Tcf1 is essential for initiation of oncogenic Notch1-driven chromatin topology in T-ALL.

NOTCH1 is a well-established lineage specifier for T cells and among the most frequently mutated genes throughout all subclasses of T cell acute lymphoblastic leukemia (T-ALL). How oncogenic NOTCH1 signaling launches a leukemia-prone chromatin landscape during T-ALL initiation is unknown. Here we demonstrate an essential role for the high-mobility-group transcription factor Tcf1 in orchestrating chromatin accessibility and topology, allowing aberrant Notch1 signaling to convey its oncogenic function. Although essential, Tcf1 is not sufficient to initiate leukemia. The formation of a leukemia-prone epigenetic landscape at the distal Notch1-regulated Myc enhancer, which is fundamental to this disease, is Tcf1-dependent and occurs within the earliest progenitor stage even before cells adopt a T lymphocyte or leukemic fate. Moreover, we discovered a unique evolutionarily conserved Tcf1-regulated enhancer element in the distal Myc-enhancer, which is important for the transition of preleukemic cells to full-blown disease.

Vitamin B12 Deficiency and the Nervous System: Beyond Metabolic Decompensation-Comparing Biological Models and Gaining New Insights into Molecular and Cellular Mechanisms.

Vitamin B12 (VitB12) is a micronutrient and acts as a cofactor for fundamental biochemical reactions: the synthesis of succinyl-CoA from methylmalonyl-CoA and biotin, and the synthesis of methionine from folic acid and homocysteine. VitB12 deficiency can determine a wide range of diseases, including nervous system impairments. Although clinical evidence shows a direct role of VitB12 in neuronal homeostasis, the molecular mechanisms are yet to be characterized in depth. Earlier investigations focused on exploring the biochemical shifts resulting from a deficiency in the function of VitB12 as a coenzyme, while more recent studies propose a broader mechanism, encompassing changes at the molecular/cellular levels. Here, we explore existing study models employed to investigate the role of VitB12 in the nervous system, including the challenges inherent in replicating deficiency/supplementation in experimental settings. Moreover, we discuss the potential biochemical alterations and ensuing mechanisms that might be modified at the molecular/cellular level (such as epigenetic modifications or changes in lysosomal activity). We also address the role of VitB12 deficiency in initiating processes that contribute to nervous system deterioration, including ROS accumulation, inflammation, and demyelination. Consequently, a complex biological landscape emerges, requiring further investigative efforts to grasp the intricacies involved and identify potential therapeutic targets.

Trimetazidine Improves Mitochondrial Dysfunction in SOD1G93A Cellular Models of Amyotrophic Lateral Sclerosis through Autophagy Activation.

Amyotrophic Lateral Sclerosis (ALS) is considered the prototype of motor neuron disease, characterized by motor neuron loss and muscle waste. A well-established pathogenic hallmark of ALS is mitochondrial failure, leading to bioenergetic deficits. So far, pharmacological interventions for the disease have proven ineffective. Trimetazidine (TMZ) is described as a metabolic modulator acting on different cellular pathways. Its efficacy in enhancing muscular and cardiovascular performance has been widely described, although its molecular target remains elusive. We addressed the molecular mechanisms underlying TMZ action on neuronal experimental paradigms. To this aim, we treated murine SOD1G93A-model-derived primary cultures of cortical and spinal enriched motor neurons, as well as a murine motor-neuron-like cell line overexpressing SOD1G93A, with TMZ. We first characterized the bioenergetic profile of the cell cultures, demonstrating significant mitochondrial dysfunction that is reversed by acute TMZ treatments. We then investigated the effect of TMZ in promoting autophagy processes and its impact on mitochondrial morphology. Finally, we demonstrated the effectiveness of TMZ in terms of the mitochondrial functionality of ALS-rpatient-derived peripheral blood mononuclear cells (PBMCs). In summary, our results emphasize the concept that targeting mitochondrial dysfunction may represent an effective therapeutic strategy for ALS. The findings demonstrate that TMZ enhances mitochondrial performance in motor neuron cells by activating autophagy processes, particularly mitophagy. Although further investigations are needed to elucidate the precise molecular pathways involved, these results hold critical implications for the development of more effective and specific derivatives of TMZ for ALS treatment.

The Combined Expression of the Nonstructural Protein NS1 and the N-Terminal Half of NS2 (NS21-180) by ChAdOx1 and MVA Confers Protection against Clinical Disease in Sheep upon Bluetongue Virus Challenge.

Bluetongue, caused by bluetongue virus (BTV), is a widespread arthropod-borne disease of ruminants that entails a recurrent threat to the primary sector of developed and developing countries. In this work, we report modified vaccinia virus Ankara (MVA) and ChAdOx1-vectored vaccines designed to simultaneously express the immunogenic NS1 protein and/or NS2-Nt, the N-terminal half of protein NS2 (NS21-180). A single dose of MVA or ChAdOx1 expressing NS1-NS2-Nt improved the protection conferred by NS1 alone in IFNAR(-/-) mice. Moreover, mice immunized with ChAdOx1/MVA-NS1, ChAdOx1/MVA-NS2-Nt, or ChAdOx1/MVA-NS1-NS2-Nt developed strong cytotoxic CD8+ T-cell responses against NS1, NS2-Nt, or both proteins and were fully protected against a lethal infection with BTV serotypes 1, 4, and 8. Furthermore, although a single immunization with ChAdOx1-NS1-NS2-Nt partially protected sheep against BTV-4, the administration of a booster dose of MVA-NS1-NS2-Nt promoted a faster viral clearance, reduction of the period and level of viremia and also protected from the pathology produced by BTV infection. IMPORTANCE Current BTV vaccines are effective but they do not allow to distinguish between vaccinated and infected animals (DIVA strategy) and are serotype specific. In this work we have develop a DIVA multiserotype vaccination strategy based on adenoviral (ChAdOx1) and MVA vaccine vectors, the most widely used in current phase I and II clinical trials, and the conserved nonstructural BTV proteins NS1 and NS2. This immunization strategy solves the major drawbacks of the current marketed vaccines.

Upper and Lower Motor Neurons and the Skeletal Muscle: Implication for Amyotrophic Lateral Sclerosis (ALS).

The relationships between motor neurons and the skeletal muscle during development and in pathologic contexts are addressed in this Chapter.We discuss the developmental interplay of muscle and nervous tissue, through neurotrophins and the activation of differentiation and survival pathways. After a brief overview on muscular regulatory factors, we focus on the contribution of muscle to early and late neurodevelopment. Such a role seems especially intriguing in relation to the epigenetic shaping of developing motor neuron fate choices. In this context, emphasis is attributed to factors regulating energy metabolism, which may concomitantly act in muscle and neural cells, being involved in common pathways.We then review the main features of motor neuron diseases, addressing the cellular processes underlying clinical symptoms. The involvement of different muscle-associated neurotrophic factors for survival of lateral motor column neurons, innervating MyoD-dependent limb muscles, and of medial motor column neurons, innervating Myf5-dependent back musculature is discussed. Among the pathogenic mechanisms, we focus on oxidative stress, that represents a common and early trait in several neurodegenerative disorders. The role of organelles primarily involved in reactive oxygen species scavenging and, more generally, in energy metabolism-namely mitochondria and peroxisomes-is discussed in the frame of motor neuron degeneration.We finally address muscular involvement in amyotrophic lateral sclerosis (ALS), a multifactorial degenerative disorder, hallmarked by severe weight loss, caused by imbalanced lipid metabolism. Even though multiple mechanisms have been recognized to play a role in the disease, current literature generally assumes that the primum movens is neuronal degeneration and that muscle atrophy is only a consequence of such pathogenic event. However, several lines of evidence point to the muscle as primarily involved in the disease, mainly through its role in energy homeostasis. Data from different ALS mouse models strongly argue for an early mitochondrial dysfunction in muscle tissue, possibly leading to motor neuron disturbances. Detailed understanding of skeletal muscle contribution to ALS pathogenesis will likely lead to the identification of novel therapeutic strategies.

Identification of small molecules capable of enhancing viral membrane fusion.

Several approaches have been developed to analyze the entry of highly pathogenic viruses. In this study, we report the implementation of a Bimolecular Multicellular Complementation (BiMuC) assay to safely and efficiently monitor SARS-CoV-2 S-mediated membrane fusion without the need for microscopy-based equipment. Using BiMuC, we screened a library of approved drugs and identified compounds that enhance S protein-mediated cell-cell membrane fusion. Among them, ethynylestradiol promotes the growth of SARS-CoV-2 and Influenza A virus in vitro. Our findings demonstrate the potential of BiMuC for identifying small molecules that modulate the life cycle of enveloped viruses, including SARS-CoV-2.

TERT Extra-Telomeric Roles: Antioxidant Activity and Mitochondrial Protection.

Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase holoenzyme, which adds telomeric DNA repeats on chromosome ends to counteract telomere shortening. In addition, there is evidence of TERT non-canonical functions, among which is an antioxidant role. In order to better investigate this role, we tested the response to X-rays and H2O2 treatment in hTERT-overexpressing human fibroblasts (HF-TERT). We observed in HF-TERT a reduced induction of reactive oxygen species and an increased expression of the proteins involved in the antioxidant defense. Therefore, we also tested a possible role of TERT inside mitochondria. We confirmed TERT mitochondrial localization, which increases after oxidative stress (OS) induced by H2O2 treatment. We next evaluated some mitochondrial markers. The basal mitochondria quantity appeared reduced in HF-TERT compared to normal fibroblasts and an additional reduction was observed after OS; nevertheless, the mitochondrial membrane potential and morphology were better conserved in HF-TERT. Our results suggest a protective function of TERT against OS, also preserving mitochondrial functionality.

Resveratrol Analogs and Prodrugs Differently Affect the Survival of Breast Cancer Cells Impairing Estrogen/Estrogen Receptor α/Neuroglobin Pathway.

Breast cancer is the first leading tumor in women in terms of incidence worldwide. Seventy percent of cases are estrogen receptor (ER) α-positive. In these malignancies, 17ß-estradiol (E2) via ERα increases the levels of neuroglobin (NGB), a compensatory protein that protects cancer cells from stress-induced apoptosis, including chemotherapeutic drug treatment. Our previous data indicate that resveratrol (RSV), a plant-derived polyphenol, prevents E2/ERα-induced NGB accumulation in this cellular context, making E2-dependent breast cancer cells more prone to apoptosis. Unfortunately, RSV is readily metabolized, thus preventing its effectiveness. Here, four different RSV analogs have been developed, and their effect on the ERα/NGB pathway has been compared with RSV conjugated with highly hydrophilic gold nanoparticles as prodrug to evaluate if RSV derivatives maintain the breast cancer cells' susceptibility to the chemotherapeutic drug paclitaxel as the original compound. Results demonstrate that RSV conjugation with gold nanoparticles increases RSV efficacy, with respect to RSV analogues, reducing NGB levels and enhancing the pro-apoptotic action of paclitaxel, even preventing the anti-apoptotic action exerted by E2 treatment on these cells. Overall, RSV conjugation with gold nanoparticles makes this complex a promising agent for medical application in breast cancer treatment.

Combined or Sequential Treatment with Immune Checkpoint Inhibitors and Car-T Cell Therapies for the Management of Haematological Malignancies: A Systematic Review.

The aim of this paper was to review the available evidence on the efficacy and safety of combined or sequential use of PD-1/PD-L1 immune checkpoint inhibitors (ICI) and CAR-T cell therapies in relapsed/refractory (R/R) haematological malignancies. A systematic literature review was performed until 21 November 2022. Inclusion criteria: cohort studies/clinical trials aimed at evaluating the efficacy and/or safety of the combination of CAR-T cell therapy with PD-1/PD-L1 inhibitors in R/R haematological malignancies, which had reported results. Those focusing only on ICI or CAR-T separately or evaluating the combination in other non-hematological solid tumours were excluded. We used a specific checklist for quality assessment of the studies, and then we extracted data on efficacy or efficiency and safety. A total of 1867 articles were identified, and 9 articles were finally included (early phase studies, with small samples of patients and acceptable quality). The main pathologies were B-cell acute lymphoblastic leukaemia (B-ALL) and B-cell non-Hodgkin's lymphoma (B-NHL). The most studied combination was tisagenlecleucel with pembrolizumab. In terms of efficacy, there is great variability: the combination could be a promising option in B-ALL, with modest data, and in B-NHL, although hopeful responses were received, the combination does not appear better than CAR-T cell monotherapy. The safety profile could be considered comparable to that described for CAR-T cell monotherapy.

Oxidative Damage and Antioxidant Response in Frontal Cortex of Demented and Nondemented Individuals with Alzheimer's Neuropathology.

Alzheimer's disease (AD) is characterized by progressive neurodegeneration in the cerebral cortex, histopathologically hallmarked by amyloid ß (Aß) extracellular plaques and intracellular neurofibrillary tangles, constituted by hyperphosphorylated tau protein. Correlation between these pathologic features and dementia has been challenged by the emergence of "nondemented with Alzheimer's neuropathology" (NDAN) individuals, cognitively intact despite displaying pathologic features of AD. The existence of these subjects suggests that some unknown mechanisms are triggered to resist Aß-mediated detrimental events. Aß accumulation affects mitochondrial redox balance, increasing oxidative stress status, which in turn is proposed as a primary culprit in AD pathogenesis. To clarify the relationship linking Aß, oxidative stress, and cognitive impairment, we performed a comparative study on AD, NDAN, and aged-matched human postmortem frontal cortices of either sex. We quantitatively analyzed immunofluorescence distribution of oxidative damage markers, and of SOD2 (superoxide dismutase 2), PGC1α [peroxisome proliferator-activated receptor (PPAR) γ-coactivator 1α], PPARα, and catalase as key factors in antioxidant response, as well as the expression of miRNA-485, as a PGC1α upstream regulator. Our results confirm dramatic redox imbalance, associated with impaired antioxidant defenses in AD brain. By contrast, NDAN individuals display low oxidative damage, which is associated with high levels of scavenging systems, possibly resulting from a lack of PGC1α miRNA-485-related inhibition. Comparative analyses in neurons and astrocytes further highlighted cell-specific mechanisms to counteract redox imbalance. Overall, our data emphasize the importance of transcriptional and post-transcriptional regulation of antioxidant response in AD. This suggests that an efficient PGC1α-dependent "safety mechanism" may prevent Aß-mediated oxidative stress, supporting neuroprotective therapies aimed at ameliorating defects in antioxidant response pathways in AD patients.

ALK-Fusion Transcripts Can Be Detected in Extracellular Vesicles (EVs) from Nonsmall Cell Lung Cancer Cell Lines and Patient Plasma: Toward EV-Based Noninvasive Testing.

BACKGROUND: ALK rearrangements are present in 5% of nonsmall cell lung cancer (NSCLC) tumors and identify patients who can benefit from ALK inhibitors. ALK fusions testing using liquid biopsies, although challenging, can expand the therapeutic options for ALK-positive NSCLC patients considerably. RNA inside extracellular vesicles (EVs) is protected from RNases and other environmental factors, constituting a promising source for noninvasive fusion transcript detection. METHODS: EVs from H3122 and H2228 cell lines, harboring EML4-ALK variant 1 (E13; A20) and variant 3 (E6a/b; A20), respectively, were successfully isolated by sequential centrifugation of cell culture supernatants. EVs were also isolated from plasma samples of 16 ALK-positive NSCLC patients collected before treatment initiation. RESULTS: Purified EVs from cell cultures were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and flow cytometry. Western blot and confocal microscopy confirmed the expression of EV-specific markers as well as the expression of EML4-ALK-fusion proteins in EV fractions from H3122 and H2228 cell lines. In addition, RNA from EV fractions derived from cell culture was analyzed by digital PCR (dPCR) and ALK-fusion transcripts were clearly detected. Similarly, plasma-derived EVs were characterized by NTA, flow cytometry, and the ExoView platform, the last showing that EV-specific markers captured EV populations containing ALK-fusion protein. Finally, ALK fusions were identified in 50% (8/16) of plasma EV-enriched fractions by dPCR, confirming the presence of fusion transcripts in EV fractions. CONCLUSIONS: ALK-fusion transcripts can be detected in EV-enriched fractions. These results set the stage for the development of EV-based noninvasive ALK testing.

Subgroup analysis in haematologic malignancies phase III clinical trials: A systematic review.

AIMS: To assess the appropriateness of the use and interpretation of subgroup analysis in haematology randomized clinical trials (RCTs). METHOD: A systematic review of Medline, including haematology phase III RCTs published between January 2013 and October 2019, was carried out to identify reported subgroup analysis. Information related to trial characteristics, subgroup analysis and claims of subgroup difference were collected. RESULTS: The initial search identified 1622 studies. A total of 98 studies reporting subgroup analyses were identified. Of those, 24 RCT reported 46 claims of subgroup difference. Among them, 44 were claims for the primary outcome, of which 25 were considered strong claims and 17 were considered suggestions of a possible effect. Authors included subgroup variables for the primary outcome measured at baseline for 38 claims (n = 86.36%), used a subgroup variable as a stratification factor at randomization for 15 (34.09%), clearly prespecified their hypothesis for 11 (25%), the subgroup effect was one of a small number of hypothesised effects tested (≤ 5) for 17 (38.64%), carried out a test of interaction that provide statistically significant for 18 (40.91%), documented replication of a subgroup effect with previously related studies for 11 (25%), identified the consistency of a subgroup effect across related outcome for 10 (22.72%) and provided a biological rationale for the effect for 8 (18.18%). Of the 44 claims for the primary outcome, 34 (77.27%) met four or fewer of the 10 credibility criteria. CONCLUSION: The subgroup claims reported in haematology RCTs lack credibility, even when the claims are strong. Information about subgroup difference should be interpreted cautiously.

A DNA Vaccine Delivery Platform Based on Elastin-Like Recombinamer Nanosystems for Rift Valley Fever Virus.

This work analyzes the immunogenicity of six genetically engineered constructs based on elastin-like recombinamers (ELRs) fused to the Gn glycoprotein from Rift Valley fever virus (RVFV). Upon transfection, all constructs showed no effect on cell viability. While fusion constructs including ELR blocks containing hydrophobic amino acids (alanine or isoleucine) did not increase the expression of viral Gn in eukaryotic cells, glutamic acid- or valine-rich fusion proteins showed enhanced expression levels compared with the constructs encoding the viral antigen alone. However, in vivo DNA plasmid immunization assays determined that the more hydrophobic constructs reduced viremia levels after RVFV challenge to a higher extent than glutamic- or valine-rich encoding plasmids and were better inducers of cellular immunity as judged by in vitro restimulation experiments. Although the Gn-ELR fusion constructs did not surpass the protective efficacy of a plasmid vaccine expressing nonfused Gn, our results warrant further experiments directed to take advantage of the immunomodulatory potential of ELR biomaterials for improving vaccines against infectious diseases.

Antioxidant Amelioration of Riboflavin Transporter Deficiency in Motoneurons Derived from Patient-Specific Induced Pluripotent Stem Cells.

Mitochondrial dysfunction is a key element in the pathogenesis of neurodegenerative disorders, such as riboflavin transporter deficiency (RTD). This is a rare, childhood-onset disease characterized by motoneuron degeneration and caused by mutations in SLC52A2 and SLC52A3, encoding riboflavin (RF) transporters (RFVT2 and RFVT3, respectively), resulting in muscle weakness, ponto-bulbar paralysis and sensorineural deafness. Based on previous findings, which document the contribution of oxidative stress in RTD pathogenesis, we tested possible beneficial effects of several antioxidants (Vitamin C, Idebenone, Coenzyme Q10 and EPI-743, either alone or in combination with RF) on the morphology and function of neurons derived from induced pluripotent stem cells (iPSCs) from two RTD patients. To identify possible improvement of the neuronal morphotype, neurite length was measured by confocal microscopy after ß-III tubulin immunofluorescent staining. Neuronal function was evaluated by determining superoxide anion generation by MitoSOX assay and intracellular calcium (Ca2+) levels, using the Fluo-4 probe. Among the antioxidants tested, EPI-743 restored the redox status, improved neurite length and ameliorated intracellular calcium influx into RTD motoneurons. In conclusion, we suggest that antioxidant supplementation may have a role in RTD treatment.

Dental Anomalies in Permanent Teeth Associated With Nonsyndromic Cleft Lip and Palate in a Group of Colombian Children.

OBJECTIVES: To assess the risk of dental anomaly presentation in permanent teeth in a group of Colombian children with nonsyndromic cleft lip and palate (NSCLP) and to determine the frequency of the anomalies according to the cleft type. METHODS: An analytical matched case-control study was conducted with 210 controls and 210 patients with NSCLP. The patients were classified into 3 groups: complete right unilateral cleft lip and palate (RCLP), complete left unilateral cleft lip and palate (LCLP), and complete bilateral cleft lip and palate (BCLP). Univariate and multivariate Poisson regression models were used to analyze paired samples (Bonferroni adjustment, P ≤ .002). RESULTS: A high risk of finding agenesis of the maxillary lateral incisors, supernumerary teeth, microdontia of the maxillary lateral incisors, and rotation of the maxillary central incisors adjacent to the cleft (P < .0001) was observed in the patients with NSCLP. One or more dental anomalies were found in 98% of patients with BCLP, in 96% of those with LCLP, and in 87% of those with RCLP. Most of the anomalies were located on the cleft area. The incidence relative risk (IRR) of anomalies was highest in patients with BCLP (IRR: 10.5; 95% confidence interval [CI]: 6.76-16.3), followed by in those with LCLP (IRR: 8.51; 95% CI: 5.64-12.8). CONCLUSIONS: Most dental anomalies were found in the cleft area; this was expected because the cleft area was the most affected in the patients included in this study.

The influence of gender roles in alcohol consumption: a qualitative study of adolescents and young adults in Asturias. / La influencia de los roles de género en el consumo de alcohol: estudio cualitativo en adolescentes y jóvenes en Asturias.

Despite the implementation of prevention policies aimed at addressing alcohol consumption among both adolescents and young adults, there has been a considerable increase in those who abuse alcohol in Spain over the last decade. Official surveys on this phenomenon show that both the prevalence and risky consumption of men and women are reaching similar levels, with even higher figures for these behaviours in the case of girls at the end of adolescence. The aim of this article is to understand the influence of gender roles in the consumption of alcohol among adolescents and young adults. To this end, focus groups have been employed to identify similarities and differences both in drinking patterns and alcohol abuse among young males and females, as well as in the social meaning that both groups attribute to these practices.   The results obtained show that the variables gender and age act in a combined way on the learning of alcohol consumption, as well as on the motivations and expectations that adolescents and young adults have regarding these practices. In addition, in this study three differentiated stages are identified: in the first, gender roles are clearly defined; in the second, there is a certain transgression of these roles mainly by young women, and in the third, there is a return to traditional gender roles.

A pesar del desarrollo de políticas de prevención dirigidas a abordar el consumo de alcohol en adolescentes y jóvenes, durante la última década se ha producido un aumento considerable de quienes realizan un consumo abusivo de esta sustancia en España. Las encuestas oficiales sobre este fenómeno muestran un acercamiento entre varones y mujeres en las prevalencias de consumo y en los consumos de riesgo, e incluso una mayor incidencia de estas conductas en el caso de las mujeres al final de la etapa adolescente. El objetivo de este artículo es conocer la influencia que ejercen los roles de género en estas pautas de consumo en adolescentes y jóvenes. Para ello, se han realizado grupos focales que han permitido identificar las similitudes y diferencias que se producen tanto en las prácticas de consumo y abuso del alcohol que desarrollan estos colectivos, como en el significado social que unos y otras atribuyen a las citadas prácticas.  Los resultados obtenidos muestran que las variables género y edad actúan de forma combinada en el aprendizaje del consumo de bebidas alcohólicas, así como en las motivaciones y expectativas que los y las adolescentes y jóvenes tienen sobre dichas prácticas. Además, en este estudio se identifican tres etapas diferenciadas, en la primera de ellas los roles de género se encuentran claramente definidos, en la segunda se observa cierta transgresión de los mismos fundamentalmente por parte de las mujeres y en la última se advierte una vuelta a los tradicionales roles de género.

Differential toxicity of TAR DNA-binding protein 43 isoforms depends on their submitochondrial localization in neuronal cells.

TAR DNA-binding protein 43 (TDP-43) is an RNA-binding protein and a major component of protein aggregates found in amyotrophic lateral sclerosis and several other neurodegenerative diseases. TDP-43 exists as a full-length protein and as two shorter forms of 25 and 35 kDa. Full-length mutant TDP-43s found in amyotrophic lateral sclerosis patients re-localize from the nucleus to the cytoplasm and in part to mitochondria, where they exert a toxic role associated with neurodegeneration. However, induction of mitochondrial damage by TDP-43 fragments is yet to be clarified. In this work, we show that the mitochondrial 35 kDa truncated form of TDP-43 is restricted to the intermembrane space, while the full-length forms also localize in the mitochondrial matrix in cultured neuronal NSC-34 cells. Interestingly, the full-length forms clearly affect mitochondrial metabolism and morphology, possibly via their ability to inhibit the expression of Complex I subunits encoded by the mitochondrial-transcribed mRNAs, while the 35 kDa form does not. In the light of the known differential contribution of the full-length and short isoforms to generate toxic aggregates, we propose that the presence of full-length TDP-43s in the matrix is a primary cause of mitochondrial damage. This in turn may cause oxidative stress inducing toxic oligomers formation, in which short TDP-43 forms play a major role.

Targeting of mTOR catalytic site inhibits multiple steps of the HIV-1 lifecycle and suppresses HIV-1 viremia in humanized mice.

HIV necessitates host factors for successful completion of its life cycle. Mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that forms two complexes, mTORC1 and mTORC2. Rapamycin is an allosteric inhibitor of mTOR that selectively inhibits mTORC1. Rapamycin interferes with viral entry of CCR5 (R5)-tropic HIV and with basal transcription of the HIV LTR, potently inhibiting replication of R5 HIV but not CXCR4 (X4)-tropic HIV in primary cells. The recently developed ATP-competitive mTOR kinase inhibitors (TOR-KIs) inhibit both mTORC1 and mTORC2. Using INK128 as a prototype TOR-KI, we demonstrate potent inhibition of both R5 and X4 HIV in primary lymphocytes (EC50 < 50 nM), in the absence of toxicity. INK128 inhibited R5 HIV entry by reducing CCR5 levels. INK128 also inhibited both basal and induced transcription of HIV genes, consistent with inhibition of mTORC2, whose activity is critical for phosphorylation of PKC isoforms and, in turn, induction of NF-κB. INK128 enhanced the antiviral potency of the CCR5 antagonist maraviroc, and had favorable antiviral interactions with HIV inhibitors of reverse transcriptase, integrase and protease. In humanized mice, INK128 decreased plasma HIV RNA by >2 log10 units and partially restored CD4/CD8 cell ratios. Targeting of cellular mTOR with INK128 (and perhaps others TOR-KIs) provides a potential strategy to inhibit HIV, especially in patients with drug resistant HIV strains.

Effect of the cross-linking agent and drying method on encapsulation efficiency of orange essential oil by complex coacervation using whey protein isolate with different polysaccharides.

Orange essential oil was microencapsulated by complex coacervation with whey protein isolate (WPI): carboxymethylcellulose (CMC), WPI:sodium alginate (SA) and WPI:chitosan (CH). Effect of pH, protein:polysaccharide ratio and solid concentration on coacervation efficiency were selected for the best coacervation conditions. Tannic acid (TA), sodium tripolyphosphate, oxidised tannic acid and transglutaminase enzyme (TG) were used as cross-linking agents. Highest encapsulation efficiency (EE) for wet coacervated microcapsules ranged from 88% to 94%. Microcapsules were freeze and spray dried to evaluate their effect on its integrity. EE was higher than 80% in freeze dried coacervated microcapsules with and without cross-linking agent, but they formed a solid cake. Spray-dried samples formed a free fluid solid (10-20 µm), where the systems WPI:CMC and WPI:CH cross-linked with TA and TG, respectively showed the highest EE (47% and 50% respectively), representing 400% improvement compared to the samples without cross-linking.

Monotherapy with either dolutegravir or raltegravir fails to durably suppress HIV viraemia in humanized mice.

Objectives: To compare the effectiveness of HIV integrase inhibitor monotherapy between raltegravir and dolutegravir as an approach to simplify therapy. Methods: We evaluated and compared the efficacy of 20 week monotherapy with dolutegravir or raltegravir in humanized mice (HSC-NSG) infected with HIVBaL. Plasma HIV RNA was measured by quantitative RT-PCR (limit of detection of 150 copies/45 µL of plasma) and drug levels by LC-MS/MS. Escape viruses were genotyped and analysed for replication capacity and drug susceptibility in tissue culture. Results: Drug-untreated control mice maintained constant viraemia throughout the study. Virus isolates from these mice were susceptible to both raltegravir (EC50 of <8 nM) and dolutegravir (EC50 of <1 nM). Mice treated with raltegravir or dolutegravir had plasma drug levels comparable to those in humans. Monotherapy with raltegravir initially suppressed HIV viraemia, but failed to maintain suppression in 4/4 mice. Viruses from raltegravir failing mice developed mutations G140G/S and Q148H/K, and were resistant to both raltegravir (EC50 values of >100 nM) and dolutegravir (EC50 values ranging from 8.8 to 13.3 nM). Monotherapy with dolutegravir suppressed viraemia in 5/5 of mice, but viraemia rebounded in one animal. The virus from this mouse had mutations E138K, G140S, Q148H, N155H and S230R, was highly resistant to both raltegravir (EC50 of >1000 nM) and dolutegravir (EC50 of 550 nM), and replicated to levels similar to those of control viruses in PBMCs. Conclusions: Monotherapy with either raltegravir or dolutegravir does not consistently maintain HIV suppression, suggesting that dual therapy may be required in simplification strategies.