BAG3 regulates cilia homeostasis of glioblastoma via its WW domain.

The multidomain protein BAG3 exerts pleiotropic oncogenic functions in many tumor entities including glioblastoma (GBM). Here, we compared BAG3 protein-protein interactions in either adherently cultured or stem-like cultured U251 GBM cells. In line with BAG3's putative role in regulating stem-like properties, identified interactors in sphere-cultured cells included different stem cell markers (SOX2, OLIG2, and NES), while interactomes of adherent BAG3-proficient cells indicated a shift toward involvement of BAG3 in regulation of cilium assembly (ACTR3 and ARL3). Applying a set of BAG3 deletion constructs we could demonstrate that none of the domains except the WW domain are required for suppression of cilia formation by full-length BAG3 in U251 and U343 cells. In line with the established regulation of the Hippo pathway by this domain, we could show that the WW mutant fails to rescue YAP1 nuclear translocation. BAG3 depletion reduced activation of a YAP1/AURKA signaling pathway and induction of PLK1. Collectively, our findings point to a complex interaction network of BAG3 with several pathways regulating cilia homeostasis, involving processes related to ciliogenesis and cilium degradation.

TBC1D2B undergoes phase separation and mediates autophagy initiation.

Small ubiquitin-like modifiers from the ATG8 family regulate autophagy initiation and progression in mammalian cells. Their interaction with LC3-interacting region (LIR) containing proteins promotes cargo sequestration, phagophore assembly, or even fusion between autophagosomes and lysosomes. Previously, we have shown that RabGAP proteins from the TBC family directly bind to LC3/GABARAP proteins. In the present study, we focus on the function of TBC1D2B. We show that TBC1D2B contains a functional canonical LIR motif and acts at an early stage of autophagy by binding to both LC3/GABARAP and ATG12 conjugation complexes. Subsequently, TBC1D2B is degraded by autophagy. TBC1D2B condensates into liquid droplets upon autophagy induction. Our study suggests that phase separation is an underlying mechanism of TBC1D2B-dependent autophagy induction.

Ubiquitination regulates ER-phagy and remodelling of endoplasmic reticulum.

The endoplasmic reticulum (ER) undergoes continuous remodelling via a selective autophagy pathway, known as ER-phagy1. ER-phagy receptors have a central role in this process2, but the regulatory mechanism remains largely unknown. Here we report that ubiquitination of the ER-phagy receptor FAM134B within its reticulon homology domain (RHD) promotes receptor clustering and binding to lipidated LC3B, thereby stimulating ER-phagy. Molecular dynamics (MD) simulations showed how ubiquitination perturbs the RHD structure in model bilayers and enhances membrane curvature induction. Ubiquitin molecules on RHDs mediate interactions between neighbouring RHDs to form dense receptor clusters that facilitate the large-scale remodelling of lipid bilayers. Membrane remodelling was reconstituted in vitro with liposomes and ubiquitinated FAM134B. Using super-resolution microscopy, we discovered FAM134B nanoclusters and microclusters in cells. Quantitative image analysis revealed a ubiquitin-mediated increase in FAM134B oligomerization and cluster size. We found that the E3 ligase AMFR, within multimeric ER-phagy receptor clusters, catalyses FAM134B ubiquitination and regulates the dynamic flux of ER-phagy. Our results show that ubiquitination enhances RHD functions via receptor clustering, facilitates ER-phagy and controls ER remodelling in response to cellular demands.

Heteromeric clusters of ubiquitinated ER-shaping proteins drive ER-phagy.

Membrane-shaping proteins characterized by reticulon homology domains play an important part in the dynamic remodelling of the endoplasmic reticulum (ER). An example of such a protein is FAM134B, which can bind LC3 proteins and mediate the degradation of ER sheets through selective autophagy (ER-phagy)1. Mutations in FAM134B result in a neurodegenerative disorder in humans that mainly affects sensory and autonomic neurons2. Here we report that ARL6IP1, another ER-shaping protein that contains a reticulon homology domain and is associated with sensory loss3, interacts with FAM134B and participates in the formation of heteromeric multi-protein clusters required for ER-phagy. Moreover, ubiquitination of ARL6IP1 promotes this process. Accordingly, disruption of Arl6ip1 in mice causes an expansion of ER sheets in sensory neurons that degenerate over time. Primary cells obtained from Arl6ip1-deficient mice or from patients display incomplete budding of ER membranes and severe impairment of ER-phagy flux. Therefore, we propose that the clustering of ubiquitinated ER-shaping proteins facilitates the dynamic remodelling of the ER during ER-phagy and is important for neuronal maintenance.

Eliciting Patient Insights on the Burden of Nontuberculous Mycobacterial Lung Disease (NTM-LD) and Healthcare Gaps in Germany Through Qualitative Semi-structured Interviews.

INTRODUCTION: Nontuberculous mycobacterial lung disease (NTM-LD) is a rare but growing health concern, particularly affecting vulnerable patients with chronic lung conditions. Understanding the patients' perspective on their disease and treatment expectations can help to identify healthcare gaps and improve overall patient care. Therefore, the main objective of the survey study was the evaluation of patient insights on the burden of the disease and healthcare gaps. METHODS: The study used an online survey as a pre-screener to facilitate recruitment followed by semi-structured qualitative interviews. The interviews were conducted by phone from April 2019 to February 2020 in German language. Only patients with a self-reported confirmed NTM-LD diagnosis, managed and insured in Germany were included in this study. RESULTS: In total, 20 semi-structured qualitative interviews were conducted. Most (85%) patients had at least one coexisting pulmonary condition with cystic fibrosis (CF, n = 9) being most common. Chronic cough, fatigue, and dyspnea were the most reported symptoms. Of all the symptoms reported, fatigue was perceived as the most burdensome and 85% of patients felt limited in their daily life. It took a median of 5 months for patients to receive an accurate diagnosis of NTM-LD and that time was doubled when excluding patients with CF (range 0-480 months). Ninety percent of interviewed patients (n = 18) received drug treatment for NTM-LD and most of them (n = 17) reported having experienced side effects from their treatment. Patients' expressed a particular need for more comprehensive and reliable patient-friendly information on NTM-LD and a better awareness of physicians as well. CONCLUSIONS: NTM-LD can considerably impair the lives of patients and their families and/or caregivers. A multidisciplinary approach and establishment of more widespread regional expert centers for NTM-LD management in Germany with well-structured referral and communication pathways accompanied by peer-to-peer support of patient advocacy groups are urgently needed.

Targeting the MYC interaction network in B-cell lymphoma via histone deacetylase 6 inhibition.

Overexpression of MYC is a genuine cancer driver in lymphomas and related to poor prognosis. However, therapeutic targeting of the transcription factor MYC remains challenging. Here, we show that inhibition of the histone deacetylase 6 (HDAC6) using the HDAC6 inhibitor Marbostat-100 (M-100) reduces oncogenic MYC levels and prevents lymphomagenesis in a mouse model of MYC-induced aggressive B-cell lymphoma. M-100 specifically alters protein-protein interactions by switching the acetylation state of HDAC6 substrates, such as tubulin. Tubulin facilitates nuclear import of MYC, and MYC-dependent B-cell lymphoma cells rely on continuous import of MYC due to its high turn-over. Acetylation of tubulin impairs this mechanism and enables proteasomal degradation of MYC. M-100 targets almost exclusively B-cell lymphoma cells with high levels of MYC whereas non-tumor cells are not affected. M-100 induces massive apoptosis in human and murine MYC-overexpressing B-cell lymphoma cells. We identified the heat-shock protein DNAJA3 as an interactor of tubulin in an acetylation-dependent manner and overexpression of DNAJA3 resulted in a pronounced degradation of MYC. We propose a mechanism by which DNAJA3 associates with hyperacetylated tubulin in the cytoplasm to control MYC turnover. Taken together, our data demonstrate a beneficial role of HDAC6 inhibition in MYC-dependent B-cell lymphoma.

BAG3 is a negative regulator of ciliogenesis in glioblastoma and triple-negative breast cancer cells.

By regulating several hallmarks of cancer, BAG3 exerts oncogenic functions in a wide variety of malignant diseases including glioblastoma (GBM) and triple-negative breast cancer (TNBC). Here we performed global proteomic/phosphoproteomic analyses of CRISPR/Cas9-mediated isogenic BAG3 knockouts of the two GBM lines U343 and U251 in comparison to parental controls. Depletion of BAG3 evoked major effects on proteins involved in ciliogenesis/ciliary function and the activity of the related kinases aurora-kinase A and CDK1. Cilia formation was significantly enhanced in BAG3 KO cells, a finding that could be confirmed in BAG3-deficient versus -proficient BT-549 TNBC cells, thus identifying a completely novel function of BAG3 as a negative regulator of ciliogenesis. Furthermore, we demonstrate that enhanced ciliogenesis and reduced expression of SNAI1 and ZEB1, two key transcription factors regulating epithelial to mesenchymal transition (EMT) are correlated to decreased cell migration, both in the GBM and TNBC BAG3 knockout cells. Our data obtained in two different tumor entities identify suppression of EMT and ciliogenesis as putative synergizing mechanisms of BAG3-driven tumor aggressiveness in therapy-resistant cancers.

Calcitriol Promotes Differentiation of Glioma Stem-Like Cells and Increases Their Susceptibility to Temozolomide.

Glioblastoma (GBM) is the most common and most aggressive primary brain tumor, with a very high rate of recurrence and a median survival of 15 months after diagnosis. Abundant evidence suggests that a certain sub-population of cancer cells harbors a stem-like phenotype and is likely responsible for disease recurrence, treatment resistance and potentially even for the infiltrative growth of GBM. GBM incidence has been negatively correlated with the serum levels of 25-hydroxy-vitamin D3, while the low pH within tumors has been shown to promote the expression of the vitamin D3-degrading enzyme 24-hydroxylase, encoded by the CYP24A1 gene. Therefore, we hypothesized that calcitriol can specifically target stem-like glioblastoma cells and induce their differentiation. Here, we show, using in vitro limiting dilution assays, quantitative real-time PCR, quantitative proteomics and ex vivo adult organotypic brain slice transplantation cultures, that therapeutic doses of calcitriol, the hormonally active form of vitamin D3, reduce stemness to varying extents in a panel of investigated GSC lines, and that it effectively hinders tumor growth of responding GSCs ex vivo. We further show that calcitriol synergizes with Temozolomide ex vivo to completely eliminate some GSC tumors. These findings indicate that calcitriol carries potential as an adjuvant therapy for a subgroup of GBM patients and should be analyzed in more detail in follow-up studies.

The endolysosomal adaptor PLEKHM1 is a direct target for both mTOR and MAPK pathways.

The lysosome is a cellular signalling hub at the point of convergence of endocytic and autophagic pathways, where the contents are degraded and recycled. Pleckstrin homology domain-containing family member 1 (PLEKHM1) acts as an adaptor to facilitate the fusion of endocytic and autophagic vesicles with the lysosome. However, it is unclear how PLEKHM1 function at the lysosome is controlled. Herein, we show that PLEKHM1 coprecipitates with, and is directly phosphorylated by, mTOR. Using a phosphospecific antibody against Ser432/S435 of PLEKHM1, we show that the same motif is a direct target for ERK2-mediated phosphorylation in a growth factor-dependent manner. This dual regulation of PLEKHM1 at a highly conserved region points to a convergence of both growth factor- and amino acid-sensing pathways, placing PLEKHM1 at a critical juncture of cellular metabolism.

Mitochondrial PCK2 Missense Variant in Shetland Sheepdogs with Paroxysmal Exercise-Induced Dyskinesia (PED).

Four female Shetland Sheepdogs with hypertonic paroxysmal dyskinesia, mainly triggered by exercise and stress, were investigated in a retrospective multi-center investigation aiming to characterize the clinical phenotype and its underlying molecular etiology. Three dogs were closely related and their pedigree suggested autosomal dominant inheritance. Laboratory diagnostic findings included mild lactic acidosis and lactaturia, mild intermittent serum creatine kinase (CK) elevation and hypoglycemia. Electrophysiological tests and magnetic resonance imaging of the brain were unremarkable. A muscle/nerve biopsy revealed a mild type II fiber predominant muscle atrophy. While treatment with phenobarbital, diazepam or levetiracetam did not alter the clinical course, treatment with a gluten-free, home-made fresh meat diet in three dogs or a tryptophan-rich, gluten-free, seafood-based diet, stress-reduction, and acetazolamide or zonisamide in the fourth dog correlated with a partial reduction in, or even a complete absence of, dystonic episodes. The genomes of two cases were sequenced and compared to 654 control genomes. The analysis revealed a case-specific missense variant, c.1658G>A or p.Arg553Gln, in the PCK2 gene encoding the mitochondrial phosphoenolpyruvate carboxykinase 2. Sanger sequencing confirmed that all four cases carried the mutant allele in a heterozygous state. The mutant allele was not found in 117 Shetland Sheepdog controls and more than 500 additionally genotyped dogs from various other breeds. The p.Arg553Gln substitution affects a highly conserved residue in close proximity to the GTP-binding site of PCK2. Taken together, we describe a new form of paroxysmal exercise-induced dyskinesia (PED) in dogs. The genetic findings suggest that PCK2:p.Arg553Gln should be further investigated as putative candidate causal variant.

Síndrome de Zika congénito en la Argentina: presentación de dos casos clínicos / Congenital Zika syndrome in Argentina: case series study

En 2015, se observó un incremento en la incidencia de microcefalia congénita en recién nacidos en Brasil. Meses más tarde, se descubrió la relación causal entre el virus del Zika y estos hallazgos. Durante el primer brote en la Argentina, se reportaron 5 casos de síndrome de Zika congénito. En 2017, hubo un nuevo brote que involucró la provincia de Salta. En este trabajo, se presentan 2 casos clínicos con síndrome de Zika congénito autóctonos: una paciente con microcefalia congénita grave con lisencefalia, calcificaciones corticosubcorticales y ventriculomegalia y otra paciente con microcefalia posnatal con polimicrogiria asimétrica y calcificaciones subcorticales y retraso en la mielinización. El real impacto de esta enfermedad aún es incierto; es necesario un adecuado seguimiento multidisciplinario de los pacientes expuestos al virus del Zika para comprender mejor la infección y su historia natural.

In 2015, there was an increase in the incidence of congenital microcephaly in newborns in Brazil. Months later, the causal relationship between Zika virus and these findings was discovered. In Argentina, during the first outbreak there were 5 cases of congenital Zika syndrome reported. In 2017, there was a new outbreak which involved Salta province. We describe 2 patients with autochthonous congenital Zika syndrome: one of the babies with severe congenital microcephaly with lissencephaly, calcifications and ventriculomegaly; and another baby with postnatal microcephaly with asymmetric polymicrogyria, calcifications and delayed myelination. The real impact of this disease is still uncertain, so it is necessary an adequate multidisciplinary monitoring of patients exposed to Zika virus to better understand the infection and its natural history.

[Congenital Zika syndrome in Argentina: case series study]. / Síndrome de Zika congénito en la Argentina: presentación de dos casos clínicos.

In 2015, there was an increase in the incidence of congenital microcephaly in newborns in Brazil. Months later, the causal relationship between Zika virus and these findings was discovered. In Argentina, during the first outbreak there were 5 cases of congenital Zika syndrome reported. In 2017, there was a new outbreak which involved Salta province. We describe 2 patients with autochthonous congenital Zika syndrome: one of the babies with severe congenital microcephaly with lissencephaly, calcifications and ventriculomegaly; and another baby with postnatal microcephaly with asymmetric polymicrogyria, calcifications and delayed myelination. The real impact of this disease is still uncertain, so it is necessary an adequate multidisciplinary monitoring of patients exposed to Zika virus to better understand the infection and its natural history.

En 2015, se observó un incremento en la incidencia de microcefalia congénita en recién nacidos en Brasil. Meses más tarde, se descubrió la relación causal entre el virus del Zika y estos hallazgos. Durante el primer brote en la Argentina, se reportaron 5 casos de síndrome de Zika congénito. En 2017, hubo un nuevo brote que involucró la provincia de Salta. En este trabajo, se presentan 2 casos clínicos con síndrome de Zika congénito autóctonos: una paciente con microcefalia congénita grave con lisencefalia, calcificaciones corticosubcorticales y ventriculomegalia y otra paciente con microcefalia posnatal con polimicrogiria asimétrica y calcificaciones subcorticales y retraso en la mielinización. El real impacto de esta enfermedad aún es incierto; es necesario un adecuado seguimiento multidisciplinario de los pacientes expuestos al virus del Zika para comprender mejor la infección y su historia natural.

Leprosy in a University Hospital in Southern Brazil.

BACKGROUND: Leprosy is an infectious disease that may lead to irreversible nerve damage, compromising patient's quality of life and leading to loss of working years. OBJECTIVES: To evaluate the epidemiological profile of patients followed at a University Hospital. MATERIALS AND METHODS: This is a retrospective observational study, based on a review of medical records. We studied the clinical and epidemiological features of patients with leprosy monitored at the Hospital de Clínicas of the Federal University of Paraná between January 2005 and January 2010. RESULTS: The mean age was 47.51, while 35.94% of patients were aged 41-60. The male:female rate was 1.8:1. The most prevalent occupations were: retired, students or rural workers. Patients came mainly from Curitiba or nearby areas, but there were also patients from the countryside. The mean diagnostic delay was 24.57 months. Multibacillary forms prevailed, with the lepromatous variety being the most common, closely followed by the borderline type. Neural enlargement was found in more than 50% of the patients and 48.44% of them developed reactional states. Hemolysis was the most commonly detected drug side effect. Initial functional evaluation was possible in 70% of patients, 55% of whom had disabilities upon diagnosis. The most prevalent associated disease was hypertension. CONCLUSIONS: This study showed an important diagnostic delay and a high rate of sequelae in this specific population. Brazil is one of the few remaining countries that has not yet eradicated leprosy and it is important to improve health policies in order to prevent sequelae and achieve eradication.

Leprosy in a University Hospital in Southern Brazil

AbstractBACKGROUND:Leprosy is an infectious disease that may lead to irreversible nerve damage, compromising patient's quality of life and leading to loss of working years.OBJECTIVES:To evaluate the epidemiological profile of patients followed at a University Hospital.MATERIALS AND METHODS: This is a retrospective observational study, based on a review of medical records. We studied the clinical and epidemiological features of patients with leprosy monitored at the Hospital de Clínicas of the Federal University of Paraná between January 2005 and January 2010.RESULTS:The mean age was 47.51, while 35.94% of patients were aged 41-60. The male:female rate was 1.8:1. The most prevalent occupations were: retired, students or rural workers. Patients came mainly from Curitiba or nearby areas, but there were also patients from the countryside. The mean diagnostic delay was 24.57 months. Multibacillary forms prevailed, with the lepromatous variety being the most common, closely followed by the borderline type. Neural enlargement was found in more than 50% of the patients and 48.44% of them developed reactional states. Hemolysis was the most commonly detected drug side effect. Initial functional evaluation was possible in 70% of patients, 55% of whom had disabilities upon diagnosis. The most prevalent associated disease was hypertension.CONCLUSIONS:This study showed an important diagnostic delay and a high rate of sequelae in this specific population. Brazil is one of the few remaining countries that has not yet eradicated leprosy and it is important to improve health policies in order to prevent sequelae and achieve eradication.

Epidural myelolipoma in a Husky-cross: a case report.

Epidural spinal myelolipoma was diagnosed in an 11.5-year-old castrated male Husky-cross that was evaluated at the veterinary teaching hospital due to progressive thoracolumbar spinal hyperaesthesia and mild proprioceptive pelvic limb ataxia. A focal, ill-defined mildly inhomogenous extradural mass lesion was detected by MRI. The dog was euthanized. At necropsy an extradurally located reddish mass of about 2.5 cm in diameter was present in the vertebral canal. The mass was identified histopathologically as an epidural myelolipoma.

Contact heat thermal threshold testing in beagle dogs: baseline reproducibility and the effect of acepromazine, levomethadone and fenpipramide.

BACKGROUND: In this methodology article a thermal threshold testing device designed to test nociception in cats was assessed in six dogs. The purpose of this study was to investigate baseline reproducibility of thermal thresholds obtained by the contact heat testing device, to assess the influence of acepromazine and levomethadone and fenpipramide in dogs. The relationship between change in nociceptive thermal threshold and the opioid's plasma concentration was determined. Six adult beagle dogs received levomethadone (0.2 mg/kg), acepromazine (0.02 mg/kg) or saline placebo by intramuscular injection (IM) in a randomized cross-over design. Three baseline nociceptive thermal threshold readings were taken at 15 minutes intervals prior to treatment. Further readings were made at 15, 30, 45, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, 420 and 480 minutes after injection. A sedation score was assigned at every reading. Four saline placebo treatments were performed to assess baseline reproducibility. Levomethadone serum concentrations were measured prior and 0.5, 1, 2, 4, 8, 12 and 24 hours after drug dosing in a separate occasion. RESULTS: Acepromazine did not seem to increase the thermal threshold at any time. After levomethadone there was a significant rise of the thermal threshold between 15 to 120 minutes at serum concentrations between 22.6-46.3 ng/mL. Baseline reproducibility was stable in adult beagle dogs. CONCLUSION: The thermal threshold testing system is a suitable device for nociceptive threshold testing in dogs.

Plasma and urine concentrations of marbofloxacin following single subcutaneous administration to cats.

The pharmacokinetic properties of marbofoxacin, a third generation fluoroquinolone, were investigated in 12 healthy adult cats after single subcutaneous (SC) administration of 2 mg/kg BW (Part I, n=8 cats) and 4 mg/kg BW (Part II, n=4 cats). In each part of the study blood and urine samples were collected before treatment and thereafter for 5 days. The plasma and urine concentrations of marbofloxacin were determined by HPLC with UV detection. Pharmacokinetic calculations were performed for each treated animal using an open one-compartment-model with first-order elimination after SC dosing. Marbofloxacin in plasma (means): Maximum concentrations (Cmax) of about 1.2 and 3.0 microg/ml were measured 2.3 and 4 hours (tmax) after dosing of 2 and 4 mg/kg BW, respectively. Elimination from the body was low with a total clearance (Cl/F) of approximately 0.1 l/h/kg for both dosages. The half-life (t 1/2) for this process was calculated with 8-10 hours. AUC increased almost proportional when doubling the dose, i.e., 19.77 +/- 6.25 microg * h/ml (2 mg/kg BW) and 51.26 +/- 11.83 microg * h/ml (4 mg/kg BW). Plasma kinetics measured were in accordance with data from literature. Marbofloxacin in urine (means): Maximum drug concentrations were detected 4 and 8 hours after dosing with 70 microg/ml (2 mg/kg BW) and 160 microg/ml (4 mg/kg BW), respectively. Inhibitory effects of the urinary matrix on the antimicrobial activity of the drug were taken into account when performing PK/PD calculations. However, a concentration-dependent bactericidal activity (Cmax/MIC > 8-10) which is claimed for fluoroquinolones was sufficiently met with focus on Escherichia (E.) coli (MIC90 0.5 microg/ml). In the same matrix a threshold value of 1.0 microg/ml was undercut 82 and 116 hours after SC dosing, respectively. Hence, a time-dependent bacteria killing kinetic (T > MIC) which may be of relevance for some Gram-positive germs like Staphylococcus spp. (MIC90 1.0 microg/ml) should be covered, too.