SARS-CoV-2 inhibition in human airway epithelial cells using a mucoadhesive, amphiphilic chitosan that may serve as an anti-viral nasal spray

There are currently no cures for coronavirus infections, making the prevention of infections the only course open at the present time. The COVID-19 pandemic has been difficult to prevent, as the infection is spread by respiratory droplets and thus effective, scalable and safe preventive interventions are urgently needed. We hypothesise that preventing viral entry into mammalian nasal epithelial cells may be one way to limit the spread of COVID-19. Here we show that N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ), a positively charged polymer that has been through an extensive Good Laboratory Practice toxicology screen, is able to reduce the infectivity of SARS-COV-2 in A549ACE2+ and Vero E6 cells with a log removal value of −3 to −4 at a concentration of 10 – 100 μg/ mL (p < 0.05 compared to untreated controls) and to limit infectivity in human airway epithelial cells at a concentration of 500 μg/ mL (p < 0.05 compared to untreated controls). GCPQ is currently being developed as a pharmaceutical excipient in nasal and ocular formulations. GCPQ’s electrostatic binding to the virus, preventing viral entry into the host cells, is the most likely mechanism of viral inhibition. Radiolabelled GCPQ studies in mice show that at a dose of 10 mg/ kg, GCPQ has a long residence time in mouse nares, with 13.1% of the injected dose identified from SPECT/CT in the nares, 24 hours after nasal dosing. With a no observed adverse effect level of 18 mg/ kg in rats, following a 28-day repeat dose study, clinical testing of this polymer, as a COVID-19 prophylactic is warranted

A nanocommunication system for endocrine diseases

Nanotechnology is a newand very promising area of research which will allow several new applications to be created in different fields, such as, biological, medical, environmental, military, agricultural, industrial and consumer goods. This paper focuses specifically on nanocommunications, which will allow interconnected devices, at the nano-scale, to achieve collaborative tasks, greatly changing the paradigm in the fields described. Molecular communication is a new communication paradigm which allows nanomachines to exchange information using molecules as carrier. This is the most promising nanocommunication method within nanonetworks, since it can use bio-inspired techniques, inherit from studied biological systems, which makes the connection of biologic and man-made systems a easier process. At this point, the biggest challenges in these type of nanocommunication are to establish feasible and reliable techniques that will allow information to be encoded, and mechanisms that ensure a molecular communication between different nodes. This paper focus on creating concepts and techniques to tackle these challenges, and establishing new foundations on which future work can be developed. The created concepts and techniques are then applied in an envisioned medical application, which is based on a molecular nanonetwork deployed inside the Human body. The goal of this medical application is to automatously monitor endocrine diseases using the benefits of nanonetworks, which in turn connects with the internet, thus creating a Internet of NanoThings system. The concepts and techniques developed are evaluated by performing several simulations and comparing with other researches, and the results and discussions are presented on the later sections of this paper

SARS-CoV-2 inhibition using a mucoadhesive, amphiphilic chitosan that may serve as an anti-viral nasal spray

There are currently no cures for coronavirus infections, making the prevention of infections the only course open at the present time. The COVID-19 pandemic has been difficult to prevent, as the infection is spread by respiratory droplets and thus effective, scalable and safe preventive interventions are urgently needed. We hypothesise that preventing viral entry into mammalian nasal epithelial cells may be one way to limit the spread of COVID-19. Here we show that N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ), a positively charged polymer that has been through an extensive Good Laboratory Practice toxicology screen, is able to reduce the infectivity of SARS-COV-2 in A549ACE2+ and Vero E6 cells with a log removal value of - 3 to - 4 at a concentration of 10-100 μg/ mL (p < 0.05 compared to untreated controls) and to limit infectivity in human airway epithelial cells at a concentration of 500 μg/ mL (p < 0.05 compared to untreated controls). In vivo studies using transgenic mice expressing the ACE-2 receptor, dosed nasally with SARS-COV-2 (426,000 TCID50/mL) showed a trend for nasal GCPQ (20 mg/kg) to inhibit viral load in the respiratory tract and brain, although the study was not powered to detect statistical significance. GCPQ's electrostatic binding to the virus, preventing viral entry into the host cells, is the most likely mechanism of viral inhibition. Radiolabelled GCPQ studies in mice show that at a dose of 10 mg/kg, GCPQ has a long residence time in mouse nares, with 13.1% of the injected dose identified from SPECT/CT in the nares, 24 h after nasal dosing. With a no observed adverse effect level of 18 mg/kg in rats, following a 28-day repeat dose study, clinical testing of this polymer, as a COVID-19 prophylactic is warranted

Glymphatic inhibition exacerbates tau propagation in an Alzheimer’s disease model

Background The aggregation and spread of misfolded amyloid structured proteins, such as tau and α-synuclein, are key pathological features associated with neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease. These proteins possess a prion-like property, enabling their transmission from cell to cell leading to propagation throughout the central and peripheral nervous systems. While the mechanisms underlying their intracellular spread are still being elucidated, targeting the extracellular space has emerged as a potential therapeutic approach. The glymphatic system, a brain-wide pathway responsible for clearing extracellular metabolic waste from the central nervous system, has gained attention as a promising target for removing these toxic proteins. Methods In this study, we investigated the impact of long-term modulation of glymphatic function on tau aggregation and spread by chronically treating a mouse model of tau propagation with a pharmacological inhibitor of AQP4, TGN-020. Thy1-hTau.P301S mice were intracerebrally inoculated with tau into the hippocampus and overlying cortex, and subsequently treated with TGN-020 (3 doses/week, 50 mg/kg TGN-020, i.p.) for 10-weeks. During this time, animal memory was studied using cognitive behavioural tasks, and structural MR images were acquired of the brain in vivo prior to brain extraction for immunohistochemical characterisation. Results Our findings demonstrate increased tau aggregation in the brain and transhemispheric propagation in the hippocampus following the inhibition of glymphatic clearance. Moreover, disruption of the glymphatic system aggravated recognition memory in tau inoculated mice and exacerbated regional changes in brain volume detected in the model. When initiation of drug treatment was delayed for several weeks post-inoculation, the alterations were attenuated. Conclusions These results indicate that by modulating AQP4 function and, consequently, glymphatic clearance, it is possible to modify the propagation and pathological impact of tau in the brain, particularly during the initial stages of the disease. These findings highlight the critical role of the glymphatic system in preserving healthy brain homeostasis and offer valuable insights into the therapeutic implications of targeting this system for managing neurodegenerative diseases characterized by protein aggregation and spread

A Rapid FACS-Based Strategy to Isolate Human Gene Knockin and Knockout Clones

Gene targeting protocols for mammalian cells remain inefficient and labor intensive. Here we describe FASTarget, a rapid, fluorescent cell sorting based strategy to isolate rare gene targeting events in human somatic cells. A fluorescent protein is used as a means for direct selection of targeted clones obviating the need for selection and outgrowth of drug resistant clones. Importantly, the use of a promoter-less, ATG-less construct greatly facilitates the recovery of correctly targeted cells. Using this method we report successful gene targeting in up to 94% of recovered human somatic cell clones. We create functional EYFP-tagged knockin clones in both transformed and non-transformed human somatic cell lines providing a valuable tool for mammalian cell biology. We further demonstrate the use of this technology to create gene knockouts. Using this generally applicable strategy we can recover gene targeted clones within approximately one month from DNA construct delivery to obtaining targeted monoclonal cell lines

A lactate shuttle system between tumour and stromal cells is associated with poor prognosis in prostate cancer

Background In a malignant tumour, cancer cells are embedded in stromal cells, namely cancer-associated fibroblasts (CAFs). These CAFs are now accepted as important players in cancer dynamics, being involved in tumour growth and progression. Although there are various reports on the interaction between tumour and stromal cells, the clinico-pathological significance of this cross-talk is still largely unknown. In this study, we aimed to characterise the expression of key metabolic proteins involved in glucose transport, pyruvate/lactate shuttle system, glycolytic metabolism and fatty acid oxidation in CAFs and tumour cells in different stages of malignant transformation. We further aimed to contextualise the clinico-pathological significance of these protein expression profiles with reference to known prognostic indicators, including biochemical recurrence in pT stage. Methods Prostate tissues were obtained from 480 patients with a median age of 64 years following radical prostatectomy with no previous hormonal therapy. Tissues were analysed for the expression of several key metabolism-related proteins in glands and surrounding fibroblasts by immunohistochemistry. Reliable markers of prognosis such as pT stage and biochemical recurrence were assessed for each case. Results We observed that prostate cancer cells did not rely mainly on glycolytic metabolism, while there was a high expression of MCT4 and CAIX - in CAFs. This corroborates the hypothesis of the "Reverse Warburg effect" in prostate cancer, in which fibroblasts are under oxidative stress and express CAIX, an established hypoxia marker. We found that alterations in the expression of metabolism-related proteins were already evident in the early stages of malignant transformation, suggesting the continuing alteration of CAFs from an early stage. Additionally, and for the first time, we show that cases showing high MCT4 expression in CAFs with concomitant strong MCT1 expression in prostate cancer (PCa) cells are associated with poor clinical outcome, namely pT3 stage of the tumour. Conclusions In summary, this work demonstrates for the first time the clinico-pathological significance of the lactate shuttle in prostate cancer. It also suggests that other alterations in CAFs may be useful prognostic factors, and further supports the use of MCT1/MCT4 as targets for PCa therapy.NPG received a fellowship from the Portuguese Foundation for Science and Technology (FCT), refs. SFRH/BD/61027/2009. This work was supported by the FCT grant ref. PTDC/SAUMET/113415/2009, under the scope of "Programa Operacional Tematico Factores de Competitividade" (COMPETE) of "Quadro Comunitario de Apoio III" and co-financed by Fundo Comunitario Europeu FEDER. JA was supported by a Boehringer Ingelheim Fonds fellowship

Prevalence of pfmdr1, pfcrt, pfdhfr and pfdhps mutations associated with drug resistance, in Luanda, Angola

<p>Abstract</p> <p>Background</p> <p>Malaria is the infectious disease causing the highest morbidity and mortality in Angola and due to widespread chloroquine (CQ) resistance, the country has recently changed its first-line treatment recommendations for uncomplicated malaria, from CQ to artemisinin combination therapies (ACT) in adults, and sulphadoxine/pyrimethamine (S/P) in pregnant women. Loss of SP sensitivity is, however, progressing rapidly in Africa and, in this study, were investigated a number of molecular markers associated to CQ and S/P.</p> <p>Methods</p> <p>Blood samples were collected from 245 children with uncomplicated malaria, admitted at the Pediatric Hospital Dr. David Bernardino (HPDB), Angola, and the occurrence of mutations in <it>Plasmodium falciparum </it>was investigated in the <it>pfmdr1 </it>(N86Y) and <it>pfcrt </it>(K76T) genes, associated with CQ resistance, as well as in <it>pfdhfr </it>(C59R) and <it>pfdhps </it>(K540E), conferring SP resistance.</p> <p>Results</p> <p>The frequencies of <it>pfmdr1 </it>mutations in codon 86 were 28.6% N, 61.3% Y and 10.1% mixed infections (NY). The frequency of <it>pfcrt </it>mutations in codon 76 were 93.9% K, 5.7% T and 0.4% mixed infections (KT). For <it>pfdhfr </it>the results were in codon 59, 60.6% C, 20.6% R and 18.8% mixed infections (CR). Concerning <it>pfdhps</it>, 6.3% of the isolates were bearers of the mutation 540E and 5.4% mixed infections (K540E).</p> <p>Conclusion</p> <p>The results of this epidemiologic study showed high presence of CQ resistance markers while for SP a much lower prevalence was detected for the markers under study.</p