Search

Microglia serve as the primary defense mechanism in the brain. Artificial sweeteners are widely used as dietary supplements, though their long‐term effects remain uncertain. In this study, we investigated the effects of sucralose on microglia during prolonged exposure via the neuroinflammatory and ferroptosis pathways. Initially, human microglial clone 3 (HMC3) cells were exposed to sucralose (0–50 mM) for 24, 48, and 72 h to investigate the short‐term effects. Subsequently, HMC3 cells were treated with 1 mM sucralose for 7, 14, and 21 days to examine long‐term effects. We measured levels of interleukin‐1β (IL‐1β), NOD‐like receptor protein 3 (NLRP3), 8‐hydroxydeoxyguanosine (8‐OHdG), Sirtuin‐1 (SIRT1), glutathione peroxidase‐4 (GPx4), reduced glutathione (GSH), malondialdehyde (MDA), ferrous iron (Fe2+), and caspase 3/7. Additionally, we analyzed the impact of sucralose on cell morphology, migration, and expression levels of IL‐1β, NLRP3, SIRT1, and GPx4. Sucralose inhibited cell viability and proliferation in HMC3 cells in a concentration‐ and time‐dependent manner and induced membrane and nuclear abnormalities. Moreover, sucralose significantly reduced the cell migration rate. Long‐term sucralose treatment decreased Fe2+, GPx4, GSH, and SIRT1 levels in HMC3 cells while increasing IL‐1β, MDA, NLRP3, 8‐OHdG, and caspase 3/7 activity. Sucralose treatment also enhanced microglial activation and neuroinflammation by upregulating IL‐1β and NLRP3 and downregulating SIRT1 and GPx4, thereby inducing ferroptosis and suppressing cell viability. Consequently, high concentrations or long‐term sucralose treatment may induce neuroinflammation and ferroptosis by targeting the SIRT1/NLRP3/IL‐1β/GPx4 pathway in HMC3 cells. [ABSTRACT FROM AUTHOR]

One of the methods of cancer treatment is radiotherapy. Although radiation resistance and toxicity in normal cells limit the use of radiotherapy in some specific anatomical situations, however, together with radiotherapy, the use of substances that increase the radiation sensitivity of cancer cells and at the same time toxicity can be useful if they do not have cells on normal cells. The present study aimed to investigate the radiosensitizing effect of tolmetin in radiotherapy treatment on human clonal cancer cells. In this study, human clone HT-29 cancer cells in different groups were exposed to X-rays and tolmetin drug and were compared with each other and with the control group by micronucleus evaluation method and nuclear division index. The effect of cytotoxicity was evaluated with the index of nuclear division and genotoxicity of cells with the method of evaluating the number of micronuclei. Based on the results, the number of micronuclei increased significantly in the radiation-receiving group compared to the control group. In the group receiving tolmetin with 75 and 100 μM concentrations, the number of micronuclei also increased compared to the control group. A significant increase in the number of micronuclei was observed in all groups treated with tolmetin that received radiation, and this increase was more noticeable in concentrations of 100 and 150 μM. Meanwhile, tolmetin did not change the nuclear division index in the studied concentrations. The present study showed that tolmetin has a radiosensitizing effect on HT-29 human colon cancer cells, which is dependent on tolmetin concentration. In addition, tolmetin does not have a cytotoxicity effect on the mentioned cell line. [ABSTRACT FROM AUTHOR]

Postoperative cognitive dysfunction is a common complication following anesthesia and surgery, and is associated with oxidative stress and apoptosis in the central nervous system. Resveratrol, an antioxidant, natural polyphenol, has been reported to have neuroprotective effect. The aim of this study was to examine whether resveratrol could attenuate the apoptosis and oxidative stress in a cell model of postoperative cognitive dysfunction. A cell model of postoperative cognitive dysfunction was constructed using human microglial clone 3 cells exposed to 1000 ng/ml lipopolysaccharide, 50 µM resveratrol, and their combination for 48 h. Then, the cell viability, proliferation, apoptosis, production of reactive oxygen species, and the expression of apoptosis-related genes and nuclear factor-E2-related factor 2 and its downstream antioxidant genes were examined. In the postoperative cognitive dysfunction cell model, lipopolysaccharide decreased the cell viability and proliferation, while it increased cell apoptosis and reactive oxygen species level. Lipopolysaccharide also upregulated the expression of pro-apoptotic genes (Bcl-2-associated X-protein and caspase-3) and genes involved in nuclear factor-E2-related factor 2-mediated antioxidant pathway (nuclear factor-E2-related factor 2, Kelch-like ECH-associated protein 1, heme oxygenase-1, and superoxide dismutase-2) in human microglial clone 3 cells. Resveratrol ameliorated the lipopolysaccharide-induced decrease of cell viability and proliferation, increase of cell apoptosis and reactive oxygen species level, and upregulation of the expression of pro-apoptotic genes. Additionally, resveratrol upregulated the expression of nuclear factor-E2-related factor 2 and its downstream antioxidant genes in lipopolysaccharide-treated human microglial clone 3 cells. Resveratrol protects human microglial clone 3 cells against lipopolysaccharide-induced injury by reducing oxidative stress through activating nuclear factor-E2-related factor 2-mediated antioxidant pathway. [ABSTRACT FROM AUTHOR]

Nancy Farmer's novel The House of the Scorpion deals with complex ethical dilemmas stemming from scientific advancements, particularly in cloning. The story unfolds in a dystopian society where clones are treated as subhuman entities. They are raised solely for the purpose of being harvested for their organs. This societal structure starkly contrasts the ethical and moral dilemmas associated with cloning. The paper highlights the stark contrast between the protected bodies of the ruling class and the exploited bodies of the clones and eejits, exposing the evils of totalitarianism and the misuse of technology. This analysis employs the ethical concepts organized in the book, The Ethics of Human Cloning, to explore the ethical dimensions surrounding human cloning. The study is targeted at stressing the necessity for implementing stringent ethical guidelines. [ABSTRACT FROM AUTHOR]

Human milk fat globule epidermal growth factor-factor VIII (MFG-E8) functions as a bridging molecule to promote the removal of dying cells by professional phagocytes. E. coli-expressed histidine-tagged recombinant human MFG-E8 (rhMFG-E8) is protective in various disease conditions. However, due to improper recombinant protein glycosylation, misfolding and the possibility of antigenicity, E. coli-expressed histidine-tagged rhMFG-E8 is unsuitable for human therapy. Therefore, we hypothesize that human cell-expressed, tag-free rhMFG-E8 will have suitable structural and functional properties to be developed as a safe and effective novel biologic to treat inflammatory diseases including radiation injury. We produced a new tag-free rhMFG-E8 protein by cloning the human MFG-E8 full-length coding sequence without any fusion tag into a mammalian vector and expressed it in HEK293-derived cells. The construct includes the leader sequence of cystatin S to maximize secretion of rhMFG-E8 into the culture medium. After purification and confirmation of the protein identity, we first evaluated its biological activity in vitro. We then determined its efficacy in vivo utilizing an experimental rodent model of radiation injury, i.e., partial body irradiation (PBI). HEK293 cell supernatant containing tag-free rhMFG-E8 protein was concentrated, purified, and rhMFG-E8 was verified by SDS-PAGE with the standard human MFG-E8 loaded as control and, mass spectrometry followed by analysis using MASCOT for peptide mass fingerprint. The biological activity of human cell-expressed tag-free rhMFG-E8 was superior to that of E. coli-expressed His-tagged rhMFG-E8. Toxicity, stability, and pharmacokinetic studies indicate that tag-free rhMFG-E8 is safe, highly stable after lyophilization and long-term storage, and with a terminal elimination half-life in circulation of at least 1.45 h. In the 15 Gy PBI model, a dose-dependent improvement of the 30-day survival rate was observed after tag-free rhMFG-E8 treatment with a 30-day survival of 89%, which was significantly higher than the 25% survival in the vehicle group. The dose modification factor (DMF) of tag-free rhMFG-E8 calculated using probit analysis was 1.058. Tag-free rhMFG-E8 also attenuated gastrointestinal damage after PBI suggesting it as a potential therapeutic candidate for a medical countermeasure for radiation injury. Our new human cell-expressed tag-free rhMFG-E8 has proper structural and functional properties to be further developed as a safe and effective therapy to treat victims of severe acute radiation injury. [ABSTRACT FROM AUTHOR]

N‑Acetyl‑d‑neuraminic acid (Neu5Ac) is the crucial compound for the chemical synthesis of antiflu medicine Zanamivir. Chemoenzymatic synthesis of Neu5Ac involves N-acetyl-d-glucosamine 2-epimerase (AGE)-catalyzed epimerization of N-acetyl-d-glucosamine (GlcNAc) to N-acetyl-d-mannosamine (ManNAc), and aldolase-catalyzed condensation between ManNAc and pyruvate. Host optimization plays an important role in the whole-cell biotransformation of value-added compounds. In this study, via single-plasmid biotransformation system, we showed that the AGE gene BT0453, cloned from human gut microorganism Bacteroides thetaiotaomicron VPI-5482, showed the highest biotransformation yield among the AGE genes tested; and there is no clear Neu5Ac yield difference between the BT0453 coupled with one aldolase coding nanA gene and two nanA genes. Next, Escherichia coli chromosomal genes involved in substrate degradation, product exportation and pH change were deleted via recombineering and CRISPR/Cas9. With the final E. coli BL21(DE3) ΔnanA Δnag ΔpoxB as host, a significant 16.5% yield improvement was obtained. Furthermore, precursor (pyruvate) feeding resulted in 3.2% yield improvement, reaching 66.8% molar biotransformation. The result highlights the importance of host optimization, and set the stage for further metabolic engineering of whole-cell biotransformation of Neu5Ac. [ABSTRACT FROM AUTHOR]

CD8+ cytotoxic T lymphocytes (CTLs) play a crucial role in anti-tumor immunity. In a previous study, we identified a subset of murine effector CTLs expressing the hepatocyte growth factor (HGF) receptor, c-Met (c-Met+ CTLs), that are endowed with enhanced cytolytic capacity. HGF directly inhibited the cytolytic function of c-Met+ CTLs, both in 2D in vitro assays and in vivo, leading to reduced T cell responses against metastatic melanoma. To further investigate the role of c-Met+ CTLs in a three-dimensional (3D) setting, we studied their function within B16 melanoma spheroids and examined the impact of cell–cell contact on the modulation of inhibitory checkpoint molecules' expression, such as KLRG1, PD-1, and CTLA-4. Additionally, we evaluated the cytolytic capacity of human CTL clones expressing c-Met (c-Met+) and compared it to c-Met− CTL clones. Our results indicated that, similar to their murine counterparts, c-Met+ human CTL clones exhibited increased cytolytic activity compared to c-Met− CTL clones, and this enhanced function was negatively regulated by the presence of HGF. Taken together, our findings highlight the potential of targeting the HGF/c-Met pathway to modulate CTL-mediated anti-tumor immunity. This research holds promise for developing strategies to enhance the effectiveness of CTL-based immunotherapies against cancer. [ABSTRACT FROM AUTHOR]

This paper addresses the issue of the loss of the real in the fiction of Kazuo Ishiguro as a contemporary author whose thought line is still in progress. His approach to this issue is anti-capitalist as he questions the so-called scientific advancement led by cash-oriented capitalists and industrialists. His writing seeks to strip the veil of the murderous nature of this kind of science. He blames it for killing the real and creating a world of simulations. He animadverts upon dystopian spaces where he holds postmodern scientific knowledge responsible for the digression of the natural course of life and lays bare the secrets behind the replacement of the real with the simulated by drawing attention to such debatable topics as human cloning, cosmetic surgeries and artificial intelligence. In terms of methodology of analysis, this paper is primarily based on a close examination of the author’s literary texts: two novels (Never Let Me Go and Klara and the Sun) and three short stories from Nocturnes (“Malvern Hills”, “Nocturne” and “Crooner”). Postmodernist concepts have been of great relevance to the analysis of these texts, for his fiction could not be approached in isolation from the realities of the postmodern era where it’s produced. Equally, bearing in mind the author’s socio-ethnic and historical background, the society where he lives and the politico-cultural transformations of the world aTher the Second World War plays an important role in the analysis of his texts.

The migratory ability of microglia facilitates their rapid transport to a site of injury to kill and remove pathogens. However, the effect of Treponema pallidum membrane proteins on microglia migration remains unclear. The effect of Tp47 on the migration ability and autophagy and related mechanisms were investigated using the human microglial clone 3 cell line. Tp47 inhibited microglia migration, the expression of autophagy‐associated protein P62 decreased, the expression of Beclin‐1 and LC3‐II/LC3‐I increased, and the autophagic flux increased in this process. Furthermore, autophagy was significantly inhibited, and microglial cell migration was significantly increased after neutralisation with an anti‐Tp47 antibody. In addition, Tp47 significantly inhibited the expression of p‐PI3K, p‐AKT, and p‐mTOR proteins, and the sequential activation of steps in the PI3K/AKT/mTOR pathways effectively prevented Tp47‐induced autophagy. Moreover, Tp47 significantly inhibited the expression of p‐FOXO1 protein and promoted FOXO1 nuclear translocation. Inhibition of FOXO1 effectively suppressed Tp47‐induced activation of autophagy and inhibition of migration. Treponema pallidum membrane protein Tp47‐induced autophagy and inhibited cell migration in HMC3 Cells via the PI3K/AKT/FOXO1 pathway. These data will contribute to understanding the mechanism by which T. pallidum escapes immune killing and clearance after invasion into the central nervous system. [ABSTRACT FROM AUTHOR]

Reservoir host associations have been observed among and within Borrelia genospecies, and host complement-mediated killing is a major determinant in these interactions. In North America, only a subset of Borrelia burgdorferi lineages cause the majority of disseminated infections in humans. We hypothesize that differential resistance to human complement-mediated killing may be a major phenotypic determinant of whether a lineage can establish systemic infection. As a corollary, we hypothesize that borreliacidal action may differ among human subjects. To test these hypotheses, we isolated primary B. burgdorferi clones from field-collected ticks and determined whether the killing effects of human serum differed among those clones in vitro and/or whether these effects were consistent among human sera. Clones associated with human invasiveness did not show higher survival in human serum compared to noninvasive clones. These results indicate that differential complement-mediated killing of B. burgdorferi lineages is not a determinant of invasiveness in humans. Only one significant difference in the survivorship of individual clones incubated in different human sera was detected, suggesting that complement-mediated killing of B. burgdorferi is usually similar among humans. Mechanisms other than differential human complement-mediated killing of B. burgdorferi lineages likely explain why only certain lineages cause the majority of disseminated human infections. [ABSTRACT FROM AUTHOR]

T-cell receptor (TR) diversity of the variable domains is generated by recombination of both the alpha (TRA) and beta (TRB) chains. The textbook process of TRB chain production starts with TRBD and TRBJ gene rearrangement, followed by the rearrangement of a TRBV gene to the partially rearranged D-J gene. Unsuccessful V-D-J TRB rearrangements lead to apoptosis of the cell. Here, we performed deep sequencing of the poorly explored pool of partial TRBD1-TRBD2 rearrangements in T-cell genomic DNA. We reconstructed full repertoires of human partial TRBD1-TRBD2 rearrangements using novel sequencing and validated them by detecting V-D-J recombination-specific byproducts: excision circles containing the recombination signal (RS) joint 5'D2-RS - 3'D1-RS. Identified rearrangements were in compliance with the classical 12/23 rule, common for humans, rats, and mice and contained typical V-D-J recombination footprints. Interestingly, we detected a bimodal distribution of D-D junctions indicating two active recombination sites producing long and short D-D rearrangements. Long TRB D-D rearrangements with two D-regions are coding joints D1-D2 remaining classically on the chromosome. The short TRB D-D rearrangements with no D-region are signal joints, the coding joint D1-D2 being excised from the chromosome. They both contribute to the TRB V-(D)-J combinatorial diversity. Indeed, short D-D rearrangements may be followed by direct V-J2 recombination. Long D-D rearrangements may recombine further with J2 and V genes forming partial D1-D2-J2 and then complete V-D1-D2-J2 rearrangement. Productive TRB V-D1-D2-J2 chains are present and expressed in thousands of clones of human antigen-experienced memory T cells proving their capacity for antigen recognition and actual participation in the immune response. [ABSTRACT FROM AUTHOR]

The online space has continued to be a platform for not only private and mundane discussions, but also a tribune for voicing critical political and national opinions. Nigerians and the international community have employed online media, as well as other media platforms to articulate their thoughts on the claims which favoured the possibility of the demise of the immediate past president of Nigeria, President Muhammadu Buhari, after a prolonged illness in the year 2007 and the ploy of a Jubril of Sudan clone in his place. This study examined the pragmatic strategies employed in the online articles on the national dilemma caused by the Buhari/Jubril claims and refutals, in response to the lacuna in the literature on such analytical investigations on the subject. An online article titled, "Buhari: The real, the fake and the dead" authored by a Nigerian writer, Tunde Odesola retrieved from the online page of The Punch Newspaper on December 3, 2018, served as data for the study. The article was analysed using insights from the Stance Theory and the Pragmatics in general. The analysis revealed eight pragmatic strategies utilised by the writer, namely Biblical allusion, evaluative stance and positioning, epistemic stance and evocation of sarcasm, metaphors, and derogative labelling as anti-Jubrilist positioning, epistemic stance, and evocation of (dis)alignment, antithetical evocations, evaluative stance and berating, evaluative stance and justifying. These result in a pragmatic reconstruction of the readers' views on the issue. [ABSTRACT FROM AUTHOR]

Ethical and theological debates concerning human cloning are getting more intense as cloning technology continues to develop. This has led to debates originating from both secular ethics and Christian theological perspectives. This paper aims to coordinate the debate according to these two perspectives. It will draw from the positions of Christian theology, the position of the United Nations Universal Declaration of Human Rights, and the positions of scientific authorities. It will conclude by stressing the importance of regulating new technologies in a manner which recognizes the shared ethico-theological concerns for human dignity, rights, freedoms, and the moral growth of humanity. [ABSTRACT FROM AUTHOR]

Expression cloning of fully human monoclonal antibodies (hmAbs) is seeing powerful utility in the field of vaccinology, especially for elucidating vaccine-induced B-cell responses and novel vaccine candidate antigen discovery. Precision of the hmAb cloning process relies on efficient isolation of hmAb-producing plasmablasts of interest. Previously, a novel immunoglobulin-capture assay (ICA) was developed, using single protein vaccine antigens, to enhance the pathogen-specific hmAb cloning output. Here, we report a novel modification of this single-antigen ICA using formalin-treated, fluorescently stained whole cell suspensions of the human bacterial invasive pathogens, Streptococcus pneumoniae and Neisseria meningitidis. Sequestration of IgG secreted by individual vaccine antigen-specific plasmablasts was achieved by the formation of an anti-CD45-streptavidin and biotin anti-IgG scaffold. Suspensions containing heterologous pneumococcal and meningococcal strains were then used to enrich for polysaccharide- and protein antigen-specific plasmablasts, respectively, during single cell sorting. Following application of the modified whole-cell ICA (mICA), ~61% (19/31) of anti-pneumococcal polysaccharide hmAbs were cloned compared to 14% (8/59) obtained using standard (non-mICA) methods - representing a ~4.4-fold increase in hmAb cloning precision. A more modest ~1.7-fold difference was obtained for anti-meningococcal vaccine hmAb cloning; ~88% of hmAbs cloned via mICA versus ~53% cloned via the standard method were specific for a meningococcal surface protein. VDJ sequencing revealed that cloned hmAbs reflected an anamnestic response to both pneumococcal and meningococcal vaccines; diversification within hmAb clones occurred by positive selection for replacement mutations. Thus, we have shown successful utilization of whole bacterial cells in the ICA protocol enabling isolation of hmAbs targeting multiple disparate epitopes, thereby increasing the power of approaches such as reverse vaccinology 2.0 (RV 2.0) for bacterial vaccine antigen discovery. [ABSTRACT FROM AUTHOR]

Answering climate change challenges, the paper proposes an intelligent decision support system (DSS) for the management of green-blue infrastructure (GBI). Addressing the gaps identified in other studies, the designed DSS incorporates four key elements: 1/interdisciplinary collaboration among all stakeholders 2/inclusion of practical operation and maintenance activities, 3/main components of distributed DSS, with practical examples of use, 4/consideration of conditions specific to the location. The multi-layered DSS architecture can be implemented as a unified platform that provides a comprehensive, customizable, and flexible framework based on AI tools, big data and analytics, edge computing, cloud, and mobile, IIoT, and biometric system tools. The use of cobots and digital clones alongside humans results in the implementation of hybrid human-machine units. DSS for GBI increases decision-making capacity and can serve as a foundation for the implementation of similar systems by governments and local communities to build sustainable and resilient communities. [ABSTRACT FROM AUTHOR]

We propose a novel approach for robotic industrial insertion tasks using the Programming by Demonstration technique. Our method allows robots to learn a high-precision task by observing human demonstration once, without requiring any prior knowledge of the object. We introduce an Imitated-to-Finetuned approach that generates imitated approach trajectories by cloning the human hand's movements and then fine-tunes the goal position with a visual servoing approach. To identify features on the object used in visual servoing, we model object tracking as the moving object detection problem, separating each demonstration video frame into the moving foreground that includes the object and demonstrator's hand and the static background. Then a hand keypoints estimation function is used to remove the redundant features on the hand. The experiment shows that the proposed method can make robots learn precision industrial insertion tasks from a single human demonstration. [ABSTRACT FROM AUTHOR]

Methicillin-resistant Staphylococcus aureus (MRSA) is a strain of pathogenic bacteria that is a major problem in the world's health. Due to their frequent interaction with humans, pets are one of the main risk factors for the spread of MRSA. The possibility for zoonotic transmission exists since frequently kept dogs and cats are prone to contract MRSA and act as reservoirs for spreading MRSA. The mouth, nose, and perineum are the primary locations of MRSA colonization, according to the findings of MRSA identification tests conducted on pets. The types of MRSA clones identified in cats and dogs correlated with MRSA clones infecting humans living in the same geographic area. A significant risk factor for the colonization or transmission of MRSA is human-pet contact. An essential step in preventing the spread of MRSA from humans to animals and from animals to humans is to keep hands, clothing, and floor surfaces clean. [ABSTRACT FROM AUTHOR]

The Department of Defense recently began an effort to improve and standardize virus challenge materials and efficacy determination strategies for testing therapeutics and vaccines. This includes stabilization of virus genome sequences in cDNA form where appropriate, use of human-derived virus isolates, and noninvasive strategies for determination of challenge virus replication. Eventually, it is desired that these approaches will satisfy the FDA "Animal Rule" for licensure, which substitutes animal efficacy data when human data are unlikely to be available. To this end, we created and examined the virulence phenotype of cDNA clones of prototypic human infection-derived strains of the alphaviruses, Venezuelan (VEEV INH9813), eastern (EEEV V105) and western (WEEV Fleming) equine encephalitis viruses, and created fluorescent and luminescent reporter expression vectors for evaluation of replication characteristics in vitro and in vivo. Sequences of minimally passaged isolates of each virus were used to synthesize full-length cDNA clones along with a T7 transcription promoter-based bacterial propagation vector. Viruses generated from the cDNA clones were compared with other "wild type" strains derived from cDNA clones and GenBank sequences to identify and eliminate putative tissue culture artifacts accumulated in the cell passaged biological stocks. This was followed by examination of aerosol and subcutaneous infection and disease in mouse models. A mutation that increased heparan sulfate binding was identified in the VEEV INH9813 biological isolate sequence and eliminated from the cDNA clone. Viruses derived from the new human isolate cDNA clones showed similar mouse virulence to existing clone-derived viruses after aerosol or subcutaneous inoculation. [ABSTRACT FROM AUTHOR]

Objective: We aimed to clone and express the human Cu, Zn superoxide dismutase (hSOD1) in Bacillus subtilis 1012. Also, we investigated the expression level of hSOD1 under different induction conditions. Result: As an essential member of the antioxidant defense system in vivo, hSOD1 has become a therapeutic agent against host diseases, such as oxygen toxicity, acute inflammation, and radiation injury. The recombinant hSOD1 was successfully secreted extracellularly into B. subtilis 1012. The expression conditions were optimized, including inoculum size, different media, temperatures, and inducer concentrations. Finally, the highest level of hSOD1 was produced as a soluble form in Super rich medium by 2% inoculum with 0.2 mM of IPTG at 37 °C after the induction for 24 h. Besides, 20 g/L of lactose also displayed the same inductive effect on hSOD1 expression as that of IPTG (0.2 mM). Finally, the specific activity of purified hSOD1 was determined to be 1625 U/mg in the presence of 800 μM of Cu2+ and 20 μM of Zn2+. Conclusions: We propose that the B. subtilis 1012-hSOD1 strain system has great potential in future industrial applications. [ABSTRACT FROM AUTHOR]

Ethical and theological debates about human cloning are getting hotter in recent times. Nowadays, cloning research is more common than ever before because of developments in biotechnologies, public outrage, and religious and ethical issues. Yet, despite concerns about its effectiveness and safety and the potential danger, studies demonstrate that human cloning does not produce a class structure. This paper aims to contribute to the research and debate on human cloning from the perspectives of ethics and Christian theology. Using the descriptive and critical analysis methodology, the paper reveals that while it is unethical, human cloning still has the prospect of benefiting future humanity by appealing to sick persons, infertile couples, those who want to avoid undesirable genetic traits, single and same-sex couples, and parents who want to clone a dead child. It concludes by calling on public authorities around the world to exercise their statutory responsibilities to maintain the unity, coherence, and organization of new technologies in society along the dictates of the United Nations Universal Declaration of Human Rights, which recognizes the dignity, rights, and freedoms, and moral growth of humanity. [ABSTRACT FROM AUTHOR]

"Reproductive" cloning and "therapeutic"or "research" cloning are both deliberate attempts to create humans that are genetically identical. Human reproductive cloning in general is prohibited by a number of international and regional agreements, including the Charter of Fukushima, the Additional Protocol of the Council of Europe to the Convention on Human Rights and Biomedicine, the World Health Organization resolutions on the implications of cloning for human health, and the Universal Declaration on the Human Genome and Human Rights. However, there are some countries that want to explore therapeutic cloning and cannot, therefore, support a general ban on cloning. This paper aims to review the legal position of human cloning in the UK and France and further compares the issue between the two countries. the legal position of human cloning in the UK and France, it is clear that both countries were initially against the idea and concept of human cloning in general. Human cloning is a much-needed technology, especially in these modern times. Every day we encounter new diseases and illnesses, so human cloning is essential to help us be better prepared for the future. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is one of the leading causes of healthcare-associated infections in humans. This bacterium is less represented in veterinary medicine, despite causing difficult-to-treat infections due to its capacity to acquire antimicrobial resistance, produce biofilms, and persist in the environment, along with its limited number of veterinary antibiotic therapies. Here, we explored susceptibility profiles to antibiotics and to didecyldimethylammonium chloride (DDAC), a quaternary ammonium widely used as a disinfectant, in 168 P. aeruginosa strains isolated from animals, mainly Equidae. A genomic study was performed on 41 of these strains to determine their serotype, sequence type (ST), relatedness, and resistome. Overall, 7.7% of animal strains were resistant to carbapenems, 10.1% presented a multidrug-resistant (MDR) profile, and 11.3% showed decreased susceptibility (DS) to DDAC. Genomic analyses revealed that the study population was diverse, and 4.9% were ST235, which is considered the most relevant human high-risk clone worldwide. This study found P. aeruginosa populations with carbapenem resistance, multidrug resistance, and DS to DDAC in equine and canine isolates. These strains, which are not susceptible to antibiotics used in veterinary and human medicine, warrant close the setting up of a clone monitoring, based on that already in place in human medicine, in a one-health approach. [ABSTRACT FROM AUTHOR]

This study sought to analyze the antimicrobial resistant phenotypes and genotypes as well as the virulence content of S. aureus isolates recovered from patients with diabetic foot infections (DFIs) in a Tunisian hospital. Eighty-three clinical samples of 64 patients were analyzed, and bacterial isolates were identified by MALDI-TOF. The antimicrobial resistance phenotypes were determined by the Kirby–Bauer disk diffusion susceptibility test. Resistance and virulence genes, agr profile, spa and SCCmec types were determined by PCR and sequencing. S. aureus was detected in 14 of the 64 patients (21.9%), and 15 S. aureus isolates were recovered. Six out of the fifteen S. aureus isolates were methicillin-resistant (MRSA, mecA-positive) (40%). The isolates harbored the following resistance genes (number of isolates): blaZ (12), erm(B) (2), erm(A) (1), msrA (2), tet(M) (2), tet(K) (3), tet(L) (1), aac(6′)-aph(2″) (2), ant(4″) (1) and fexA (1). The lukS/F-PV and tst genes were detected in three isolates. Twelve different spa-types were identified and assigned to seven clonal complexes with the predominance of agr-type III. Furthermore, the SCCmec types III, IV and V were found among the MRSA isolates. Moreover, one MSSA CC398-t571-agr-III isolate was found; it was susceptible to all antimicrobial agents and lacked luk-S/F-PV, tst, eta and etb genes. This is the first report on the prevalence and molecular characterization of S. aureus from DFIs and also the first detection of the MSSA-CC398-t571 clone in human infections in Tunisia. Our findings indicated a high prevalence S. aureus in DFIs with genetic diversity among the MSSA and MRSA isolates. [ABSTRACT FROM AUTHOR]

Microglia activation and neuroinflammation have been extensively studied in murine models of neurodegenerative diseases; however, to overcome the genetic differences between species, a human cell model of microglia able to recapitulate the activation profiles described in patients is needed. Here we developed human models of Parkinson's like neuroinflammation by using the human microglia clone 3 (HMC3) cells, whose activation profile in response to classic inflammatory stimuli has been controversial and reported only at mRNA levels so far. In fact, we showed the increased expression of the pro-inflammatory markers iNOS, Caspase 1, IL-1β, in response to IFN-γ plus high glucose, a non-specific disease stimulus that emphasized the dynamic polarization and heterogenicity of the microglial population. More specifically, we demonstrated the polarization of HMC3 cells through the upregulation of iNOS expression and nitrite production in response to the Parkinson's like stimuli, 6-hydroxidopamine (6-OHDA) and 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine (MPTP), the latter depending on the NF-κB pathway. Furthermore, we identified inflammatory mediators that promote the pro-inflammatory activation of human microglia as function of different pathways that can simulate the phenotypic transition according to the stage of the pathology. In conclusion, we established and characterized different systems of HMC3 cells activation as in vitro models of Parkinson's like neuroinflammation. [ABSTRACT FROM AUTHOR]

During disease progression and bone metastasis, breast tumor cells interact with various types of bystander cells residing in the tumor microenvironment. Such interactions prompt tumor cell heterogeneity. We used successive co-culture as an experimental model to examine cancer–bystander cell interaction. RMCF7-2, a clone of the human breast cancer MCF-7 cells tagged with a red fluorescent protein, was tracked for morphologic, behavioral, and gene expression changes. Co-cultured with various types of hematopoietic cells, RMCF7-2 adopted stable changes to a rounded shape in suspension growth of red fluorescent cells, from which derivative clones displayed marked expressional changes of marker proteins, including reduced E-cadherin and estrogen receptor α, and loss of progesterone receptor. In a successive co-culture with bone marrow-derived mesenchymal stem/stromal cells, the red fluorescent clones in suspension growth changed once more, adopting an attachment growth, but in diversified shapes. Red fluorescent clones recovered from the second-round co-culture were heterogeneous in morphology, but retained the altered marker protein expression while displaying increased proliferation, migration, and xenograft tumor formation. Interaction with bystander cells caused permanent morphologic, growth behavioral, and gene expressional changes under successive co-culture, which is a powerful model for studying cancer cell heterogeneity during breast cancer progression and metastasis. [ABSTRACT FROM AUTHOR]

The emergence of pathogens is conferring resistance to last-resort therapies such as tigecycline, colistin, and carbapenems, limiting the therapeutic options, and raising concerns about the emergence of new "superbugs." This study reports the first incident of a blaNDM􀀀5 and tet(X4) co-harboring Escherichia coli with resistance to carbapenem and tigecycline recovered as the causative agent of a urinary tract infection in a 94-year-old patient. The E. coli strain ECCL209 carries multiple resistance genes [i.e., blaTEM􀀀1B, blaNDM􀀀5, blaCMY􀀀2, aadA22, florR, erm(B), mph(A), erm(42), lnuG, qnrS1, and sul2] and exhibits resistance to almost all clinically used antibiotics. MLST analysis found that the strain belongs to ST648, considered a worldwide highrisk pandemic clone. Moreover, multiple plasmid incompatibility types were detected, i.e., IncHI1A, IncHI1B, IncFII, IncFIA, IncFIB, IncQ1, Col, and IncX4. Genetic analysis revealed that blaNDM􀀀5 and tet(X4) genes were localized on two hybrid plasmids with multiple replicons. Continuous monitoring studies are suggested to quantify the antimicrobial resistance and assess the dissemination of such superbugs into a human healthcare setting. [ABSTRACT FROM AUTHOR]

The Klebsiella species present a remarkable genetic and ecological diversity, being ubiquitous in nature. In particular, the Klebsiella pneumoniae species complex (KpSC) has emerged as a major public health threat in the world, being an interesting model to assess the risk posed by strains recovered from animals and the environment to humans. We therefore performed a genomic surveillance analysis of the KpSC using every public genome in Brazil, aiming to show their local and global relationships, and the connectivity of antibiotic resistance and virulence considering human, animal, and environmental sources. The 390 genomes from distinct sources encompassed the K. pneumoniae, Klebsiella quasipneumoniae subsp. quasipneumoniae, Klebsiella quasipneumoniae subsp. similipneumoniae, Klebsiella variicola subsp. variicola, Klebsiella variicola subsp. tropica, and Klebsiella grimontii species and subspecies. K. pneumoniae harbored dozens of antibiotic resistance genes, while most of the genomes belong to the high-risk pandemic CC258 occurring in humans, animals, and the environment. In K. pneumoniae ST11, a high prevalence of the virulence determinants yersiniabactin, colibactin, and T6SS was revealed in association with multi-drug resistance (MDR), including carbapenem resistance. A diversity of resistance genes is carried by plasmids, some shared between strains from different STs, regions, and sources. Therefore, here were revealed some factors driving the success of KpSC as a pathogen. [ABSTRACT FROM AUTHOR]

Programmed cell death protein 1 (PD-1) plays a significant role in suppressing antitumor immune responses. Cancer treatment with immune checkpoint inhibitors (ICIs) targeting PD-1 has been approved to treat numerous cancers and is the backbone of cancer immunotherapy. Anti-PD-1 molecule is necessary for next-generation cancer immunotherapy to further improve clinical efficacy and safety as well as integrate into novel treatment combinations or platforms. We developed a highly efficient hybridoma generation and screening strategy to generate high-potency chimeric anti-PD-1 molecules. Using this strategy, we successfully generated several mouse hybridoma and mouse/human chimeric clones that produced high-affinity antibodies against human PD-1 with high-quality in vitro PD-1/PD-L1 binding blockade and T cell activation activities. The lead chimeric prototypes exhibited overall in vitro performance comparable to commercially available anti-PD-1 antibodies and could be qualified as promising therapeutic candidates for further development toward immuno-oncology applications. [ABSTRACT FROM AUTHOR]

Insulin-producing cells derived from induced pluripotent stem cells (iPSCs) are promising candidates for β cell replacement in type 1 diabetes. However, the risk of teratoma formation due to residual undifferentiated iPSCs contaminating the differentiated cells is still a critical concern for clinical application. Here, we hypothesized that pretreatment of iPSC-derived insulin-producing cells with an anti-CD30 antibody–drug conjugate could prevent in vivo teratoma formation by selectively killing residual undifferentiated cells. CD30 is expressed in all human iPSCs clones tested by flow cytometry (n = 7) but not in iPSC-derived β cells (iβs). Concordantly, anti-CD30 treatment in vitro for 24 h induced a dose-dependent cell death (up to 90%) in human iPSCs while it did not kill iβs nor had an impact on iβ identity and function, including capacity to secrete insulin in response to stimuli. In a model of teratoma assay associated with iβ transplantation, the pretreatment of cells with anti-CD30 for 24 h before the implantation into NOD-SCID mice completely eliminated teratoma development (0/10 vs. 8/8, p < 0.01). These findings suggest that short-term in vitro treatment with clinical-grade anti-CD30, targeting residual undifferentiated cells, eliminates the tumorigenicity of iPSC-derived β cells, potentially providing enhanced safety for iPSC-based β cell replacement therapy in clinical scenarios. [ABSTRACT FROM AUTHOR]

Novel approaches for malaria prophylaxis remain important. Synthetic DNA-encoded monoclonal antibodies (DMAbs) are a promising approach to generate rapid, direct in vivo host-generated mAbs with potential benefits in production simplicity and distribution coupled with genetic engineering. Here, we explore this approach in a malaria challenge model. We engineered germline-reverted DMAbs based on human mAb clones CIS43, 317, and L9 which target a junctional epitope, major repeat, and minor repeat of the Plasmodium falciparum circumsporozoite protein (CSP) respectively. DMAb variants were encoded into a plasmid vector backbone and their expression and binding profiles were characterized. We demonstrate long-term serological expression of DMAb constructs resulting in in vivo efficacy of CIS43 GL and 317 GL in a rigorous mosquito bite mouse challenge model. Additionally, we engineered an Fc modified variant of CIS43 and L9-based DMAbs to ablate binding to C1q to test the impact of complement-dependent Fc function on challenge outcomes. Complement knockout variant DMAbs demonstrated similar protection to that of WT Fc DMAbs supporting the notion that direct binding to the parasite is sufficient for the protection observed. Further investigation of DMAbs for malaria prophylaxis appears of importance. [ABSTRACT FROM AUTHOR]

The positive effects on the career progress of the successful MSCA applicants can be partly attributed to the training aspects that go along with the MSCA Fellowship. Rule 1: Familiarise yourself with the MSCA funding programme and do not hesitate to ask for h... Make sure that you understand the overall logic as well as specific requirements of the MSCA programme. Further, free seminars and webinars that explain the current EU framework programme and the MSCA funding scheme are offered frequently from the regional to the EU level. The MSCA is part of the ninth European research and innovation framework programme I Horizon Europe i and is the European Union's (EU) flagship career development support programme with a budget totalling €6.6 billion from 2021 to 2027. [Extracted from the article]

Background: The CAAX-prenyltransferases farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I) are heterodimers with a common α- (FTα) and unique β-subunits. Recently, α-subunits of species (e.g., human) that harbour an N-terminal proline-rich region (PRR) showed different dimerization behaviours than α-subunits without PRR (e.g., yeast). However, the specific function of the PRR has not been elucidated so far. Methods: To determine whether the PRR is a conserved motif throughout eukaryotes, we performed phylogenetics. Elucidating the impact of the PRR on enzyme properties, we cloned human as well as rat PRR deficient FTα, expressed them heterologously and compared protein–protein interaction by pull-down as well as crosslinking experiments. Substrate binding, enzyme activity and sensitivity towards common FTase inhibitors of full length and PRR-deletion α-subunits and their physiological partners was determined by continuous fluorescence assays. Results: The PRR is highly conserved in mammals, with an exception for marsupials harbouring a poly-alanine region instead. The PRR shows similarities to canonical SH3-binding domains and to profilin-binding domains. Independent of the PRR, the α-subunits were able to dimerize with the different physiological β-subunits in in vitro as well as in yeast two-hybrid experiments. FTase and GGTase I with truncated FTα were active. The KM values for both substrates are in the single-digit µM range and show no significant differences between enzymes with full length and PRR deficient α-subunits within the species. Conclusions: Our data demonstrate that an N-terminal PRR of FTα is highly conserved in mammals. We could show that the activity and inhibitability is not influenced by the truncation of the N-terminal region. Nevertheless, this region shows common binding motifs for other proteins involved in cell-signalling, trafficking and phosphorylation, suggesting that this PRR might have other or additional functions in mammals. Our results provide new starting points due to the relevant but only partly understood role of FTα in eukaryotic FTase and GGTase I. 83PbrUwLcXbuUPceRJ4Uzr Video Abstract [ABSTRACT FROM AUTHOR]

Biofilm synthesizing multi-drug resistant Staphylococcus pseudintermedius bacteria has been recognized as the human infectious agent. It has been detected in the diseases of skin, ear, and postoperative infections. Its infections are becoming a major health problem due to its multi-drug resistance capabilities. However, no commercial vaccine for the treatment of its infections is currently available in the market. Here we employed the subtractive proteomics and reverse vaccinology approach to determine the potential novel drug and vaccine targets against S. pseudintermedius infections in humans. After screening the core-proteome of the 39 complete genomes of S. pseudintermedius, 2 metabolic pathways dependent and 34 independent proteins were determined as novel potential drug targets. Two proteins were found and used as potential candidates for designing the chimeric vaccine constructs. Depending on the properties such as antigenicity, toxicity and solubility, multi-epitope based vaccines constructs were designed. For immunogenicity enhancement, different specific sequences like linkers, PADRE sequences and molecular adjuvants were added. Molecular docking and molecular dynamic simulation analyses were performed to evaluate the prioritized vaccine construct's interactions with human immune cells HLA and TLR4. Finally, the cloning and expression ability of the vaccine construct was determined in the bacterial cloning system and human body immune response was predicted through immune simulation analysis. In conclusion, this study proposed the potential drug and vaccine targets and also designed a chimera vaccine to be tested and validated against infectious S. pseudintermedius species. [ABSTRACT FROM AUTHOR]

The article analyzes the increase in the use of assisted reproduction techniques and their legal regulation in Latin America, focusing on the context of Sinaloa, Mexico. It highlights the need for less restrictive and conservative regulation that protects human rights and legal principles and values. The diversity in assisted reproduction legislation in Latin America is mentioned, as well as the influence of non-legal criteria in its regulation. Additionally, the lack of regulation in some countries is mentioned, which has led to the commodification of human genetics. The document provides a list of references related to infertility treatment, heterologous artificial insemination, surrogacy, semen donor selection, and fundamental rights in legal philosophy. It also includes international declarations and agreements on the human genome, human cloning, intergenerational responsibility, and bioethics. [Extracted from the article]

Objective: To determine whether the width of the shoulder and the size of the bystander effect are correlated using clonal lineages derived from a cultured cell line. Methods: HCT 116 (p53 wildtype) cells were grown at cloning density and individual viable colonies were picked off and grown to establish a series of cell lines from both unirradiated and irradiated progenitors. These cell lines were then irradiated to generate full survival curves. Highly variant clones were then tested to determine the level of the bystander effect using a medium transfer protocol. Results: The multi-target model gave the best fit in these experiments and size of the shoulder n is assessed in terms of radiosensitivity. The parent cell line has an n value of 1.1 while the most variant clones have n values of 0.88 (Clone G) and 5.5 (Clone A). Clonal lines subject to irradiation prior to isolation differed in bystander signal strength in comparison to clonal lines which were not initially irradiated (P =.055). Conclusions: Based on these experiments we suggest there may be a link between shoulder size of a mammalian cell line and the strength of a bystander effect produced in vitro. This may have implications for radiotherapy related to out-of-field effects. [ABSTRACT FROM AUTHOR]

Aquaculture is a rapidly expanding sector in which it is important to monitor the occurrence of multi-drug resistant (MDR) bacteria. The presence of extended-spectrum β-lactamase (ESBL-) or carbapenemase-producing Enterobacterales is a commonly used indicator of the resistance burden in a given sector. In this study, 641 pieces of farmed fish (sea bream and sea bass), as well as 1075 Mediterranean clams, were analyzed. All ESBL- and carbapenemase-producing Enterobacterales collected were whole-genome sequenced. The proportion of ESBL-producing Enterobacterales was 1.4% in fish and 1.6% in clams, carried by Escherichia coli (n = 23) and Klebsiella pneumoniae (n = 4). The ESBL phenotype was exclusively due to the presence of blaCTX-M genes, the most frequent one being blaCTX-M-15. The blaCTX-M-1 gene was also identified in six E. coli, among which four were carried by IncI1/pST3 plasmids, possibly betraying an animal origin. Carbapenemases were absent in fish but identified in two K. pneumoniae isolates from clams (blaNDM-1 and blaOXA-48). Several sequence types (STs) identified were associated with human MDR clones such as E. coli ST131 and ST617, or K. pneumoniae ST307 and ST147. Our results might indicate that bacteria from hospital or farm effluents can reach the open sea and contaminate seafood and fish that are living or raised nearby. Therefore, monitoring the quality of water discharged to the sea and the presence of MDR bacteria in seafood is mandatory to ensure the quality of fishery products. [ABSTRACT FROM AUTHOR]

In 2001, the DNA Copyright Institute sought to capitalize on the fear of human cloning by offering celebrities the opportunity to use copyright to secure exclusive rights in their DNA. At the time, a Copyright Office spokesperson pointed out that a person's DNA "is not an original work of authorship." That statement is no longer self-evident. A scientist claims to have used CRISPR technology to create a pair of twin girls with human-altered DNA that may provide immunity to HIV infection and improved cognitive function. Through gene therapy, doctors can "author" changes to patients' DNA to cure disease. Scientists "edit" bacterial cell DNA to produce medicines and industrial enzymes. Researchers have "written" original DNA encoding a GIF of a running horse. Does copyright grant exclusive rights to these creations? For decades, scholars have argued that DNA sequences, like computer programs, are copyrightable "works" encompassed by the Copyright Act's definition of "literary works." So far, the Copyright Office is unconvinced and continues to list DNA sequences and compounds as "works" that do not constitute copyrightable subject matter. This Article takes a new approach by proposing that DNA is not a "work" at all. Rather, DNA is a medium in which information is stored. In the words of the Copyright Act, DNA compounds are "copies" in which an original copyrightable work or a functional creation may be fixed. Under this framework, literature is entitled to copyright protection whether it exists as a copy printed on paper or encoded into DNA. Genetic DNA, which functions as a component of cellular machinery to produce useful chemicals, is entitled to no more copyright protection than any other machine component. Rejecting this approach and continuing to treat DNA as a "work" rather than a "copy" has real world consequences. The recent history of copyright protection for computer programs provides a cautionary tale. Mischaracterizing DNA in the way that computer programs have been mischaracterized -- as a type of "work" under the Copyright Act -- could lead to the extension of exclusive copyrights to the functional DNA in living organisms in the same way that copyright protection has been extended to some functional aspects of computer programs. [ABSTRACT FROM AUTHOR]

A number of AI-generated tools are used today to clone human voices, leading to a new technology known as Audio Deepfakes (ADs). Despite being introduced to enhance human lives as audiobooks, ADs have been used to disrupt public safety. ADs have thus recently come to the attention of researchers, with Machine Learning (ML) and Deep Learning (DL) methods being developed to detect them. In this article, a review of existing AD detection methods was conducted, along with a comparative description of the available faked audio datasets. The article introduces types of AD attacks and then outlines and analyzes the detection methods and datasets for imitation- and synthetic-based Deepfakes. To the best of the authors' knowledge, this is the first review targeting imitated and synthetically generated audio detection methods. The similarities and differences of AD detection methods are summarized by providing a quantitative comparison that finds that the method type affects the performance more than the audio features themselves, in which a substantial tradeoff between the accuracy and scalability exists. Moreover, at the end of this article, the potential research directions and challenges of Deepfake detection methods are discussed to discover that, even though AD detection is an active area of research, further research is still needed to address the existing gaps. This article can be a starting point for researchers to understand the current state of the AD literature and investigate more robust detection models that can detect fakeness even if the target audio contains accented voices or real-world noises. [ABSTRACT FROM AUTHOR]

Urinary tract infections (UTI) caused by Escherichia coli are frequently diagnosed in humans and companion animals. Extended-spectrum beta-lactamase (ESBL)- and cephalosporinase (pAmpC)-producing Escherichia coli are worldwide-disseminated and frequently multidrug-resistant, hence leading to treatment failure and public health concerns. This study aimed to characterize and compare ESBL/pAmpC-producing E. coli strains causing community-acquired UTI in companion animals and non-related humans. Third-generation cephalosporin (3GC)-resistant E. coli (companion animals n = 35; humans n = 85) isolated from patients with UTI were tested against 14 antimicrobials following CLSI guidelines. PCR-based assays were used to detect the major E. coli phylogenetic groups, pathogenicity associated-islands (PAIs), virulence genes, and ESBLs/pAmpC resistance genes. ESBL/pAmpC-producing E. coli isolates were typed by multi-locus sequence typing (MLST) and PCR. E. coli strains from companion animals and humans shared two MDR high-risk clonal lineages: ST131 and ST648. To the best of our knowledge, this study reports the first description of E. coli ST131 clade C1-M27 and the clonal lineage ST131 clade A in humans with community-acquired UTI in Portugal. Considering that companion animals with UTI are generally treated at home by the owners, measures should be implemented to avoid the spread of multidrug-resistant high-risk clones to humans and their household environment. [ABSTRACT FROM AUTHOR]

• Infectious ratHEV was generated based on sequence data of a human patient strain. • Comparison with HEV-GT3 and rat-derived ratHEV replication in different cell lines. • All strains replicated efficiently in the human hepatoma cell line HuH-7-Lunet BLR. • The results underline the zoonotic potential of ratHEV. • The genomic clone can be used for further investigation on the emerging ratHEV. The hepatitis E virus (HEV) can cause acute and chronic hepatitis in humans. Whereas HEV genotypes 1–4 of species Paslahepevirus balayani are commonly found in humans, infections with ratHEV (species Rocahepevirus ratti) were previously considered to be restricted to rats. However, several cases of human ratHEV infections have been described recently. To investigate the zoonotic potential of this virus, a genomic clone was constructed here based on sequence data of ratHEV strain pt2, originally identified in a human patient with acute hepatitis from Hongkong. For comparison, genomic clones of ratHEV strain R63 from a rat and of HEV genotype 3 strain 47832mc from a human patient were used. After transfection of in vitro -transcribed RNA from the genomic clones into the human hepatoma cell line HuH-7-Lunet BLR, virus replication was shown for all strains by increasing genome copy numbers in cell culture supernatants. These cells developed persistent virus infections, and virus particles in the culture supernatant as well as viral antigen within the cells were demonstrated. All three generated virus strains successfully infected fresh HuH-7-Lunet BLR cells. In contrast, the human hepatoma cell lines HuH-7 and PLC/PRF/5 could only be infected with the genotype 3 strain and to a lesser extent with ratHEV strain R63. Infection of the rat-derived hepatoma cell lines clone 9, MH1C1 and H-4-II-E did not result in efficient virus replication for either strain. The results indicate that ratHEV strains from rats and humans can infect human hepatoma cells. The replication efficiency is strongly dependent on the cell line and virus strain. The investigated rat hepatoma cell lines could not be infected and other rat-derived cells should be tested in future to identify permissive cell lines from rats. The developed genomic clone can represent a useful tool for future research investigating pathogenicity and zoonotic potential of ratHEV. [ABSTRACT FROM AUTHOR]

Background: NOS2 expression is mostly found in bacteria-exposed or cytokine-treated tissues and is mostly connected to innate immune reactions. There are three isoforms of NOS2 (NOS2-1 to -3). In RNA-seq data sets, analyzing inflammatory gene expression, only expression of the NOS2-1 mRNA isoform is detected. However, the expression of NOS2 in differentiating human pluripotent stems (hPSCs) has not been analyzed yet. Methods: Public available RNA-seq databases were screened for data of hPSCs during differentiation to different target cells. An isoform specific algorithm was used to analyze NOS2 mRNA isoform expression. In addition, we differentiated four different human iPSC cell lines toward cortical neurons and analyzed NOS2 mRNA expression by qRT-PCR and 5′-RACE. The functionality of the NOS2-2 protein was analyzed by transient transfection of expression clones in human DLD1 cells and nitrate measurement in the supernatant of these cells. Results: In RNA-seq databases we detected a transient expression of the NOS2 mRNA during the differentiation of hPSCs to cardiomyocytes, chondrocytes, mesenchymal stromal cells, neurons, syncytiotrophoblast cells, and trophoblasts. NOS2 mRNA isoform specific analyses showed, that the transiently expressed NOS2 mRNA in differentiating hPSC (NOS2-2; "diff-iNOS") differ remarkably from the already described NOS2 transcript found in colon or induced islets (NOS2-1; "immuno-iNOS"). Also, analysis of the NOS2 mRNA- and protein expression during the differentiation of four different hiPSC lines towards cortical neurons showed a transient expression of the NOS2 mRNA and NOS2 protein on day 18 of the differentiation course. 5′-RACE experiments and isoform specific qRT-PCR analyses revealed that only the NOS2-2 mRNA isoform was expressed in these experiments. To analyze the functionality of the NOS2-2 protein, we transfected human DLD-1 cells with tetracycline inducible expression clones encoding the NOS2-1- or -2 coding sequence. After induction of the NOS2-1 or -2 mRNA expression by tetracycline a similar nitrate production was measured proofing the functionality of the NOS2-2 protein isoform. Conclusions: Our data show that a differentiation specific NOS2 isoform (NOS2-2) is transiently expressed during differentiation of hPSC. 1uj3Epc_eyJHiftngx9Qfc Video Abstract [ABSTRACT FROM AUTHOR]

Introduction: The role of wildlife in the transmission of antimicrobial resistant (AMR) is suspected but scarcely reported in current studies. Therefore, we studied the dynamics and prevalence of antibiotic-resistant Enterobacterales in antibiotic-limited areas of Senegal. Materials and Methods: We collected fecal samples from monkeys and apes (N = 226) and non-fecal environmental samples (N = 113) from Senegal in 2015 and 2019. We grew the samples on selective media, subsequently isolated AMR Enterobacterales , and then sequenced their genomes. Results: We isolated 72 different Enterobacterales among which we obtained a resistance rate of 65% for colistin (N = 47/72) and 29% for third generation-cephalosporin (C3G) (29%, N = 21/72). Interestingly, almost 46% of our isolates, among Enterobacter sp., Citrobacter cronae and Klebsiella aerogenes , belong to 34 new STs. Moreover, the genes bla CTX – M –15, bla TEM 1 B , sul2 , dfrA14 , qnrs , aph (3′′), aph (6), tetA , and tetR harbored within a transposon on the IncY plasmid of ST224 Escherichia coli were transferred and inserted into a ST10 E. coli phage coding region. Conclusion: Wildlife constitutes a rich, unexplored reservoir of natural microbial diversity, AMR genes and international resistant clones pathogenic in humans. The presence of a transposon that carries AMR genes is intriguing since no antibiotics are used in the non-human primates we studied. [ABSTRACT FROM AUTHOR]

MAPK-organizer 1 (MORG1) is a molecular scaffold for prolyl-hydroxylase-3 containing a domain (PHD3) protein linking MORG1 to mechanisms of adaptation in hypoxic conditions. In this paper, we report the cloning of the promoter region of the murine and human MORG1 gene. Among other transcriptional factors binding sites, we identified that both (mouse and human) promoter regions contained several putative hypoxia-inducible factor binding motifs. Analyses of the human MORG1 promoter by reporter assays revealed that hypoxia and pharmacological inhibitors of prolyl-hydroxylases under in vitro conditions in HEK 293 cells differentially regulate the MORG1 promoter reporter activity. The exposure of the cells to 10% hypoxia showed inhibition of MORG1 promotor activity at 6 and 12 h, but stimulation after 24 h while treated with prolyl-hydroxylase inhibitors led to a time-independent MORG1 promoter activation. Mutational analyses of the individual HIF binding sites on human MORG1 promoter suggest that the binding sites work in a complex corporation because single mutations were not sufficient to abolish completely the MORG1 reporter activation by PHD inhibitors. Our data provide the first evidence that not only MORG1 regulate HIF stabilization through a PHD complex, but also that, vice versa, HIFs control MORG1 expression directly or indirectly by a complex regulatory mechanism. [ABSTRACT FROM AUTHOR]

Background: Human cloning is a recent occurrence that is not confined to bio-issues; rather, it has provoked numerousquestions worldwide and presented scientific and religious challenges. These series of articles aim to examine theproposed approaches and analyze the aspects of human cloning in terms of tenets, morals, jurisprudence, and laws. Inthis paper, we analyze the ideological and theological evidences, regardless of scientific, ethical and legal problemsthat exist in the reproduction method.Materials and Methods: We used a descriptive-analytical method to consider the challenges of human cloning accordingto the “system of Divine creativity” and “the will of God”, as well as the “pairing system” and “diversity innature” with emphasis on the Holy Qur’an and Qur’anic commentaries.Results: According to the Qur’an, although any type of physical changes and retouching of the human body are forbidden,the alteration of God's creation may not prove the prohibition of cloning. Cloning is not contradictory to theprinciple, precedent, and rule of coupling, and it may be one of the hidden precedents of creation. Therefore, not onlydoes a clone not contradict the precedent of the variety of men, but this variety is a sign for men and not a precedentpredominated over the order of nature.Conclusion: It is proven that cloning does not give life; instead, it utilizes the life bestowed by God. This techniquedoes not contradict the precedents of existence. It is a way to discover some precedents of God and is under the orderof cause and effect of the world. Cloning is not considered as a challenge to human beliefs, nor is it a change in Divinecreation. Moreover, cloning does not contradict the theological teachings and concepts of the Holy Qur’an and ShiiteMuslims.

The relevance of the research is due to the invariance of approaches to the legal regulation of public relations in the field of human cloning, the need for semantic differentiation of the concept of cloning, which ensures formation of an adequate attitude of society and the state to human cloning and development of an optimal model of legal regulation of public relations in this area. The purpose of the research is to select and justify a promising model of legal regulation of public relations in the field of human cloning in modern Russia. Achieving this goal calls for analysis of international legal acts and the system of legislation of foreign countries, as well as approaches to regulating public relations that have been formed in legal doctrine and medical practice. Based on the obtained empirical data, the author conducts a General theoretical characterization of the type of legal regulation, suggest recommendations for improving the legal regime, and substantiates the prospects of the proposed model of legal regulation of public relations in the field of human cloning. The level of philosophical methodology in the study is represented by the General principles of knowledge and categorical structure of science as a whole. The level of General scientific principles and forms of research is characterized by applying the systemic analysis techniques, the method of ascent from the abstract to the concrete, as well as General logical methods: analysis, synthesis, abstraction, and analogy. At the level of concrete scientific methodology, this study employs the comparative method and the special legal method.