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SARS-CoV-2 spike requires proteolytic processing for viral entry. A polybasic furin-cleavage site (FCS) in spike, and evolution toward an optimized FCS by dominant variants of concern (VOCs), are linked to enhanced infectivity and transmission. Here we show interferon-inducible restriction factors Guanylate-binding proteins (GBP) 2 and 5 interfere with furin-mediated spike cleavage and inhibit the infectivity of early-lineage isolates Wuhan-Hu-1 and VIC. By contrast, VOCs Alpha and Delta escape restriction by GBP2/5 that we map to the spike substitution D614G present in these VOCs. Despite inhibition of spike cleavage, these viruses remained sensitive to plasma membrane IFITM1, but not endosomal IFITM2 and 3, consistent with a preference for TMPRSS2-dependent plasma membrane entry. Strikingly, we find that Omicron is unique among VOCs, being sensitive to restriction factors GBP2/5, and also IFITM1, 2, and 3. Using chimeric spike mutants, we map the Omicron phenotype and show that the S1 domain determines Omicron’s sensitivity to GBP2/5, whereas the S2’ domain determines its sensitivity to endosomal IFITM2/3 and preferential use of TMPRSS2- independent entry. We propose that evolution of SARS-CoV-2 for the D614G substitution has allowed for escape from GBP restriction factors, but the selective pressures on Omicron for spike changes that mediate antibody escape, and altered tropism, have come at the expense of increased sensitivity to innate immune restriction factors that target virus entry.

Variants in genes encoding for voltage-gated K + (Kv) channels are frequent cause of drug-resistant pediatric epilepsies. Obtaining a molecular diagnosis gives the opportunity to assess the efficacy of pharmacological strategies based on in vitro features of mutant channels. In this retrospective observational study, we selected patients with drug-resistant pediatric epilepsies caused by variants in potassium channel encoding genes, followed at the Fondazione IRCCS Istituto Neurologico Carlo Besta of Milan, Italy. After the experimental characterization of variants’ functional properties in transiently transfected Chinese Hamster Ovary (CHO) cells, we identified drugs to be used as pharmacological approaches. We recruited six patients carrying different missense variants in four Kv channels (Kv7.2, Kv7.3, Kv3.1, and K Na 1.1). In vitro experiments demonstrated that variants in Kv7 channels induced loss-of-function (LoF) effects, while those affecting Kv3.1 or K Na 1.1 led to gain-of-function (GoF). Moreover, we found that the Kv7 channels activator gabapentin was able to revert the LoF effects caused by Kv7.2/Kv7.3 variants, and the potassium channel-blocker fluoxetine counteracted the GoF effects in Kv3.1 or K Na 1.1 variants. According to experimental data, patients carrying Kv7 variants were treated with gabapentin. While this treatment resulted successful in two patients (#1, Kv7.2 G310S variant; #3, Kv7.3 V359L + Kv7.3 D542N), it resulted detrimental in the remaining case (#2, Kv7.2 D535E), requiring drug withdrawal. The application in vivo of fluoxetine to counteract GoF effects induced by Kv3.1 or K Na 1.1 variants determined a significant reduction of both seizure frequency and behavior disturbances in patient #4 (Kv3.1 V425M), and in both subjects carrying K Na 1.1 variants (#5, S937G and #6, R262Q). However, for the latter case, this drug was halted due to severe behavioral side effects. For most of the patients herein reported, pharmacological strategies, selected according to the in vitro functional properties of Kv-channels pathogenic variants, resulted in a significant improvement of both epileptic and cognitive features.

Background Guanylate-binding proteins (GBPs) belong to the large guanosine triphosphatases (GTPases) family and have specialised in host defence in vivo against a broad spectrum of invading pathogens. This ancient evolutionary group of genes was first studied in humans and rodents, but its evolution remained largely unknown for nearly 20 years. In recent years, more studies have emerged deepening the knowledge of GBP evolution in specific mammalian groups: Primates, Tupaia , Muroids (Rodents), Bats and Lagomorphs. Results Here, we aimed to present a comprehensive analysis of mammals GBP evolution. Our phylogenetic analysis demonstrates that mammals’ GBPs share a common ancestor and that each major mammalian group has evolved a specific GBP repertoire. Two Monotreme GBP groups, GBP8 and GBP9 , cluster independently in the phylogenetic tree and do not share the synteny of the other mammalian GBP genes. The other two Monotreme GBP groups, GBP1/2/3/5 and GBP4/6/7 , are at the basal position of the main mammalian groups. Marsupials have two GBP groups, Marsupial GBP1/2/3/5 , basal to Placental GBP1/2/3/5 , and Marsupial GBP4/6/7 , basal to Placental GBP4/6/7 . Marsupial GBP1/2/3/5 can be subdivided into three sub-groups, similarly to what is observed in the Placental GBPs, whereas Marsupial GBP4/6/7 underwent several duplication events across species. We also examined the GBP tissue expression pattern in Monodelphis domestica and found that GBPs are ubiquitously expressed in most tissues, with some differences. Noteworthy was the presence of GBP transcripts in late foetal and newborn opossum tissues. Conclusions The GBP genes revealed a distinct evolutionary pattern in each main mammalian group. Phylogenetic analysis shows that Monotremes and Marsupials have specific GBPs. Particularly intriguing is the presence of GBP8 and GBP9 only in Monotremes.

Abstract Guanylate-Binding Proteins are interferon-inducible GTPases that play a key role in cell autonomous responses against intracellular pathogens. Despite sharing high sequence similarity, subtle differences among GBPs translate into functional divergences that are still largely not understood. A key GBP feature is the formation of supramolecular GBP complexes on the bacterial surface. Such complexes are observed when GBP1 binds lipopolysaccharide (LPS) from Shigella and Salmonella and further recruits GBP2-4. Here, we compared GBP recruitment on two cytosol-dwelling pathogens, Francisella novicida and S. flexneri. Francisella novicida was coated by GBP1 and GBP2 and to a lower extent by GBP4 in human macrophages. Contrary to S. flexneri, F. novicida was not targeted by GBP3, a feature independent of T6SS effectors. Multiple GBP1 features were required to promote targeting to F. novicida while GBP1 targeting to S. flexneri was much more permissive to GBP1 mutagenesis suggesting that GBP1 has multiple domains that cooperate to recognize F. novicida atypical LPS. Altogether our results indicate that the repertoire of GBPs recruited onto specific bacteria is dictated by GBP-specific features and by specific bacterial factors that remain to be identified. Human Guanylate Binding Proteins target the cytosolic pathogen Francisella novicida in a different way as other cytosolic bacteria such as Shigella flexneri.

Toxoplasma gondii ( T. gondii ) virulence in mice depends on different multiprotein complexes that assemble at the parasitophorous vacuole membrane (PVM) of the parasite. Individual rhoptry proteins within these complexes inhibit different Immunity-Related GTPases (IRG proteins). The rhoptry pseudokinase ROP5 is a central element to achieve IRG-specific rhoptry kinase activity and/or efficient complex formation. The rop5 locus of each of the canonical T. gondii strains encodes three major isoforms, ROP5A, ROP5B and ROP5C, and was shown to have the largest impact on virulence. By reverse genetics, we have generated T. gondii strains expressing either ROP5A, ROP5B or ROP5C in a RHΔ rop5 genetic background and demonstrate that ROP5B is mainly responsible for heightened virulence of type I T. gondii in laboratory strains of mice. In vivo virulence correlates with diminished vacuolar IRG protein loading and parasite control in vitro only in presence of ROP5B but not ROP5A or ROP5C. Our results suggest that ROP5A and ROP5C isoforms might have co-evolved with IRG proteins or other host cell resistance factors in evolutionarily important intermediate hosts beyond Mus musculus . The same parasite effectors that inhibit IRG protein accumulation and function reduce the vacuolar amount of Guanylate Binding Proteins (GBP proteins). However, a parasite effector targeting a GBP protein at the PVM has not been described yet. Using two different approaches, Yeast Two-Hybrid analysis and Protein-fragment complementation assay, we here identified three heterologous IRG:GBP pairs, GBP6:Irgb10, GBP5:Irgb10, GBP5:Irgb6, and demonstrate that the accumulation of these GTPases at the PVM is interdependent. Our results offer a novel perspective on the IRG and GBP protein-mediated control of T. gondii infections and may further advance the investigation of GBP-specific T. gondii effectors.

With the continuous promotion and popularity of the digitalization of big data information, it has gradually penetrated various industries and played an increasingly important role in people’s lives. In this paper, the design of a literary poetry appreciation system is studied using a perceptron model, focusing on the construction of a literary poetry appreciation system through a generalized multi-layer perceptron model structure and the evaluation of metrics on the poetry appreciation system dataset using the GBP training algorithm and genetic training method. In the generalized multilevel perceptron model, the data indicators are classified into four categories: poetry music, poetry sound effects, poetry vocals, and other types, and the accuracy rates of the four categories of indicators are analyzed. The analysis shows that the accuracy rate of all four categories of indicators is between 92% and 97%, indicating the feasibility of the poetry appreciation system. The generalized multilevel perceptron model was used to analyze and evaluate the students from the ability perspective. It can be seen that the students’ data showed only a small and insignificant improvement in the dimensions of literacy, comprehension, and analysis, while the scores in the dimensions of reasoning, strategy, and expression improved significantly. It provides a good research perspective for perceptual machine model-assisted teaching and cultivating students’ quality from a competence perspective, which is of historical importance for developing and transmitting Chinese literature.

The recent spread of the monkeypox virus among humans has heightened concerns regarding orthopoxvirus infections. Consequently, conducting a comprehensive study on the immunobiology of the monkeypox virus is imperative for the development of effective therapeutics. Ectromelia virus (ECTV) closely resembles the genetic and disease characteristics of monkeypox virus, making it a valuable research tool for studying orthopoxvirus–host interactions. Guanylate-binding proteins (GBPs), highly expressed interferon-stimulated genes (ISGs), have antagonistic effects against various intracellular pathogenic microorganisms. Our previous research has shown that GBP2 has a mild but statistically significant inhibitory effect on ECTV infection. The presence of a significant number of molecules in the poxvirus genome that encode the host immune response raises questions about whether it also includes proteins that counteract the antiviral activity of GBP2. Using IP/MS and co-IP technology, we discovered that the poly(A) polymerase catalytic subunit (PAPL) protein of ECTV is a viral regulatory molecule that interacts with GBP2. Further studies have shown that PAPL antagonizes the antiviral activity of GBP2 by reducing its protein levels. Knocking out the PAPL gene of ECTV with the CRISPR/Cas9 system significantly diminishes the replication ability of the virus, indicating the indispensable role of PAPL in the replication process of ECTV. In conclusion, our study presents preliminary evidence supporting the significance of PAPL as a virulence factor that can interact with GBP2.

Guanylate binding proteins (GBPs) are paramount in the host immunity by providing defense against invading pathogens. Multigene families related to the immune system usually show that the duplicated genes can either undergo deletion, gain new functions, or become non-functional. Here, we show that in muroids, the Gbp genes followed an unusual pattern of gain and loss of genes. Muroids present a high diversity and plasticity regarding Gbp synteny, with most species presenting two Gbp gene clusters. The phylogenetic analyses revealed seven different Gbps groups. Three of them clustered with GBP2 , GBP5 and GBP6 of primates. Four new Gbp genes that appear to be exclusive to muroids were identified as Gbpa , b , c and d . A duplication event occurred in the Gbpa group in the common ancestor of Muridae and Cricetidae (~20 Mya), but both copies were deleted from the genome of Mus musculus , M. caroli and Cricetulus griseus . The Gbpb gene emerged in the ancestor of Muridae and Cricetidae and evolved independently originating Gbpb1 in Muridae, Gbpb2 and Gbpb3 in Cricetidae. Since Gbpc appears only in three species, we hypothesize that it was present in the common ancestor and deleted from most muroid genomes. The second Gbp gene cluster, Gbp6 , is widespread across all muroids, indicating that this cluster emerged before the Muridae and Cricetidae radiation. An expansion of Gbp6 occurred in M. musculus and M. caroli probably to compensate the loss of Gbpa and b . Gbpd is divided in three groups and is present in most muroids suggesting that a duplication event occurred in the common ancestor of Muridae and Cricetidae. However, in Grammomys surdaster and Mus caroli, Gbpd2 is absent, and in Arvicanthis niloticus, Gbpd1 appears to have been deleted. Our results further demonstrated that primate GBP1 , GBP3 and GBP7 are absent from the genome of muroids and showed that the Gbp gene annotations in muroids were incorrect. We propose a new classification based on the phylogenetic analyses and the divergence between the groups. Extrapolations to humans based on functional studies of muroid Gbps should be re-evaluated. The evolutionary analyses of muroid Gbp genes provided new insights about the evolution and function of these genes.

Gabapentin (GBP) is widely prescribed to older patients for pain management. Recent clinical studies highlight that GBP adversely affect cognitive function in older patients. GBP binds to the α2δ1 subunit of L-type voltage-gated Ca channels to inhibit Ca channel current. It is being increasingly recognized that GBP affects neuronal activity in multifaceted ways. However, the molecular mechanism underlying GBP's impact on cognitive function in older subjects remains unelucidated. Aged mice (18-month-old, female) were subjected to spared nerve injury (SNI) or sham surgery and treated with GBP for 60 days. Learning and memory were assessed using novel object recognition (NOR) test and contextual and cued fear conditioning test (FCT). Adeno-associated viral vector (AAV) was used for gene overexpression in the brain. Brain tissue was analyzed by Western blot, qRT-PCR, and protein activity assay. Long-term GBP treatment impaired learning and memory in aged mice with or without nerve injury-induced pain as GBP-treated aged mice had lower novel object recognition index in NOR test and shorter freezing time in FCT, respectively. In the hippocampus of GBP-treated mice, increased levels of p-tau (S416) and p-tau (S262) were observed, together with increased CaMKIIα and decreased Sirt1 expression. AAV-mediated Sirt1 overexpression in the hippocampus or systemic administration of the Sirt1 activator resveratrol prevented cognitive impairment and tau hyperphosphorylation via enhancing Sirt1 activity in GBP-treated mice. Long-term GBP treatment is detrimental to cognitive function in aged mice. GBP suppressed Sirt1 expression, leading to elevated CaMKIIα level and hyperphosphorylation of tau, and boosting Sirt1 activity curbed the adverse effect of GBP on memory in aged mice.

Objective: Previous studies have shown that gabapentin or pregabalin use is associated with cognitive decline. Herein, we aimed to evaluate the association between gabapentin or pregabalin use and the risk of dementia. Methods: In this retrospective, population-based matched cohort study, all research data were collected from the 2005 Longitudinal Health Insurance Database, which contains data of 2 million people randomly selected from the National Health Insurance Research Database of Taiwan in 2005. The study extracted data from 1 January 2000, to 31 December 2017. Adult patients taking gabapentin or pregabalin were included in the exposure group, and patients not using gabapentin or pregabalin matched to exposure subjects in a 1:5 ratio by propensity scores composed of age, sex and index date were included in the non-exposure group. Results: A total of 206,802 patients were enrolled in the study. Of them, 34,467 gabapentin- or pregabalin-exposure and 172,335 non-exposure patients were used for analysis. The mean follow-up day (±standard deviation) after the index date was 1724.76 (±1282.32) and 1881.45 (±1303.69) in the exposure and non-exposure groups, respectively; the incidence rates of dementia were 980.60 and 605.48 per 100,000 person-years, respectively. The multivariate-adjusted hazard ratio of risk of dementia for gabapentin or pregabalin exposure versus the matched non-exposed group was 1.45 (95% confidence interval [CI], 1.36–1.55). The risk of dementia increased with higher cumulative defined daily doses during the follow-up period. Moreover, the stratification analysis revealed that the risk of dementia associated with gabapentin or pregabalin exposure was significant in all age subgroups; however, it was higher in younger patients (age <50) than in the older patients (hazard ratio, 3.16; 95% CI, 2.23–4.47). Conclusion: Patients treated with gabapentin or pregabalin had an increased risk of dementia. Therefore, these drugs should be used with caution, particularly in susceptible individuals.