth Factor Receptor (EGFR) is overexpressed on a number of human cancers, and often is indicative of a poor outcome. Treatment of EGFR/HER2 overexpressing cancers includes monoclonal antibody therapy (cetuximab/trastuzumab) either alone or in conjunction with other standard cancer therapies. While monoclonal antibody therapy has been proven to be efficacious in the treatment of EGFR/HER2 overexpressing tumors, drawbacks include the lack of long-lasting immunity and acquired resistance to monoclonal therapy. An alternative approach is to induce a polyclonal anti-EGFR/HER2 tumor antigen response by vaccine therapy. In this phase I/II open-label study, we examined anti-tumor immunity in companion dogs with spontaneous EGFR expressing tumors. Canine cancers represent an outbred population in which the initiation, progression of disease, mutations and growth factors closely resemble that of human cancers. Dogs with EGFR expressing tumors were immunized with a short peptide of the EGFR extracellular domain with sequence homology to HER2. Serial serum analyses demonstrated high titers of EGFR/HER2 binding antibodies with biological activity similar to that of cetuximab and trastuzumab. Canine antibodies bound both canine and human EGFR on tumor cell lines and tumor tissue. CD8 T cells and IgG deposition were evident in tumors from immunized dogs. The antibodies inhibited EGFR intracellular signaling and inhibited tumor growth in vitro. Additionally, we illustrate objective responses in reducing tumors at metastatic sites in host animals. The data support the approach of amplifying anti-tumor immunity that may be relevant in combination with other immune modifying therapies such as checkpoint inhibitors.

Fatal familial insomnia (FFI) is a dominantly inherited prion disease linked to the D178N mutation in the gene encoding the prion protein (PrP). Symptoms, including insomnia, memory loss and motor abnormalities, appear around 50 years of age, leading to death within two years. No treatment is available. A ten-year clinical trial of doxycycline (doxy) is under way in healthy individuals at risk of FFI to test whether presymptomatic doxy prevents or delays the onset of disease. To assess the drug's effect in a tractable disease model, we used Tg(FFI-26) mice, which accumulate aggregated and protease-resistant PrP in their brains and develop a fatal neurological illness highly reminiscent of FFI. Mice were treated daily with 10 mg/kg doxy starting from a presymptomatic stage for twenty weeks. Doxy rescued memory deficits and restored circadian motor rhythmicity in Tg(FFI-26) mice. However, it did not prevent the onset and progression of motor dysfunction, clinical signs and progression to terminal disease. Doxy did not change the amount of aggregated and protease-resistant PrP, but reduced microglial activation in the hippocampus. Presymptomatic doxy treatment rescues cognitive impairment and the motor correlates of sleep dysfunction in Tg(FFI-26) mice but does not prevent fatal disease., Highlights • Early doxycycline treatment improves memory in fatal familial insomnia mice. • Doxycycline restores normal circadian motor rhythmicity. • Doxycycline reduces microglial activation in the hippocampus. • Doxycycline does not reduce mutant prion protein aggregation or protease resistance. • Doxycycline does not delay disease onset or prolong survival of the mice.

International audience; The rigid core of intracellular tau filaments from Alzheimer’s disease (AD), Pick’s disease (PiD), and Corticobasal disease (CBD) brains has been shown to differ in their cryo-EM atomic structure. Despite providing critical information on the intimate arrangement of a fraction of htau molecule within the fibrillar scaffold, the cryo-EM studies neither yield a complete picture of tau fibrillar assemblies structure nor contribute insights into the surfaces that define their interactions with numerous cellular components. Here, using proteomic approaches such as proteolysis and molecular covalent painting, we mapped the exposed amino acid stretches at the surface and those constituting the fibrillar core of in vitro-assembled fibrils of human htau containing one N-terminal domain and three (1N3R) or four (1N4R) C-terminal microtubule-binding repeat domains as a result of alternative splicing. Using limited proteolysis, we identified the proteolytic fragments composing the molecular “bar-code” for each type of fibril. Our results are in agreement with structural data reported for filamentous tau from AD, PiD, and CBD cases predigested with the protease pronase. Finally, we report two amino acid stretches, exposed to the solvent in 1N4R not in 1N3R htau, which distinguish the surfaces of these two kinds of fibrils. Our findings open new perspectives for the design of highly specific ligands with diagnostic and therapeutic potential.

Background Studying human ageing is of increasing importance due to the worldwide ageing population. However, it faces the challenge of lengthy experiments to produce an ageing phenotype. Often, to recreate the hallmarks of ageing requires complex empirical conditions that can confound data interpretation. Indeed, many studies use whole organisms with relatively short life spans, which may have little, or limited, relevance to human ageing. There has been extensive use of cell lines to study ageing in human somatic cells, but the modelling of human neuronal ageing is somewhat more complex in vitro. New Method We cultured the well-characterised SH-SY5Y human neural cell line to produce high purity cultures of cells differentiated to express a neuronal phenotype, and designed a protocol to maintain these cells in culture until they accumulated biomarkers of cellular ageing. Results Our data validate a novel and simple technique for the efficient differentiation and long-term maintenance of SH-SY5Y cells, expressing markers of neuronal differentiation and demonstrating electrical activity in culture. Over time in vitro, these cells progressively accumulate markers of ageing such as enhanced production of reactive oxygen species and accumulation of oxidative damage. Comparison to Existing Methods In comparison to existing techniques to model neuronal ageing our method is cost effective, requiring no specialist equipment or growth factors. Conclusions We demonstrate that SH-SY5Y cells, grown under these culture conditions, represent a simple model of neuronal ageing that is amenable to cell biological, biochemical and electrophysiological investigation., Highlights • Ageing study is often hindered by the need for complex and lengthy experiments. • SH-SY5Y cells underwent neuronal differentiation and were cultured until they were of an aged phenotype. • These cells were electrically active and acquired oxidative damage. • This is a novel technique to model neuronal ageing in vitro.

Highlights • Ribosome display used for recombinant antibody selection and scFv generation targeting CCK-BR. • Engineered scFv have stronger affinity and increased tissue penetrability due to smaller size. • Single dose scFv reduces hypersensitivity, anxiety- and depression-like behavior in two models. • Reduced neuronal firing frequency in TG primary neuronal cultures treated in vitro. • CCK-BR scFv is ideal for neuropathic pain with both nociceptive and emotional components., The cholecystokinin B receptor and its neuropeptide ligand are upregulated in chronic neuropathic pain models. Single-chain Fragment variable antibodies were generated as preferred non-opioid targeting therapy blocking the cholecystokinin B receptor to inhibit chronic neuropathic pain models in vivo and in vitro. Engineered antibodies of this type feature binding activity similar to monoclonal antibodies but with stronger affinity and increased tissue penetrability due to their smaller size. More importantly, single-chain Fragment variable antibodies have promising biotherapeutic applications for both nervous and immune systems, now recognized as interactive in chronic pain. A mouse single-chain Fragment variable antibody library recognizing a fifteen amino acid extracellular peptide fragment of the cholecystokinin B receptor was generated from immunized spleens. Ribosome display, a powerful cell-free technology, was applied for recombinant antibody selection. Antibodies with higher affinity, stability, solubility, and binding specificity for cholecystokinin B not A receptor were selected and optimized for in vivo and in vitro efficacy. A single dose of the lead candidate reduced mechanical and cold hypersensitivity in two rodent models of neuropathic pain for at least seven weeks. Continuing efficacy was evident with either intraperitoneal or intranasal dosing. Likewise, the lead single-chain Fragment variable antibody totally prevented development of anxiety- and depression-like behaviors and cognitive deficits typical in the models. Reduction of neuronal firing frequency was evident in trigeminal ganglia primary neuronal cultures treated in vitro with the cholecystokinin B receptor antibody. Immunofluorescent staining intensity in the trigeminal neuron primary cultures was significantly reduced incrementally after overnight binding with increasingly higher dilutions of the single-chain Fragment variable antibody. While it is reported that single-chain Fragment variable antibodies are removed systemically within 2–6 h, Western blot evidence indicates the His-tag marker remained after 7 weeks in the trigeminal ganglia and in the dorsolateral medulla, providing evidence of brain and ganglia penetrance known to be compromised in overactivated states. This project showcases the in vivo efficacy of our lead single-chain Fragment variable antibody indicating its potential for development as a non-opioid, non-addictive therapeutic intervention for chronic pain. Importantly, studies by others have indicated treatments with cholecystokinin B receptor antagonists suppress maintenance and reactivation of morphine dependence in place preference tests while lowering tolerance and dose requirements. Our future studies remain to address these potential benefits that may accompany the cholecystokinin B receptor biological therapy. Both chronic sciatic and orofacial pain can be unrelenting and excruciating, reducing quality of life as well as diminishing physical and mental function. An effective non-opiate, non-addictive therapy with potential to significantly reduce chronic neuropathic pain long term is greatly needed.

Long-term cannabis use during adolescence has deleterious effects in brain that are largely ascribed to the activation of cannabinoid-1 receptors (CB1Rs) by delta-9-tetrahydrocannabinol (∆9-THC), the primary psychoactive compound in marijuana. Systemic administration of ∆9-THC inhibits acetylcholine release in the prelimbic-prefrontal cortex (PL-PFC). In turn, PL-PFC acetylcholine plays a role in executive activities regulated by CB1R-targeting endocannabinoids, which are generated by cholinergic stimulation of muscarinic-1 receptors (M1Rs). However, the long-term effects of chronic administration of increasing doses of ∆9-THC in adolescent males on the distribution and function of M1 and/or CB1 receptors in the PL-PFC remains unresolved. We used C57BL\6J male mice pre-treated with vehicle or escalating daily doses of ∆9-THC to begin filling this gap. Electron microscopic immunolabeling showed M1R-immunogold particles on plasma membranes and in association with cytoplasmic membranes in varying sized dendrites and dendritic spines. These dendritic profiles received synaptic inputs from unlabeled, CB1R- and/or M1R-labeled axon terminals in the PL-PFC of both treatment groups. However, there was a size-dependent decrease in total (plasmalemmal and cytoplasmic) M1R gold particles in small dendrites within the PL-PFC of mice receiving ∆9-THC. Whole cell current-clamp recording in PL-PFC slice preparations further revealed that adolescent pretreatment with ∆9-THC attenuates the hyperpolarization and increases the firing rate produced by local muscarinic stimulation. Repeated administration of ∆9-THC during adolescence also reduced spontaneous alternations in a Y-maze paradigm designed for measures of PFC-dependent memory function in adult mice. Our results provide new information implicating M1Rs in cortical dysfunctions resulting from adolescent abuse of marijuana., Highlights • Cholinergic M1 receptors are located on dendritic plasma and cytoplasmic membranes of in the PL-PFC of adult Mice. • Repeated administration of ∆9-THC during adolescents impacts the dendritic density of M1Rs in the PL-PFC of adults. • Dendritic profiles expressing M1 receptors receive input from inhibitory- and excitatory-type terminals containing CB1Rs. • Chronic ∆9-THC in adolescence attenuates muscarinic evoked hyperpolarization and increases the firing rate of PL-PFC neurons. • Adolescent ∆9-THC reduces PFC-dependent memory function in adult mice.

Monoclonal antibodies can acquire the property of engagement of a second antigen via fusion methods or modification of their CDR loops, but also by modification of their constant domains, such as in the mAb2 format where a set of mutated amino acid residues in the CH3 domains enables a high-affinity specific interaction with the second antigen. We tested the possibility of introducing multiple binding sites for the second antigen by replacing the Fab CH1/CL domain pair with a pair of antigen-binding CH3 domains in a model scaffold with trastuzumab variable domains and VEGF-binding CH3 domains. Such bispecific molecules were produced in a “Fab-like” format and in a full-length antibody format. Novel constructs were of expected molecular composition using mass spectrometry. They were expressed at a high level in standard laboratory conditions, purified as monomers with Protein A and gel filtration and were of high thermostability. Their high-affinity binding to both target antigens was retained. Finally, the Her2/VEGF binding domain-exchanged bispecific antibody was able to mediate a potentiated surface Her2-internalization effect on the Her2-overexpressing cell line SK-BR-3 due to improved level of cross-linking with the endogenously secreted cytokine. To conclude, bispecific antibodies with Fabs featuring exchanged antigen-binding CH3 domains offer an alternative solution in positioning and valency of antigen binding sites., Graphical abstract Image 1, Highlights • Fab constant domains can be efficiently exchanged for antigen-binding CH3 domains. • Such mutagenesis results in bispecific antibodies with correct chain pairing. • Domain-exchanged bispecific Fab- and IgG-like formats are of favorable biophysical properties. • Resulting bispecific antibodies show high-affinity binding to both target antigens.

Highlights • Hyperglycemia is known to accelerate oxidative stress-induced myocardial injury. • Mitochondrial energetics is an important mechanism to explore in the diabetic heart. • NAC protects against hyperglycemia-induced cardiomyocyte toxicity. • NAC improves mitochondrial energetics and enhances endogenous CoQ levels. • CoQ supports the process of bioenergetics in addition to its antioxidant activities., The diabetic heart has been linked with reduced endogenous levels of coenzyme Q9/10 (CoQ), an important antioxidant and component of the electron transport chain. Although CoQ has displayed cardioprotective potential in experimental models of diabetes, the impact of N-acetyl cysteine (NAC) on mitochondrial energetics and endogenous levels of CoQ remains to be clarified. To explore these effects, high glucose-exposed H9c2 cardiomyocytes were used as an experimental model of hyperglycemia-induced cardiac injury. The results showed that high glucose exposure caused an increased production of reactive oxygen species (ROS), which was associated with impaired mitochondrial energetics as confirmed by a reduction of maximal respiration rate and depleted ATP levels. These detrimental effects were consistent with significantly reduced endogenous CoQ levels and accelerated cell toxicity. Although metformin demonstrated similar effects on mitochondrial energetics and cell viability, NAC demonstrated a more pronounced effect in ameliorating cytosolic and mitochondrial ROS production. Interestingly, the ameliorative effects of NAC against hyperglycemia-induced injury were linked with its capability to enhance endogenous CoQ levels. Although such data are to be confirmed in other models, especially in vivo studies, the overall findings provide additional evidence on the therapeutic mechanisms by which NAC protects against diabetes-induced cardiac injury.

The assembly of tau protein into abnormal filaments and brain cell degeneration are characteristic of a number of human neurodegenerative diseases, including Alzheimer's disease and frontotemporal dementia and parkinsonism linked to chromosome 17. Several murine models have been generated to better understand the mechanisms contributing to tau assembly and neurodegeneration. Taking advantage of the more elaborate central nervous system and higher cognitive abilities of the rat, we generated a model expressing the longest human tau isoform (2N4R) with the P301S mutation. This transgenic rat line, R962-hTau, exhibits the main features of human tauopathies, such as: age-dependent increase in inclusions comprised of aggregated-tau, neuronal loss, global neurodegeneration as reflected by brain atrophy and ventricular dilation, alterations in astrocytic and microglial morphology, and myelin loss. In addition, substantial deficits across multiple memory and learning paradigms, including novel object recognition, fear conditioning and Morris water maze tasks, were observed at the time of advanced tauopathy. These results support the concept that progressive tauopathy correlates with brain atrophy and cognitive impairment., Highlights • A transgenic rat model expressing the full length human tau with the P301S mutation. • Progressive human-like tau brain pathology with recruitment of endogenous rodent tau. • Pathology-induced gliosis, myelin breakdown, neuronal loss and ventricular dilation. • Widespread behavioural deficits occur at end-stage tau pathology.

Background DNA damage repair (DDR) alterations are important events in cancer initiation, progression, and therapeutic resistance. However, the involvement of DDR alterations in glioma malignancy needs further investigation. This study aims to characterize the clinical and molecular features of gliomas with DDR alterations and elucidate the biological process of DDR alterations that regulate the cross talk between gliomas and the tumor microenvironment. Methods Integrated transcriptomic and genomic analyses were undertaken to conduct a comprehensive investigation of the role of DDR alterations in glioma. The prognostic DDR-related cytokines were identified from multiple datasets. In vivo and in vitro experiments validated the role of p53, the key molecule of DDR, regulating M2 polarization of microglia in glioma. Findings DDR alterations are associated with clinical and molecular characteristics of glioma. Gliomas with DDR alterations exhibit distinct immune phenotypes, and immune cell types and cytokine processes. DDR-related cytokines have an unfavorable prognostic implication for GBM patients and are synergistic with DDR alterations. Overexpression of MDK mediated by p53, the key transcriptional factor in DDR pathways, remodels the GBM immunosuppressive microenvironment by promoting M2 polarization of microglia, suggesting a potential role of DDR in regulating the glioma microenvironment. Interpretation Our work suggests that DDR alterations significantly contribute to remodeling the glioma microenvironment via regulating the immune response and cytokine pathways. Fund This study was supported by: 1. The National Key Research and Development Plan (No. 2016YFC0902500); 2. National Natural Science Foundation of China (No. 81702972, No. 81874204, No. 81572701, No. 81772666); 3. China Postdoctoral Science Foundation (2018M640305); 4. Special Fund Project of Translational Medicine in the Chinese-Russian Medical Research Center (No. CR201812); 5. The Research Project of the Chinese Society of Neuro-oncology, CACA (CSNO-2016-MSD12); 6. The Research Project of the Health and Family Planning Commission of Heilongjiang Province (2017–201); and 7. Harbin Medical University Innovation Fund (2017LCZX37, 2017RWZX03)., Highlights • Gliomas with DNA damage repair alterations had distinct genomic variation spectrum. • DDR alterations exhibit distinct immune phenotypes, cytokine processes and immune cell types in glioma. • DDR-related cytokines in GME have an unfavorable prognostic implication for GBM patients. • P53-mediated midkine expression derived from glioma cells promotes M2 polarization of microglia.

Background Nitrite is reduced by heme-proteins and molybdenum-containing enzymes to form the important signaling molecule nitric oxide (NO), mediating NO signaling. Substantial evidence suggests that deoxygenated hemoglobin within red blood cells (RBCs) is the main erythrocytic protein responsible for mediating nitrite-dependent NO signaling. In other work, infrared and far red light have been shown to have therapeutic potential that some attribute to production of NO. Here we explore whether a combination of nitrite and far red light treatment has an additive effect in NO-dependent processes, and whether this effect is mediated by RBCs. Methods and results Using photoacoustic imaging in a rat model as a function of varying inspired oxygen, we found that far red light (660 nm, five min. exposure) and nitrite feeding (three weeks in drinking water at 100 mg/L) each separately increased tissue oxygenation and vessel diameter, and the combined treatment was additive. We also employed inhibition of human platelet activation measured by flow cytometry to assess RBC-dependent nitrite bioactivation and found that far red light dramatically potentiates platelet inhibition by nitrite. Blocking RBC-surface thiols abrogated these effects of nitrite and far-red light. RBC-dependent production of NO was also shown to be enhanced by far red light using a chemiluminescence-based nitric oxide analyzer. In addition, RBC-dependent bioactivation of nitrite led to prolonged lag times for clotting in platelet poor plasma that was enhanced by exposure to far red light. Conclusions Our results suggest that nitrite leads to the formation of a photolabile RBC surface thiol-bound species such as an S-nitrosothiol or heme-nitrosyl (NO-bound heme) for which far red light enhances NO signaling. These findings expand our understanding of RBC-mediated NO production from nitrite. This pathway of NO production may have therapeutic potential in several applications including thrombosis, and, thus, warrants further study., Graphical abstract Potential mechanism of RBC-mediated nitrite bioactivation and its potentiation by far red light. Nitrite reacts with deoxygenated Hb to make NO which then binds other vacant hemes or forms a nitrosothiol (RSNO). The nitrosyl-heme or nitrosothiol is exported and binds a surface thiol. Another potential NO species that may form is a DNIC (not shown). The NO congener can then be transported in plasma and interact with platelets and other blood components and this action is potentiated by photolysis using far red light.fx1, Highlights • Nitrite and far red light increase tissue oxygenation under hypoxia. • Far red light enhances nitrite-mediated inhibition of platelet activation by red cells. • Blocking red cell surface thiols abrogates nitrite-mediated effects. • NO production from nitrite and red cells is enhanced by far red light. • Lag time in clotting is prolonged by nitrite plus far red light.

Erythrocytes (red blood cells, RBCs) are the most abundant circulating cells in the blood and have been widely used in drug delivery systems (DDS) because of their features of biocompatibility, biodegradability, and long circulating half-life. Accordingly, a “camouflage” comprised of erythrocyte membranes renders nanoparticles as a platform that combines the advantages of native erythrocyte membranes with those of nanomaterials. Following injection into the blood of animal models, the coated nanoparticles imitate RBCs and interact with the surroundings to achieve long-term circulation. In this review, the biomimetic platform of erythrocyte membrane-coated nano-cores is described with regard to various aspects, with particular focus placed on the coating mechanism, preparation methods, verification methods, and the latest anti-tumor applications. Finally, further functional modifications of the erythrocyte membranes and attempts to fuse the surface properties of multiple cell membranes are discussed, providing a foundation to stimulate extensive research into multifunctional nano-biomimetic systems., Graphical abstract In this review, the biomimetic platform of erythrocyte membrane-coated nano-cores is described with regard to coating mechanism, preparation methods, verification methods, and the latest anti-tumor applications. Finally, further functional modifications of the erythrocyte membranes and attempts to fuse the surface properties of multiple cell membranes are discussed.fx1

Monoamine oxidases (MAOs) are located on the outer mitochondrial membrane and are drug targets for the treatment of neurological disorders. MAOs control the levels of neurotransmitters in the brain via oxidative deamination and contribute to reactive oxygen species (ROS) generation through their catalytic by-product H2O2. Increased ROS levels may modulate mitochondrial function and mitochondrial dysfunction is implicated in a vast array of disorders. However, the downstream effects of MAO-A mediated ROS production in a neuronal model has not been previously investigated. In this study, using MAO-A overexpressing neuroblastoma cells, we demonstrate that higher levels of MAO-A protein/activity results in increased basal ROS levels with associated increase in protein oxidation. Increased MAO-A levels result in increased Lysine-63 linked ubiquitination of mitochondrial proteins and promotes autophagy through Bcl-2 phosphorylation. Furthermore, ROS generated locally on the mitochondrial outer membrane by MAO-A promotes phosphorylation of dynamin-1-like protein, leading to mitochondrial fragmentation and clearance without complete loss of mitochondrial membrane potential. Cellular ATP levels are maintained following MAO-A overexpression and complex IV activity/protein levels increased, revealing a close relationship between MAO-A levels and mitochondrial function. Finally, the downstream effects of increased MAO-A levels are dependent on the availability of amine substrates and in the presence of exogenous substrate, cell viability is dramatically reduced. This study shows for the first time that MAO-A generated ROS is involved in quality control signalling, and increase in MAO-A protein levels leads to a protective cellular response in order to mediate removal of damaged macromolecules/organelles, but substrate availability may ultimately determine cell fate. The latter is particularly important in conditions such as Parkinson's disease, where a dopamine precursor is used to treat disease symptoms and highlights that the fate of MAO-A containing dopaminergic neurons may depend on both MAO-A levels and catecholamine substrate availability., Graphical abstract fx1, Highlights • Increased MAO-A levels result in increased ROS and protein oxidation. • Increased MAO-A promotes protective autophagy and mitochondrial clearance. • MAO-A modulates mitochondrial health and function. • Availability of amine substrate regulates the effects of increased MAO-A levels. • MAO-A has a role in autophagy-apoptosis crosstalk and regulates cell survival.