CryoET shows cofilactin filaments inside the microtubule lumen.

Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identities of these objects and mechanisms for their accumulation have not been conclusively established. Here, we used cryogenic electron tomography of Drosophila S2 cell protrusions and found filaments inside the microtubule lumen, which resemble those reported recently in human HAP1 cells. The frequency of these filaments increased upon inhibition of the sarco/endoplasmic reticulum Ca2+ ATPase with the small molecule drug thapsigargin. Subtomogram averaging showed that the luminal filaments adopt a helical structure reminiscent of cofilin-bound actin (cofilactin). Consistent with this, we observed cofilin dephosphorylation, an activating modification, in cells under the same conditions that increased luminal filament occurrence. Furthermore, RNA interference knock-down of cofilin reduced the frequency of luminal filaments with cofilactin morphology. These results suggest that cofilin activation stimulates its accumulation on actin filaments inside the microtubule lumen.

Differential Effects of Cerebellar Transcranial Direct Current Stimulation with Gait Training on Functional Mobility, Balance, and Ataxia Symptoms.

Cerebellar transcranial direct current stimulation (ctDCS) has emerged as a promising, non-invasive, and safe neuromodulatory intervention capable of reducing ataxia symptoms and restoring cerebellum-motor connectivity. However, previous studies have only applied ctDCS in isolation, without association with specific training. This study aimed to assess the effect of ctDCS combined with gait training on functional mobility, balance, and symptoms and severity of ataxia. A randomized, triple-blind, sham-controlled, bi-center clinical trial was conducted with forty-four adults with cerebellar ataxia. Volunteers were randomized to receive five daily sessions of either real ctDCS (n = 11; 2 mA for 25 min) or sham ctDCS (n = 11) during gait training. Functional mobility, balance, and symptoms and severity of ataxia were assessed using the Time Up and Go test, the MiniBESTest, and the Scale for the Assessment and Rating of Ataxia (SARA), respectively, before and after the interventions. Both groups showed improvement in functional mobility, but there was no significant difference between the ctDCS and sham groups. However, the ctDCS group demonstrated significant improvements in cerebellar ataxia severity as reflected by SARA scores, particularly in tests of stance, sitting, speech disturbance, nose-finger test, and heel-shin slide test. Notably, no improvements were observed in balance. This study indicates that while ctDCS combined with gait training may improve specific symptoms of cerebellar ataxia, it does not significantly enhance overall functional mobility compared to sham treatment.

COVID-19 recurrence is related to disease-early profile T cells while detection of anti-S1 IgG is related to multifunctional T cells.

COVID-19 is caused by SARS-CoV-2 infection and leads from asymptomatic to severe outcomes. The recurrence of the COVID-19 has been described, however, mechanisms involved remains unclear. Thus, the work aimed to investigate the role of multifunctional T cells in patients with recurrent COVID-19. We evaluated clinical characteristics, presence of anti-S1 and anti-Nucleocapsid IgG in patients' sera, and multifunctional T cells (for IFN-γ, IL-2, and TNF-α) in patients with multiple episodes of COVID-19 and controls. Data demonstrate that patients with recurrent COVID-19 have a T cell pattern predominantly related to IFN-γ production. Also, patients with COVID-19 history and absence of anti-S1 IgG had lower levels of CD4+ IFN + IL-2 + TNF + T cells independently of number of disease episodes. Complementary, vaccination changed the patterns of T cells phenotypes and induced IgG seroconversion, despite not induce higher levels of multifunctional T cells in all patients. In conclusion, the data suggest that recurrent disease is related to early-disease T cell profile and absence of anti-S1 IgG is related to lower multifunctional CD4 T cell response, what suggests possibility of new episodes of COVID-19 in these patients.

Role of the YehD fimbriae in the virulence-associated properties of enteroaggregative Escherichia coli.

The interaction of enteroaggregative Escherichia coli (EAEC) strains with the colonic gut mucosa is characterized by the ability of the bacteria to form robust biofilms, to bind mucin, and induce a local inflammatory response. These events are mediated by a repertoire of five different aggregative adherence fimbriae variants (AAF/I-V) typically encoded on virulence plasmids. In this study, we report the production in EAEC strains of a new YehD fimbriae (YDF), which is encoded by the chromosomal gene cluster yehABCD, also present in most E. coli strains. Immuno-labelling of EAEC strain 042 with anti-AAF/II and anti-YDF antibodies demonstrated the presence of both AAF/II and YDF on the bacterial surface. We investigated the role of YDF in cell adherence, biofilm formation, colonization of spinach leaves, and induction of pro-inflammatory cytokines release. To this aim, we constructed yehD deletion mutants in different EAEC backgrounds (strains 17-2, 042, 55989, C1010, 278-1, J7) each harbouring one of the five AAFs. The effect of the YDF mutation was strain dependent and AAF independent as the lack of YDF had a different impact on the phenotypes manifested by the different EAECs tested. Expression of the yehABCD operon in a E. coli K12 ORN172 showed that YDF is important for biofilm formation but not for adherence to HeLa cells. Lastly, screening of pro-inflammatory cytokines in supernatants of Caco-2 cells infected with EAEC strains 042 and J7 and their isogenic ΔyehD mutants showed that these mutants were significantly defective in release of IL-8 and TNF-α. This study contributes to the understanding of the complex and diverse mechanisms of adherence of EAEC strains and identifies a new potential target for preventive measures of gastrointestinal illness caused by EAEC and other E. coli pathogroups.

Antimony resistance associated with persistence of Leishmania (Leishmania) infantum infection in macrophages.

Visceral leishmaniasis is a severe disease caused by protozoan parasites that include Leishmania (L.) infantum. The disease is established when parasites subvert the immune response of the host. Notably, chemotherapy-based use of antimonial compounds can partially alleviate disease burden. Unfortunately, the resistance to drug treatments is increasing in areas endemic to the disease. In this report, we investigated immune responses within macrophages infected with antimony-resistant L. infantum isolates from patients with a relapse in the disease. Results revealed that antimony-resistant parasites persist in the first 24 h of infection. Activation of macrophage or blocking of thiol production during infection shows enhanced clearance of parasites, which is coordinately associated with increased production of pro-inflammatory cytokines. Taken together, these results suggest that the mechanism of antimony resistance in L. infantum isolates may be related to a decrease in macrophage microbicidal functions.

Association Between Zika Virus Microcephaly in Newborns With the rs3775291 Variant in Toll-Like Receptor 3 and rs1799964 Variant at Tumor Necrosis Factor-α Gene.

Congenital Zika syndrome (CZS) is a cluster of malformation, and the mechanisms that lead it are still unclear. Using hypothesis-driven candidate genes and their function in viral infections, single-nucleotide polymorphisms (SNPs) were genotyped by quantitative polymerase chain reaction in a sample population from Sergipe State, Brazil. This study shows that rs3775291 SNP at Toll-like receptor 3, which triggers type I interferon antiviral responses in mothers infected by Zika virus during pregnancy, is associated with CZS occurrence (odds ratio [OR], 2.19; 95% confidence interval [CI], 1.158-4.148). Moreover, rs1799964 SNP at tumor necrosis factor-α gene in CZS babies is associated with severe microcephaly (OR, 2.63; 95% CI, 1.13-6.21).

Antimicrobial activity of Melaleuca alternifolia nanoparticles in polymicrobial biofilm in situ.

Microbial biofilms represent a challenge in the treatment of infections, due to the low efficacy of the antimicrobials. This study evaluated the antimicrobial effect of nanoparticles of Melaleuca alternifolia (TTO) in dental biofilm. Thirty-eight volunteers used an oral device in situ in situ including four bovine enamel specimens for 07 days. From the fifth day four solutions were applied randomly for each specimen: Physiological Saline Solution (0.85% NaCl) (C+), Chlorhexidine 0.12% (CHX), M. alternifolia oil 0.3% (TTO), and a nanoparticle solution of 0.3% M. alternifolia oil (NPTTO). The nanoparticles of TTO were characterized for pH, IPD, medium size, zeta potential and Transmission Electron Microscopy. Antimicrobial activity was evaluated by viable microorganisms count and the structure of the biofilm by atomic force microscopy. The NPTTO presented pH 6.4, particle diameter of 197.9 ± 1 nm, polydispersion index of 0.242 ± 0.005, zeta potential of -7.12 mV and ±0:27 spherical shape. The C+ resulted in 100% of bacterial vitality, while CHX, TTO and NPTTO showed 34.2%, 51.4% and 25.8%, respectively. The AFM images showed biofilms with an average roughness of 350 nm for C+, 275 nm for CHX, 500 nm for TTO and 100 nm for NPTTO. The NPTTO demonstrated excellent antimicrobial activity in the biofilm formed in situ and will possibly be used in future for the treatment/prevention of oral biofilms.

Debugging Eukaryotic Genetic Code Expansion for Site-Specific Click-PAINT Super-Resolution Microscopy.

Super-resolution microscopy (SRM) greatly benefits from the ability to install small photostable fluorescent labels into proteins. Genetic code expansion (GCE) technology addresses this demand, allowing the introduction of small labeling sites, in the form of uniquely reactive noncanonical amino acids (ncAAs), at any residue in a target protein. However, low incorporation efficiency of ncAAs and high background fluorescence limit its current SRM applications. Redirecting the subcellular localization of the pyrrolysine-based GCE system for click chemistry, combined with DNA-PAINT microscopy, enables the visualization of even low-abundance proteins inside mammalian cells. This approach links a versatile, biocompatible, and potentially unbleachable labeling method with residue-specific precision. Moreover, our reengineered GCE system eliminates untargeted background fluorescence and substantially boosts the expression yield, which is of general interest for enhanced protein engineering in eukaryotes using GCE.

Sensitivity and specificity of the EAT-10 and SDQ-DP in identifying the risk of dysphagia in Parkinson's disease.

BACKGROUND: The early identification of risk for dysphagia in patients with Parkinson's disease (PD) is essential for the prevention of nutritional and pulmonary complications. OBJECTIVE: To analyze the sensitivity and specificity of the Swallowing Disturbance Questionnaire (SDQ-PD) and the Eating Assessment Tool (EAT-10) in identifying dysphagia risk in patients with early and intermediate stages of PD. METHODS: Twenty-nine patients with PD participated in the study. EAT-10 and SDQ-PD questionnaires were applied, and a videofluoroscopic swallowing study. Dysphagia Outcome and Severity Scale (DOSS) was used to classify the presence and severity of dysphagia, and the Penetration-Aspiration Scale (PAS) was used to identify the presence of penetration/aspiration. In the statistical analysis, the sensitivity and specificity of the risk questionnaires were calculated, as well as positive predictive value, negative predictive value, and accuracy. RESULTS: EAT-10 to identify the risk of penetration/aspiration revealed a sensitivity of 71.42% and specificity of 45.45%; in the identification of the presence of dysphagia, the sensitivity was 47.61%, and the specificity was 12.5%. The SDQ-PD questionnaire for risk of penetration/aspiration demonstrated a sensitivity of 28.57%, and a specificity of 68.18%. In terms of identifying the presence of dysphagia, the sensitivity was 20%, while the specificity was 44.44%. CONCLUSION: The SDQ-PD revealed low sensitivity and low specificity to identify the presence of dysphagia and/or penetration/aspiration in patients with early and intermediate stages of PD in this sample. Despite its low specificity, the EAT-10 exhibited good sensitivity in indicating the risk of penetration/aspiration.

ANTECEDENTES: A identificação precoce de risco para disfagia nos pacientes com doença de Parkinson (DP) é fundamental para a prevenção de complicações nutricionais e pulmonares. OBJETIVO: Analisar a sensibilidade e especificidade dos questionários Swallowing Disturbance Questionnaire (SDQ-PD) e Eating Assessment Tool (EAT-10) para a identificação do risco de disfagia em pacientes com DP nos estágios iniciais e intermediários da doença. MéTODOS: Participaram 29 pacientes com DP. Foi realizado a aplicação dos questionários EAT-10 e SDQ-PD e o exame de videofluoroscopia da deglutição. Para a classificação da presença e gravidade da disfagia foi utilizada a escala Dysphagia Outcome and Severity Scale e, para identificação da presença de penetração/aspiração, a escala Penetration-Aspiration Scale (PAS). Na análise estatística, calcularam-se a sensibilidade e a especificidade dos questionários de risco EAT-10 e SDQ-DP e o valor preditivo positivo, o valor preditivo negativo e a acurácia. RESULTADOS: A análise do EAT-10 para identificar o risco de penetração/aspiração revelou sensibilidade de 71.42% e especificidade de 45.45%; para identificar a presença de disfagia, a sensibilidade foi de 47,61% e a especificidade de 12.5%. Em relação ao questionário SDQ-PD, para identificar risco de penetração/aspiração, a sensibilidade foi de 28.57% e a especificidade de 68.18% e, para identificar a presença de disfagia, a sensibilidade foi de 20% e a especificidade de 44.44%. CONCLUSãO: O questionário SDQ-PD revelou baixa sensibilidade e baixa especificidade para identificar presença de disfagia e/ou penetração/aspiração em pacientes com DP em estágios iniciais e intermediários para essa amostra. O EAT-10 revelou boa sensibilidade na indicação de risco de penetração/aspiração, apesar de baixa especificidade.

Assessing Mechanisms of Potential Local Adaptation Through a Seascape Genomic Approach in a Marine Gastropod, Littoraria flava.

Understanding the combined effects of environmental heterogeneity and evolutionary processes on marine populations is a primary goal of seascape genomic approaches. Here, we utilized genomic approaches to identify local adaptation signatures in Littoraria flava, a widely distributed marine gastropod in the tropical West Atlantic population. We also performed molecular evolution analyses to investigate potential selective signals across the genome. After obtaining 6,298 and 16,137 single nucleotide polymorphisms derived from genotyping-by-sequencing and RNA sequencing, respectively, 69 from genotyping-by-sequencing (85 specimens) and four from RNA sequencing (40 specimens) candidate single nucleotide polymorphisms were selected and further evaluated. The correlation analyses support different evolutionary pressures over transcribed and non-transcribed regions. Thus, single nucleotide polymorphisms within transcribed regions could account for the genotypic and possibly phenotypic divergences in periwinkles. Our molecular evolution tests based on synonymous and non-synonymous ratio (kN/kS) showed that genotype divergences containing putative adaptive single nucleotide polymorphisms arose mainly from synonymous and/or UTR substitutions rather than polymorphic proteins. The distribution of genotypes across different localities seems to be influenced by marine currents, pH, and temperature variations, suggesting that these factors may impact the species dispersion. The combination of RNA sequencing and genotyping-by-sequencing derived datasets provides a deeper understanding of the molecular mechanisms underlying selective forces responses on distinct genomic regions and could guide further investigations on seascape genomics for non-model species.

The role of neurotrophin polymorphisms and susceptibility to neural damage in leprosy.

OBJECTIVES: Mycobacterium leprae is able to infect Schwann cells leading to neural damage. Neurotrophins are involved in nervous system plasticity and impact neural integrity during diseases. Investigate the association between single nucleotide polymorphisms in neurotrophin genes and leprosy phenotypes, especially neural damage. DESIGN: We selected single nucleotide polymorphisms in neurotrophins or their receptors genes associated with neural disorders: rs6265 and rs11030099 of brain-derived neurotrophic factor (BDNF), rs6330 of BDNF, rs6332 in NT3 and rs2072446 of P75NTR. The association of genetic frequencies with leprosy phenotypes was investigated in a case-control study. RESULTS: An association of the BDNF single nucleotide polymorphism rs11030099 with the number of affected nerves was demonstrated. The "AA+AC" genotypes were demonstrated to be protective against nerve impairment. However, this variation does not affect BDNF serum levels. BDNF is an important factor for myelination of Schwann cells and polymorphisms in this gene can be associated with leprosy outcome. Moreover, rs11030099 is located in the binding region for micro-RNA (miRNA) 26a that could be involved in control of BDNF expression. We demonstrated different expression levels of this miRNA in polar forms of leprosy. CONCLUSION: Our findings demonstrate for the first time an association between the polymorphism rs11030099 in the BDNF gene and neural commitment in leprosy and may indicate a possible role of miRNA-26a acting synergistically to these genetic variants in neural damage development.

The genome sequence of the giant clam, Tridacna gigas (Linnaeus, 1758).

We present a chromosomal-level genome assembly from an individual Tridacna gigas (the giant clam; Mollusca; Bivalvia; Veneroida; Cardiidae). The genome sequence is 1,175.9 megabases in span. Most of the assembly is scaffolded into 17 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 25.34 kilobases in length. Gene annotation of this assembly on Ensembl identified 18,177 protein coding genes.

The genome sequence of a heart cockle, Fragum fragum (Linnaeus, 1758).

We present a genome assembly from an individual specimen of Fragum fragum (a heart cockle; Mollusca; Bivalvia; Veneroida; Cardiidae). The genome sequence is 1,153.1 megabases in span. Most of the assembly is scaffolded into 19 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 22.36 kilobases in length. Gene annotation of this assembly on Ensembl identified 17,262 protein coding genes.

The genome sequences of the marine diatom Epithemia pelagica strain UHM3201 (Schvarcz, Stancheva & Steward, 2022) and its nitrogen-fixing, endosymbiotic cyanobacterium.

We present the genome assembly of the pennate diatom Epithemia pelagica strain UHM3201 (Ochrophyta; Bacillariophyceae; Rhopalodiales; Rhopalodiaceae) and that of its cyanobacterial endosymbiont (Chroococcales: Aphanothecaceae). The genome sequence of the diatom is 60.3 megabases in span, and the cyanobacterial genome has a length of 2.48 megabases. Most of the diatom nuclear genome assembly is scaffolded into 15 chromosomal pseudomolecules. The organelle genomes have also been assembled, with the mitochondrial genome 40.08 kilobases and the plastid genome 130.75 kilobases in length. A number of other prokaryote MAGs were also assembled.

CryoET shows cofilactin filaments inside the microtubule lumen.

Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identity of these objects and what causes their accumulation has not been conclusively established. Here, we used cryogenic electron tomography (cryoET) of Drosophila S2 cell protrusions and found filaments inside the microtubule lumen, which resemble those reported recently in human HAP1 cells. The frequency of these filaments increased upon inhibition of the sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) with the small-molecule drug thapsigargin. Subtomogram averaging showed that the luminal filaments adopt a helical structure reminiscent of cofilin-bound actin (cofilactin). Consistent with this, cofilin was activated in cells under the same conditions that increased luminal filament occurrence. Furthermore, RNAi knock-down of cofilin reduced the frequency of luminal filaments with cofilactin morphology. These results suggest that cofilin activation stimulates its accumulation on actin filaments inside the microtubule lumen.

Industry Perspective on Temperature Cycling Studies to Meet Regulatory Temperature Excursion Support Requirements: Survey Outcome and Recommendations.

Temperature cycling stability studies can be appropriately designed and utilized to ensure that drug product quality, efficacy, and safety are not compromised when materials are subjected to short term temperature excursions from intended storage that may occur during e.g., shipping, transport, or patient use. Some countries, such as Australia and Brazil, impose specific regulations that specify the need to conduct stability studies that are supportive of "real world" excursions as part of licensing approval requirements. These temperature cycling stability studies extend beyond what is described in ICH Guidelines Q1A(R2) and Q5C, and companies may be challenged in designing studies that not only satisfy country specific regulations, but also satisfy all global regulatory health authority expectations. This article focuses on responses to a cross-industry survey conducted within the International Consortium for Innovation and Quality (iqconsortium.org) member companies, regarding practices related to temperature cycling stability studies, in order to determine how these requirements are being interpreted and met. The results indicate that while there is no one-size-fits-all approach to performing temperature cycling stability studies, there are common and best practices that can be followed to satisfy global health authority regulatory guidelines and requirements. PURPOSE: The purpose of this paper is to describe the outcome of an industry survey and common/best practices on temperature cycling stability studies performed on drug product (DP) to satisfy the requirements established for marketing authorizations in Australia and Brazil or any other countries that may have similar requirements. The framework is proposed within the context of late phase and commercial development of common biological and/or large molecule modalities, such as monoclonal antibodies (mAbs, including bispecific antibodies), fusion proteins, complex proteins, oligonucleotides, and antibody-drug conjugates (ADCs), but many of the general principles involved may be applied to other therapeutics, such as Virus Like Particles (VLP), gene or cell therapies (GTx or CTx), or vaccines. For the purposes of this paper, temperature cycling stability studies refer to studies that are designed, in part, to support short term temperature excursions that drug product may be subjected to during shipping and storage activities and is outside of the labeled storage condition of the product.

Transcription factor EB regulates phosphatidylinositol-3-phosphate levels that control lysosome positioning in the bladder cancer model.

Lysosomes orchestrate degradation and recycling of exogenous and endogenous material thus controlling cellular homeostasis. Little is known how this organelle changes during cancer. Here we investigate the intracellular landscape of lysosomes in a cellular model of bladder cancer. Employing standardized cell culture on micropatterns we identify a phenotype of peripheral lysosome positioning prevailing in bladder cancer cell lines but not normal urothelium. We show that lysosome positioning is controlled by phosphatidylinositol-3-phosphate (PtdIns3P) levels on endomembranes which recruit FYVE-domain containing proteins for lysosomal dispersion. We identify transcription factor EB (TFEB) as an upstream regulator of PtdIns3P production by VPS34 that is activated in aggressive bladder cancer cells with peripheral lysosomes. This conceptually clarifies the dual role of TFEB as regulator of endosomal maturation and autophagy, two distinct processes controlled by PtdIns3P. Altogether, our findings uncover peripheral lysosome positioning, resulting from PtdIns3P production downstream of TFEB activation, as a potential biomarker for bladder cancer.

From in vivo to in vitro: exploring the key molecular and cellular aspects of human female gametogenesis.

Human oogenesis is a highly complex and not yet fully understood process due to ethical and technological barriers that limit studies in the field. In this context, replicating female gametogenesis in vitro would not only provide a solution for some infertility problems, but also be an excellent study model to better understand the biological mechanisms that determine the formation of the female germline. In this review, we explore the main cellular and molecular aspects involved in human oogenesis and folliculogenesis in vivo, from the specification of primordial germ cells (PGCs) to the formation of the mature oocyte. We also sought to describe the important bidirectional relationship between the germ cell and the follicular somatic cells. Finally, we address the main advances and different methodologies used in the search for obtaining cells of the female germline in vitro.

FHL1 promotes chikungunya and o'nyong-nyong virus infection and pathogenesis with implications for alphavirus vaccine design.

Arthritogenic alphaviruses are positive-strand RNA viruses that cause debilitating musculoskeletal diseases affecting millions worldwide. A recent discovery identified the four-and-a-half-LIM domain protein 1 splice variant A (FHL1A) as a crucial host factor interacting with the hypervariable domain (HVD) of chikungunya virus (CHIKV) nonstructural protein 3 (nsP3). Here, we show that acute and chronic chikungunya disease in humans correlates with elevated levels of FHL1. We generated FHL1-/- mice, which when infected with CHIKV or o'nyong-nyong virus (ONNV) displayed reduced arthritis and myositis, fewer immune infiltrates, and reduced proinflammatory cytokine/chemokine outputs, compared to infected wild-type (WT) mice. Interestingly, disease signs were comparable in FHL1-/- and WT mice infected with arthritogenic alphaviruses Ross River virus (RRV) or Mayaro virus (MAYV). This aligns with pull-down assay data, which showed the ability of CHIKV and ONNV nsP3 to interact with FHL1, while RRV and MAYV nsP3s did not. We engineered a CHIKV mutant unable to bind FHL1 (CHIKV-ΔFHL1), which was avirulent in vivo. Following inoculation with CHIKV-ΔFHL1, mice were protected from disease upon challenge with CHIKV and ONNV, and viraemia was significantly reduced in RRV- and MAYV-challenged mice. Targeting FHL1-binding as an approach to vaccine design could lead to breakthroughs in mitigating alphaviral disease.